uncovering bacterial diversity on and below the surface of
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Louisiana State UniversityLSU Digital Commons
LSU Master's Theses Graduate School
2006
Uncovering bacterial diversity on and below thesurface of a hyper-arid environment, the AtacamaDesert, ChileDanielle Rene BagaleyLouisiana State University and Agricultural and Mechanical College
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Recommended CitationBagaley, Danielle Rene, "Uncovering bacterial diversity on and below the surface of a hyper-arid environment, the Atacama Desert,Chile" (2006). LSU Master's Theses. 3474.https://digitalcommons.lsu.edu/gradschool_theses/3474
UNCOVERING BACTERIAL DIVERSITY ON AND BELOW THE SURFACE OF A HYPER-ARID ENVIRONMENT, THE ATACAMA DESERT, CHILE
A Thesis
Submitted to the Graduate Faculty of the Louisiana State University and
Agricultural and Mechanical College in partial fulfillment of the
requirements for the degree of Master of Science
in
The Department of Biological Sciences
by Danielle Rene Bagaley
B.S., Louisiana State University, 2002 May 2006
ii
DEDICATION
I dedicate this composition to my parents Robbie and Daniel Bagaley. Without their
unconditional love and support this work would never have been possible. I also would like to
dedicate this work to my fellow thesis writing buddy, Jennifer Movassaghi. Her companionship
during this experience helped make this accomplishment more enjoyable.
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ACKNOWLEDGEMENTS
During my Master of Science study at Louisiana State University I have significantly
advanced in skill and knowledge as a result of determination, diligence, and immeasurable
attributions from professors and fellow graduate students. For that reason, I would first like to
give my utmost thanks to my major professor Dr. Fred A. Rainey for all of his patience, advice,
assistance, and support. I would also like to express my gratitude to the other members of my
graduate committee, Dr. Greg Pettis and Dr. Annette S. Engel.
I also am very appreciative of a Ryan P. Callegan, a fellow graduate student, and Brian
A. Rash, a postdoctoral researcher. They have both contributed considerable amounts of their
time and knowledge to questions I had concerning this research. Current and former student
workers of the RaineyLab were very helpful throughout the course of my research. These people
include Monica Morgan, Mallory Durel, Claire Ponson, and Jeff Cutrera. I also thank Abhishek
Satyendranath who created the RaineyLab webpage and the isolate database for this study.
I am very grateful to Dr. Mark A. Batzer and Dr. Randy Garber, a postdoctoral researcher
in the Batzer laboratory, for their aid in the process of 16S rRNA gene sequencing.
I could never have even begun this research if it were not for support from the NASA-
Headquarters (NASA-Ames/LSU Cooperative Agreement NCC 2-5469 and NCC 2-5528) and
the NASA - Ames Research Center, especially Chris Mckay. I also am grateful to those who
helped to collect the numerous soil samples, mainly the subsurface samples, from the Atacama
Desert.
I am thankful for our collaboration with the National Autonomous University of Mexico,
in particularly Dr. Rafael Navarro-González, who preformed the organic analyses for the
Atacama Desert soil samples.
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I appreciate Dr. Bob Gambrell of the Wetland Biogeochemistry Institute within the
Department of Oceanography and Coastal Sciences at Louisiana State University for his
contribution of the Atacama Desert soil elemental analysis. Also, I would like to express my
gratitude to Sarah Jones of the Institute for Ecological Infrastructure Engineering and Water
Quality Laboratory at the Louisiana State University College of Engineering who performed
anion analysis on all Atacama Desert soil samples.
I must thank the Socolofsky Microscopy Center, particularly Cindy Henk, for all of the
hours that she spent assisting me with fluorescence microscopy, scanning electron microscopy,
and many other microscopy related issues.
Lastly, I express thanks to the Department of Biological Sciences and the Office of
Graduate Studies, Priscilla M. Milligan, Dr. Thomas S. Moore, and Chimene Boyd for their time,
efforts, and direction throughout my graduate school career.
v
TABLE OF CONTENTS
DEDICATION………………………………………………………………………….....ii ACKNOWLEDGEMENTS…………………………………………………………….....iii
LIST OF TABLES……………………………………………………………..................vii
LIST OF FIGURES…………………………………………………………………….…viii
LIST OF ABBREVIATIONS………………………………………………………….….x
ABSTRACT…………………………………………………………………………….…xiv
CHAPTER 1 INTRODUCTION……………………………………………………………..1
The Driest Place on Earth, the Atacama Desert of Chile………………….1 Methods for Microbial Community Analysis……………………………...4
2 MATERIALS AND METHODS………………………………………….......14 Samples Collection………………………………………………………...14 Naming of Sites, Samples, and Isolates……………………………………14
Culture Media………………………………………………………….......15 Soil Dilution Plating………………………………………………….........15 Enrichment Cultures…………………………………………………….....16 Plate Counting and Colony Forming Unit Determination………………....16 Isolation of Pure Cultures………………………………………………….17 Preservation………………………………………………………………...17
DNA Extraction…………………………………………………………....18 Polymerase Chain Reaction (PCR) and PCR Purification………………...18 Sequencing, Sequence Purification, and Data Analysis…………………...19 Isolate Database…………………………………………………………....20 Direct Counts Using Fluorescence Microscopy…………………………...20 Determining Limit of Direct Counts on Atacama Desert Soils…..….….....22 Lake Water Sample Preparations for DAPI Staining Control……………..25 DAPI Staining Preparations for Control Soil Sample……………………...26 Determination of Salinity in Atacama Desert Soil Samples……………….27 pH Determination…………………………………………………………..27 Detection of Carbonate Presence in Selected Samples…………………….28 Elemental Assay on Atacama Desert Soils………………………………...28 Inorganic Anion Analysis………………………………………………….29
3 QUANTIFYING AND IDENTIFYING CULTURABLE HETEROTROPHIC BACTERIA IN THE SURFACE SOILS OF THE ATACAMA DESERT…...31
Results……………………………………………………………………...31
vi
Colony Forming Units……………………...……………………...31 Diversity of Culturable Bacteria………………...…………………39
Discussion………………………………………………………………….42 4 HETEROTROPHIC BACTERIA BENEATH THE SURFACE……………...48
Results……………………………………………………………………...48 Colony Forming Units...……………………………………………48
Diversity of Culturable Heterotrophic Bacteria Isolated from the Soil Pits…………………………………………………………......52
Discussion…………………………………………………………………..61
5 DIRECT CELL COUNTS USING FLUORESCENCE MICROSCOPY……..67 Results……………………………………………………………………...67
DAPI Direct Count Detection Limit ………………………………67 DAPI Direct Cell Count Determination for AT04-166…………….75 Sample Sites Having Cell Totals below DAPI Direct Count Detection Limit…………………………………………………….80
Discussion………………………………………………………………….85
6 ASSOCIATION BETWEEN VALUES OF CFUS AND SOIL CHEMICAL COMPOSITION…………………………………………………97
Results…………………………………………………… ………………...97 Elemental Cation Analysis of Soil Samples….……..……...……….97
Inorganic Anion Analysis of Soil Samples………………….……..104 Detection of Carbonates in Atacama Desert Soils…………………109
Discussion………………………………………………………………….110 7 CONCLUSIONS……………………………………………………................112
REFERENCES………………………………………………………………….................118 APPENDIX
A MEDIA INSTRUCTIONS …………………………………………..……….123 B TABLE OF SURFACE AND SOIL PIT ISOLATES ..………………...….....126
C TABLE OF PHOSPHATE, NITRITE, AND BROMIDE CONCENTRATIONS (MG/L)……………………………………………….156 D MEASUREMENTS OF SALINTY, TDS, AND CONDUCTIVITY IN TABLE OF SOLUBLE SALTS WITHIN SOILS…..………………………..157
VITA………………………………………………………………………………………158
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LIST OF TABLES
Table 3.1: Degree minute decimal coordinates for surface sites within the core arid region of Yungay…………………………….……………......33
Table 3.2: Surface sample pH values and CFUs/g of soil on five media after 20
days incubation…………..…………………………………….…..35 Table 3.3: Values of CFU/g recovered on various culture media for the 33 sites
sampled…………………………………………………………….37 Table 4.1: CFUs/g of soil for all four soil pits sampled on five media………………..50
Table 4.2: Values of CFU/g recovered on various culture media for the AT04-159 southern soil pit…………………………………………………….53
Table 5.1: Direct count detection limit trials displaying cell numbers per field,
average cells per field, and known versus calculated cells/g for stock and 1/10 diluted cell suspension after overnight and one month drying experiment……………………….………….………73
Table 5.2: Direct count trials displaying cell numbers per field, average cells per
field, and calculated cells/g for each tested sample site…….……...84 Table 6.1: Cation concentrations (mg/L) for Atacama Desert soils……………...........98 Table 6.2: Major anion concentrations (mg/L) and CFU/g values for Atacama
Desert soils………………………………………………………....105
viii
LIST OF FIGURES
Figure 3.1: Scatter plot illustrating proximity of surface sites in core arid region and distribution of CFUs…………………….…………………….34 Figure 3.2: CFUs/g of soil for each of the 33 surface samples on five media………....36 Figure 3.3: Diversity at the phyla level within surface samples…………………….....40
Figure 3.4: Closest relatives level diversity within surface sites………………….…...41
Figure 3.5: Photomicrographs depicting cell morphology of Blastococcus aggregatus……………………………………………….…….…..45 Figure 4.1: CFUs/g of soil for layers within each soil pit on five media…………..….51 Figure 4.2: Diversity at the phyla and closest relative levels in soil pit AT04-150
and AT04-150B enrichment cultures……………………………...54
Figure 4.3: Diversity at the phyla and closest relative levels in soil pit AT04-152 and AT04-152B enrichment cultures………………….…………..55
Figure 4.4: Diversity at the phyla and closest relative levels in soil pit AT04-153 and AT04-153B enrichment cultures……………………………...56
Figure 4.5: Diversity at the phyla and closest relative levels in soil pit AT04-159 (the Altamira soil pit)……………………………………………...58
Figure 5.1: Photomicrographs of AT04-170 spiked with cultured bacterial cells and dried overnight to determine DAPI direct count detection limit….70
Figure 5.2: Detection limit photomicrographs of AT04-170 spiked with cultured bacterial cells and dried for one month……….…………………..74
Figure 5.3: Photomicrographs from the fluorescence direct count method
employing DAPI in surface site AT04-166……………..………...76 Figure 5.4: Photomicrographs of AT04-165 soils unable to be truly quantified by
DAPI direct counts………………………………………………..81 Figure 5.5: Photomicrographs of AT04-170 soils unable to be quantified by DAPI
direct counts……………………………………………………....86 Figure 5.6: Comparison photomicrographs of cornfield soil sample……………….....94
ix
Figure 5.7: Comparison photomicrographs of LSU lake water sample…..…………...95 Figure 6.1: CFU/g values and cation concentrations for Yungay surface soils….…....102 Figure 6.2: Charts displaying relationship between CFU/g values and cation
concentrations for Atacama Desert soil pits………………………103 Figure 6.3: CFU/g values anion concentrations for Yungay surface soils....……….....107 Figure 6.4: CFU/g values and anion concentrations at depth for Atacama Desert
soil pits……………………………………………..………..….....108
x
LIST OF ABBREVIATIONS
1X IO 1 times Instant Ocean®
2X IO 2 times Instant Ocean®
3X IO 3 times Instant Ocean®
Al aluminum
APTs agar precipitation tests
As arsenic
ATP adenosine triphosphate
BCECF 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein
BLAST basic local alignments search tool
bp base pairs
Ca calcium
CaCO3 calcium carbonate
Cd cadmium
CFU colony forming unit
Cl chloride
cm centimeters
Cr chromium
Cu copper
DAPI 4'-6-Diamidino-2-phenylindole
DIC differential interference contrast
DNA deoxyribonucleic acid
DVC direct microscopic viable count
xi
ECe Electrical Conductivity of the extract EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay
eV electron volt F fluoride
FC flow cytometry FDA fluorogenic ester fluorescein diacetate Fe Iron
FISH fluorescence in situ hybridization
FS filter sterilized
g grams
GCMS gas chromatograph/mass spectrometer
HCl hydrochloric acid
I.D. inner diameter
IF immunofluorescence
IMC immunomagnetic capture
K potassium
m meters
M Molarity
MA Marine Agar
MB Marine Broth
MA+IO Marine Agar + Instant Ocean®
MB+IO Marine Broth + Instant Ocean®
xii
MER Mars Exploration Rovers
Mg magnesium
min. minutes
µL microliter
mL milliliter
mm millimeter
mM millimolar
µm micrometer
Mn manganese
MPN most probable number
mS/g milliSiemens per gram m/z mass-to-charge ratio Na sodium
NA Nutrient Agar NB Nutrient Broth ng nanogram Ni nickel
NO3 nitrate
P phosphorous
Pb lead
PBS phosphate buffered saline PCA Plate Count Agar PCB Plate Count Broth
xiii
PCR polymerase chain reaction ppt parts per trillion pyr-GC-MS pyrolysis-gas chromatography-mass spectrometry RFLP restriction fragment length polymorphism RNA ribonucleic acid rRNA ribosomal RNA SDS sodium dodecyl (lauryl) sulfate sec. seconds SEM scanning electron microscopy Si silicon
spp. species
SO4 sulfate
TDS total dissolved solids Z zinc
xiv
ABSTRACT
The Atacama Desert in Chile extends from latitudes 17º S to 28º S between longitudes
69º W and 71º W. It has been reported that surface soils in the hyper-arid region contain low
numbers of culturable heterotrophic bacteria. These soils are considered Mars-like and offer an
ideal setting to investigate the application of life detection systems. Thirty-three surface sites
were sampled to examine further the extent of the hyper-arid region. We also excavated four soil
pits, 40 cm to 90 cm in depth, to explore subsurface microbial communities. One pit was dug in
a southern region of the desert compared to the location of the pits in the hyper-arid region.
Samples were examined using culture-dependent techniques, including serial dilution plating
methods on five media for the cultivation of heterotrophic bacteria. Using 16S rRNA gene
sequence comparisons, 1,260 organisms have been recovered and identified. Fifty-four percent
of the samples obtained from within the hyper-arid region show numbers of culturable bacteria
above the detection limit, yet there is evidence of microbial patchiness in surface and subsurface
soils. In some samples, no bacterial colonies were retrieved; in the majority, less than ten were
recovered. One pit showed an increase in CFUs/g at 40 cm then dropped back to levels near and
below the detection limit. A second pit showed an increase at 10 cm with numbers near and
below the detection limit at further depths. The southern soil pit had CFUs/g up to four orders of
magnitude greater than those in the hyper-arid region. Direct count methods employing DAPI
epifluorescence microscopy were applied to samples, but proved suitable only for a sample
having the highest CFUs/g of soil (7.4 x 105) due to the determined detection limit for the
technique utilized on the minimal-life containing soils. A chemical composition analysis was
performed on all soil samples and showed that elevated ion concentrations may correlate with
low numbers of culturable bacteria. The data obtained for the desert samples point to the
xv
importance of developing surface and subsurface sampling protocols for future missions to Mars
searching for evidence of past or present life.
1
CHAPTER 1 INTRODUCTION
THE DRIEST PLACE ON EARTH, THE ATACAMA DESERT OF CHILE
The Atacama Desert of northern Chile is a sparsely populated, virtually rainless plateau,
extending from the Pacific Ocean to the Andes Mountains and encompassing an area from
latitudes 17º to 28º S and 69º to 71º W. The northern desert region is considered to be one of the
driest places on Earth. The Pacific Coastal Range, the mountains on the western side of the
desert, has an average elevation of approximately 1,000 m (Hickman 1998). In the core of the
desert, rain is measured in millimeters (mm) per decade. There are some areas where rain has
never been recorded, at least as long as humans have measured it (Hickman 1998).
The collective effects of five different factors result in the hyper-aridity the desert: (1)
precipitation is reduced by a high-pressure system that produces descending stable air because of
the Hadley circulation, (2) the cold north-flowing Peruvian Current prevents the ability of
onshore winds and generates a constant layer of warm air that prevents the rise of cooling air and
traps moisture beneath it, (3) the oceanic cloud barrier effect of the coastal range, (4) the
rainshadow effect of the Andes Mountains to the east prevents moisture transfer from the east,
(5) and the substantial distance (≤ 2000 km) from the Amazon basin and Atlantic Ocean
moisture supply (Houston and Hartley 2003).
The Atacama Desert is also a unique source for nitrate (NO3) minerals, which are
traditionally used in agricultural production systems as a fertilizer (NOSB TAP 2002). Chilean
NO3 is a naturally occurring inorganic mineral salt that is derived from caliche ore, which is a
crude mineral conglomerate of salts comprised of: NO3, SO4, Cl of Na, Ca, K, Mg, and various
micronutrients (Ericksen 1983). Darwin (1871) put forward that the NO3 deposits may have
2
formed at the inland border of the oceanic expansion. It was proposed by Ericksen (1983),
however, that the NO3 can be accredited to nitrogen fixing and nitrifying microorganisms in the
soils or in nearby Andean salars, which are present due to preexisting salt-water lakes, 10-15
million years ago. The NO3 is proposed to make its way into the Atacama Desert via wind or
groundwater (Ericksen 1983). Other possibilities for the origin of the NO3 deposits include the
nitrification of ammonia from seaweed, or vegetation in salt lakes, or bird guano (Ericksen
1983). Atmospheric NO3 accumulation from sea spray, ammonia deposition, or nitric acid
deposition may also contribute to the NO3 mineral abundance observed in the Atacama Desert
(Ericksen 1981). Chong (1994) states that the NO3 deposition was caused by magmatic
processes that produced NO3 through unknown catalytic reactions. The real reason for the NO3
and other soluble salts that exist in the Atacama Desert has yet to be agreed upon by researchers;
most attribute these characteristics to a combination of sources (Michalski et al. 2004).
The Atacama Desert provides excellent testing grounds for Mars research due to three
similarities that exist between the Atacama Desert and Mars; the similarities between the two
environments are compared and contrasted throughout the remainder of this section.
The existence of Mars-like soils in the core hyper-arid region, the area known as Yungay,
of the Atacama Desert has been described (Navarro-González et al. 2003). The Yungay region is
located around 24º S and 69º W. This region consists of surface-soils containing organic
materials at trace concentrations and extremely low levels of culturable bacteria (Navarro-
González et al. 2003). In the Navarro-González et al. (2003) study the organic analysis was
determined for surface soils from the Atacama Desert using pyrolysis-gas chromatography-mass
spectrometry (pyr-GC-MS). In addition, numbers of colony forming unit (CFU) of heterotrophic
bacteria below detection limits of the dilution plating technique were observed. In a number of
3
cases no bacterial colonies formed from the lowest dilution. The aim of this study was to
investigate further the CFUs/g and diversity of heterotrophic bacteria in additional soils collected
from the Yungay region.
The 1976 Viking missions to Mars carried pyrolysis gas chromatograph/mass
spectrometers (GCMS), and provided evidence that the Martian soils were depleted in organic
compounds, at only a few parts per billion, in the upper 10 cm of surface soil. This suggested
that there was an absence of Martian life (Glavin et al. 2001). In the Yungay region of the
Atacama Desert, high ratios of formic acid/benzene were discovered from of pyrolysis of organic
matter, suggesting that this region is depleted in organic matter due to highly oxidizing
characteristics (Navarro-González et al. 2003). Martian soils and Atacama Desert soils appear to
be similar because both appear to be depleted of organic matter due to strong soil oxidants which
result in the fast isolation of extinct or existing life forms. The Atacama Desert is considered to
be a Mars analog because it has attributes similar to those found on Mars, in terms of chemistry,
mineralogy, geology, topography, etc.
The Atacama Desert is a Mars analog for other reasons. The findings of Navarro-
González et al. (2003), initially demonstrated that the Atacama Desert is similar to Mars, which
is considered to be a hyper-arid environment. The data obtained by Mars Exploration Rovers
(MER), however, suggest that at one time water may have been present on Mars (Baldridge and
Calvin 2003, Herkenhoff et al. 2004, Haskin et al. 2005). The Atacama Desert is one of the
oldest and driest deserts in the world; it has been said to be at least 15 million years old (Eriksen
1981). At the end of the last glacial maximum (approximately 12,000 years ago) the Atacama
existed in a less arid state than today (Bortman 2003). The salars of the Atacama Desert were
once saline lakes that eventually dried out up over time. Any life that existed in the Atacama
4
Desert prior to its hyper-aridity either died or adapted to the hyper-arid environment. If the MER
are accurate in revealing that Mars was wetter in the past, then the Atacama Desert is similar to
Mars in the sense that a previously wetter environment once existed where today exists a hyper-
arid environment; therefore, the Atacama Desert may reveal possibilities of life on Mars.
The core hyper-arid region of the Atacama Desert provides an ideal setting to investigate
the survival of microorganisms under extremely arid conditions. Areas with little or no
culturable bacteria provide sites for the testing of life detection systems as well as for the
development of planetary exploration protocols. In order to search for evidence of water and
oxidants at some depths, NASA’s Mars Science Laboratory is preparing to send a Lander/Rover
that is capable of drilling and retrieving soil samples at Martian shallow subsurface in 2009
(NASA 2003 Strategic Plan). Preparations for a 2018 Deep Drill mission are also underway to
search 3 to 10 m of Mars’ subsurface for evidence of past or present life, although the landing
site depends on outcomes of earlier expeditions (Mars Science Program Synthesis Group 2003).
With regards to the 2009 and 2018 missions, this thesis not only focuses on the surface soil, but
also the subsurface soil in the hyper-arid core region of the Atacama Desert. This study uses
both culture-dependent and culture-independent methods to analyze bacterial communities that
exist above and below the surface of the Atacama Desert.
METHODS FOR MICROBIAL COMMUNITY ANALYSIS
Culture-dependent and culture-independent approaches are used to study prokaryotic
populations existing in environmental samples. The culture-dependent approach employs
artificial media in order to culture or enrich for groups of microorganisms inhabiting
environmental samples. This approach is also used simply to isolate single colonies. Until
recently, this method was considered standard and was carried out in most microbial ecology
5
studies. In recent years it has been discovered that the majority of microorganisms in the
environment are not culturable by traditional plate culture methods. With this, the culture-
independent approach is essential to evaluate microbial diversity in various types of
environments (Amann et al. 1995). With the culture-independent approach, scientists have been
able to identify unculturable prokaryotes in situ (Brock 1987). This method commonly
concentrates on the ability to extract total nucleic acids from cells occupying a sample and
employing biomarker-based techniques, such as DNA analysis (reviewed in Head et al. 1998).
The use of ribosomal gene sequences as indicators of bacterial diversity is most common,
although other genes, including protein-coding genes, have also been used (reviewed in
Wintzingerode et al. 1997). Although the 16S rRNA gene research is more widely utilized, there
are various culture-independent techniques that target other genes that are responsible for a
number of metabolic actions. Culture-independent approaches using the 16S rRNA gene as a
marker neglect to consider any detail regarding the differences in cellular metabolic activity, or
to ensure that ecologically significant microbes are analyzed and not microbial cells existing in
quiescent forms that do not add to the function of the environmental system (Ellis et al. 2003).
By incorporating both of these analytical strategies, it is possible to target only those cells that
are actively undergoing the specific metabolic function coded for by the targeted gene.
In culture-dependent methods, viable culturable cells can be recovered with the plate
count method, which is thoroughly described in chapter 2. After incubation, colonies that form
are assumed to result from a single cell, thus total CFUs per volume of sample can be calculated.
The most probable number (MPN) technique is similar to the plate count method, except that the
method depends on detection of certain qualitative characteristics of the organism of interest, in
particular on growth of the target organisms in liquid medium. Microorganisms capable of being
6
cultured are preserved in laboratory culture and strain collections. These organisms are
classified by phenotypic and genotypic characteristics and are ultimately assigned to a specific
taxonomic group.
Although culture-dependent techniques provide an excellent understanding of the
physiological potential of isolated organisms, they do not necessarily offer insight into the
complete makeup of microbial communities (Horner-Devine 2003). Culture-dependent methods
are biased in that a microorganism can only be cultivated after its physiological niche is evident
and experimentally reproduced. The biases that culture-dependent analyses demonstrate are
evident in the inconsistencies that exist between total cells viewed microscopically versus total
CFUs determined for environmental samples (Staley and Konopka 1985). It is widely accepted
that culture-dependent studies can only assess between 0.001% and 15% of the actual microbial
communities in a sample (Amann et al. 1995). Most of the viable cells that can be viewed
microscopically, but are not considered culturable, can be classified into one of two groups. The
first of which are known species that have entered an unculturable state or for which the
cultivation conditions utilized are not adequate. The second group consists of unknown species
that as of now have never been able to be cultured due to a lack of appropriate processes (Amann
et al. 1995).
The culture-dependent method when applied to soil microbiology is relevant because it
offers an overall fraction of the microbes residing in a specific soil. Although the research
executed for this study focuses mainly on these culture-dependent techniques, and in turn is
influenced by the biases mentioned above, it is possible that in environments having extreme
conditions, such as the Atacama Desert, culturable organisms tell more of the community than in
less extreme environments. Culture-dependent methods provide an undeniable knowledge of
7
any previously undescribed environments, and with regards to this study, it is necessary to
perform the culture-dependent studies, because it provides a solid backbone to future research
that applies culture-independent protocols.
Culture-independent methods were developed to understand better the complete
microbial populations existing in natural habitats. The culture-independent approach involves
the extraction of total environmental nucleic acids, but the extraction method employed depends
on the type of environment being studied. Soil samples require a harsh extraction method due to
the tight bonds that can usually exist between soil particles and inhabiting microorganisms
(Priemé et al. 1996). In order to lyse microbial cell walls, extraction methods can utilize both
physical and chemical processes. Physical methods include freeze/thaw protocols and/or bead
beating; chemical procedures usually entail lysozyme and sodium dodecyl (lauryl) sulfate.
Extraction of total nucleic acids from soil samples yields better results when chemical and
physical methods are combined due to the strong attachment of microbial cells to soil particles.
Often, extraction kits designed specifically for soil join together bead beating and chemical lysis
methods. Once the microbial cell has been disrupted and the DNA/RNA has been extracted, the
nucleic acids must be purified, usually with proteinase K and phenol/chloroform washes.
Because nucleic acids carry a fixed negative charge per unit length of molecule, for purification
purposes nucleic acids are collected using binding material, which is positively charged, and
washed to discard of unnecessary compounds.
Gene clone library construction is a widely used procedure that employs purified nucleic
acids from an environmental sample and amplifies a specific sequence on the various genomes
present using Polymerase Chain Reaction (PCR), for which oligonucleotide primers specific to
the preferred amplicon gene sequence can be designed. The amplified gene sequences are then
8
inserted into engineered plasmids containing an antibiotic resistant gene, resulting in
recombinant plasmids, which can then be taken up by competent Escherichia coli host cells. The
cells are then grown on nutrient media containing the antibiotic, thus only cells that have the
plasmid survive and replicate. Resulting colonies, or clones, contain copies of the same
recombinant plasmid with its fragment of foreign DNA. A variety of assay methods can be used
on the bacterial clones to determine which colonies contain particular DNA sequences.
The gene sequence most commonly used for the construction of gene clone libraries in
microbial ecology studies is the 16S rRNA gene because it is highly conserved in all prokaryotes
(Hillis and Dixon 1991). The 16S rRNA gene is conservative because it is the slowest gene
sequence to evolve, indicating that research based on the 16S rRNA gene can be used to study
ancient evolutionary events, also the 16S rRNA gene sequences are all similar in length
(approximately 1500 nucleotides). The slow rate of change that has been observed in the 16S
rRNA gene allows for the construction of universal primers (Hillis and Dixon 1991). The first
study that used the 16S rRNA gene for the construction of gene clone libraries was done by
Liesack and Stackebrandt (1992). The research was performed on an Australian soil sample to
determine the total microbial diversity from the cloned and retrieved 16S rRNA gene sequences.
A total of 116 clones were created, which significantly revealed diversity in nature that had never
before been discovered. The technique opened new doors in microbiology, especially soil
microbiology, and has since been used extensively, although it is expensive and extremely time-
consuming. Therefore, most 16S rRNA gene clone libraries usually consist of between 100 and
300 clones. Gene clone libraries having considerable more clones have an overall higher degree
of retrieved diversity, in terms of community composition, richness, and structure (Dunbar et al.
2002).
9
An inexpensive technique that is based on the PCR amplification of a mixture of genes
representing different microorganisms from environmental nucleic acid extracts is Restriction
Fragment Length Polymorphism (RFLP). This method employs the digestion of amplicons by
restriction enzymes that nick DNA at specific sites, and forms an assortment of DNA fragments
(Lee et al. 1993; Gundersen et al. 1994; Muyzer et al. 1995). The fragments are separated via gel
electrophoresis; the ultra-pure agarose used is capable of resolving fragments that vary by < 25
bp. The gel is stained with ethidium bromide and viewed under ultra-violet illumination; only
the fluorescently labeled restriction fragments are visualized. It is useful to apply multiple
restriction enzymes because ordinarily this will allow for better resolution since it will increase
the number of different sized DNA fragments.
Denaturing or temperature gradient gel electrophoresis (DGGE/TGGE) is a culture-
independent technique first developed by Myers et al. (1987) that involves separation of DNA in
solution by various melting properties. DNA molecules melt in distinct segments, known as
melting domains, when the temperature or denaturant (usually urea) concentration is increased.
The melting temperature (Tm) of a melting domain depends on the nucleotide sequence. As
DNA fragments are separated in an electrical field through a linear gradient of increasing
denaturing concentration or increased temperature, the separation of double-stranded DNA into
single-stranded segments increases causing the DNA segments to form a less uniform three-
dimensional structure that moves through a polyacrylamide matrix at a slower rate (Muyzer et al.
1993). Profiles that are produced are also known as fingerprints, which can be compared and
evaluated to match up any similar banding patterns across environmental samples. Unique
banding patterns can be cut out of the gel and sequenced for additional testing. One
disadvantage to this method is that DGGE and TGGE techniques cannot provide any quantitative
10
data (van Hannen et al. 1999). Another downside to this method is that as the complexity of a
community increases, it may be more difficult to excise individual bands for sequence analysis.
Thus, this technique could limit the extent of the environment’s diversity.
Although the extraction of total nucleic acids from environmental samples is widely used,
there are many other conventional methods that do not involve these extraction techniques and
are considered culture-independent. One commonly used approach includes DNA-binding
fluorochromes such as 4’,6-diamidino-2-phenylindole (DAPI) and acridine orange (3,6-
bis[dimethylamino] acridinium chloride. Direct counts using acridine orange or DAPI to
examine microbial populations in most natural environments usually surpass counts derived from
plate counts by some order of magnitudes because not only culturable microbes are revealed, but
viable unculturable and nonviable microbes are also uncovered (Hartmann et al. 1997).
The direct microscopic viable count (DVC) method determines viable microbes in
environmental samples by preventing cells to replicate using a DNA gyrase inhibitor, nalidixic
acid, but promoting cell development with yeast extract (Kogure et al. 1979). Epifluorescence
microscopy employing DNA-binding fluorochromes immediately ensues for quantification
purposes of active cells (Hartmann et al. 1997).
Fluorescence nucleic acid hybridization probes are rRNA directed oligonucleotides that
can be made and used to label targeted microorganisms for in situ detection. This technique is
able to detect a single targeted cell and can give some clue to an organism’s specific genera or
higher taxonomic classification (Hartmann et al. 1997). By using probes that target regions of
rRNA that have been conserved, measurements of total rRNA in an environmental sample can be
determined.
11
Flow cytometry is a culture-independent technique that can also be used to study
microbial cells (Melamed et al. 1990). The advantages of using flow cytometry (FC) are
because it is possible to analyze cells that stream across a laser beam, according to several
parameters at the same time, for example, most flow cytometers feature light scatterers and
fluorescent detectors. Universal oligonucleotide probes can be combined with FC to investigate
diverse prokaryotic communities. The FC method could possibly generate exceptional results
for microbial studies if the method was further developed because it can analyze masses of cells
in a short amount of time, but the problem at hand is that the size of microbes is generally too
close to the detection limit of these instruments (Davey 1994).
There are culture-independent approaches that utilize fluorescent dyes to specifically find
active microorganisms in natural environments; some are described at the Invitrogen webpage.
Cell viability can be assessed using fluorogenic esterase substrates, such as fluorescein diacetate
(FDA), calcein AM, 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) AM and
various other FDA derivatives. Membrane integrity and activity are good indicators of viability
and can be evaluated by the cell-permeant esterase substrates. This method is commonly used in
conjunction with FC (Diaper and Edwards 1994). Flourescein and fluorescein derivatives easily
diffuse into viable microbial cells and most respiring cells can break down fluorogenic ester to
create the fluorescein molecule (Breeuwer et al. 1995). This method does not detect all active
cells because some microbes are not able to capable of taking up FDA or FDA derivatives
efficiently, therefore it does not necessarily reflect the growth potential of microbial cells within
the given natural environment.
Enzymatic activity of microorganisms can also be revealed and measured by using other
artificial substrates (Gazenko et al. 1998). Special enzymes, such as lipases, phosphatases, and
12
dehydrogenases, of live microbial cells attach directly with fluorogenic substrates through
biochemical reactions and turn to fluorescent substances either intracellular or extracellular. The
amount of the resulting fluorescence depends on the enzymatic activity of the cell (Gazenko et
al. 1998). This method, which has the same disadvantage as mentioned above, does not recover
all active cells because some microbes cannot take up the substrate-enzyme complex
proficiently. Fluorescent redox probes are also useful in direct fluorescence microscopy because
they allow for the visualization of metabolically active bacteria that are competent to reduce the
redox dye (Rodriguez et al. 1992).
Immunological techniques are culture-independent methods that include
immunofluorescence (IF) and ELISA (enzyme-linked immunosorbent assay). IF staining uses
epifluorescence microscopy and involves antibodies either directly joined with a fluorescence
marker or marked with a secondary antibody. The disadvantage to IF is that the antibodies
produced are specific for distinct taxonomic groups and cannot detect entire microbial
population; however, IF can be used in soil extracts for in situ research or in PCR for locating
target microbes (Hartmann et al. 1997). ELISA, a technique based on enzyme-linked antibodies,
remains the most widely used immunological method for detecting microbes in soil (Lee et al.
1990). The primary specific antibody is added first and then the secondary antibody, which
carries one enzyme molecule per antibody, is added. ELISA is an effective method because
fairly low numbers of microbial cells in soil can be detected (Hartmann et al. 1997).
Immunomagnetic capture (IMC) is an alternative procedure that involves microscopic
magnetic beads that are coated with monoclonal or polyclonal antibodies specific for the
prokaryotes of importance. Both direct and indirect IMC methods have been successful in
recovering organisms from soil (Wellington et al. 1997).
13
Microbial biomass can be also be approximated using culture-independent methods such
as microbial respiration, metabolic heat production, adenylate energy content, adenosine
triphosphate (ATP) levels, or soil fumigation techniques. These methods only provide data for
the active organisms in the community, and therefore cannot provide an accurate microbial
biomass enumeration (Hartmann et al. 1997).
Culture-dependent and culture-independent microbiological studies of the Atacama
Desert’s core hyper-arid region of Yungay were the focus for this thesis. The first study was of
surface soils collected from within the Yungay region, and the second study was of soils sampled
from subsurface soil pits that were excavated to a depth of up to one meter. CFUs were
determined for all soil samples using five nutrient media. Isolates recovered from the soil
samples were identified by 16S rRNA gene sequencing in order to examine the diversity of
culturable heterotrophic bacteria isolated from these soils. The direct count technique using
DAPI epifluorescence microscopy was done to enumerate the unculturable fraction of the
microbial population. The results of these studies are presented in chapters 3, 4 and 5 that deal
with surface CFUs and diversity, subsurface CFUs and diversity and direct counts approaches
using DAPI epifluorescence microscopy.
14
CHAPTER 2 MATERIALS AND METHODS
SAMPLES COLLECTION
Soil samples were collected from the Atacama Desert in 2003 and 2004 from 33
accessible surface sites within the core hyper-arid region and from four soil pits that were
excavated and sampled. Three pits were excavated within the core arid region, while one soil pit
was dug at a site further south and was considered a control pit. The four soil pits ranged in
depth from 50 to 90 cm. The coordinates of each sample site were recorded using a Global
Positioning System (GPS) device. The top 3 cm were collected for surface samples using sterile
plastic scoops and sterile Whirl-Pak® bags. A surface sample for all four pits was acquired
before pit excavation. Pits were excavated using standard shovels and after construction was
complete, the pit wall was cleaned by scraping soil from the wall using a plastic Sterileware®
scoop (Bel-Art Products®). Samples were taken every 10 cm, beginning with the deepest layer,
and were extracted using a Sterileware® sampling spatula (Bel-Art Products®). To prevent
contamination of the soil samples in the laboratory, approximately 10 g of each soil were
transferred to a clean sterile 50 mL centrifuge tube to be used as a stock sample.
NAMING OF SITES, SAMPLES, AND ISOLATES
Soil pits and surface samples were labeled according the year and the collection site,
beginning with the letters AT for Atacama Desert. The numbers that follow denote the
collection year and the last digits represent the sample site number. Isolate names were derived
from the soil sample designation from which the original microbial colony was cultured. Each
colony isolated from a given soil sample was numbered consecutively.
15
CULTURE MEDIA
Five nutrient media were used to culture heterotrophic microbes from each soil sample,
including (all Difco products) Plate Count Agar (PCA), Marine Agar (MA), Nutrient Agar (NA),
1/10 strength Plate Count Agar (1/10 PCA), and 1/100 strength Plate Count Agar (1/100 PCA).
Each culture medium was also used in the liquid form without agar added for dilution
preparation. In addition soils from sites AT04-158, AT04-163, and AT04-164 were plated on
MA and 1/10 PCA that had been adjusted to pH 5.0. Soils from sites AT03-35 and AT04-170
were also plated on media varying in marine salt concentrations at pH 8.0 using a combination of
Marine Agar and Instant Ocean®, 2X Instant Ocean®, and 3X Instant Ocean®. Recipes for all
culture media are provided in Appendix A.
SOIL DILUTION PLATING
A serial dilution plating method was used to plate each soil sample on the five nutrient
media, as well as selected soil samples on media with varying marine salt concentrations or
media prepared at pH 5.0. Serial dilutions were prepared by adding 9 mL of each nutrient
medium broth to individual 15 mL dilution tubes. Using a Sterileware® sampling spatula (Bel-
Art Products®), 1 g of soil was weighed into the first dilution tube. The first dilution tubes were
vortexed at maximum speed for 30 sec. One milliliter was transferred from the first dilution tube
to the second dilution tube containing 9 mL of a culture medium broth. The second dilution was
vortexed at maximum speed and 1 mL was transferred to the third dilution tube, and so forth.
After dilutions were prepared, an aliquot of 150 µL was transferred onto the corresponding
dilution agar plate and distributed over the surface of the agar using a sterile plastic 60 mm cell
spreader. Plating was performed in triplicate. Plates were sealed with Parafilm®, inverted and
incubated at 28ºC for 20 days. Plates on the media containing varying salt concentrations were
16
incubated for 35 days. Sites AT03-35 and AT04-170 were plated an additional time using the
serial dilution plating method on the five media and incubated for 45 days to determine whether
slow-growing organisms would appear after the 20th day.
ENRICHMENT CULTURES
The tubes that were used for the serial dilutions (except AT04-159) were incubated at
28ºC for 20 days for the purpose of creating enrichment cultures. After incubation, the
enrichment cultures were plated in the same manner as the dilution plating method. Enrichment
tubes were vortexed at maximum speed for 30 sec. An aliquot of 150 µL was transferred onto
the corresponding dilution agar plate. Plates were sealed with Parafilm®, inverted and incubated
at 28ºC for 20 days. Microbial colonies grown from the enrichment cultures were isolated,
preserved, and treated in the same way as isolates retrieved in the dilution plating method.
Isolates recovered from enrichment cultures have been named similarly to dilution plating
isolates except that a capital B precedes the isolate number; for example, AT04-150-B11.
PLATE COUNTING AND COLONY FORMING UNIT DETERMINATION
CFUs/g of soil on plates were counted after incubation times of 5, 10, and 20 days. The
second plating of AT03-35 and AT04-170 were counted after the incubation days of 5, 10, 20,
35, and 45. Plates on the media containing varying salt concentrations were counted after 5, 10,
20, and 35 days. Plate counts were recorded along with a description of all visible colonies.
CFUs/g of soil were calculated from the countable dilution plate (30 to 300 colonies) by
multiplying the number of colonies by the dilution factor and 6.67 to account for the 150 µL
aliquot.
17
ISOLATION OF PURE CULTURES
Colonies were transferred to agar culture media from dilution plates on the five media
after the 20 day counts were recorded. Each colony was transferred to the same nutrient medium
on which it was originally isolated. The streak plate method was used to isolate individual
colonies. Colonies were selected from all plates of the triplicate set. If there were multiple
colonies with similar morphological characteristics on a dilution plate, two or three were selected
for isolation. In an attempt to isolate sufficient representatives of the culturable heterotrophic
bacteria from all sample sites, all unique colonies from each dilution plate for each medium were
transferred. Records for each isolate included a colony description, medium type, source
dilution, and depth (for soil pits). Each transfer plate was inverted and incubated at 28ºC until
pure cell growth was observed. Colonies on transfer plates was divided into three to four
sections; cell material from two to three were used for preservation and the remaining cells were
used for DNA extraction.
PRESERVATION
All five nutrient media were prepared as preservation solutions by adding 15% (v/v)
glycerol and 750 µL was aliquoted into three sterile Eppendorf® 1.5 mL Safe Lock Tubes.
Saline EDTA was prepared for the DNA extractions; 300 µL of saline EDTA was added to one
clean sterile Eppendorf® 1.5 mL Safe Lock Tube for each isolate. Cell material was transferred
to saline EDTA, and to tubes containing 750 µL of the culture medium plus 15% (v/v) glycerol.
The tubes with saline EDTA plus bacterial cell material were labeled and stored in the freezer (-
20ºC) for future DNA extraction. The tubes with the bacterial cell material plus 15% (v/v)
glycerol media were labeled and preserved in a Thermo Forma® ultra low temperature freezer at
-80ºC.
18
DNA EXTRACTION
DNA was extracted and purified from isolated cells using the UltraClean™ Microbial
DNA Isolation Kit (Mo Bio Laboratories, Inc.). The first five steps of the manufacturer’s
instructions were slightly modified as provided. The samples were thawed and the tubes were
centrifuged at 16,100 x g for 5 min. to pellet cells. The supernatant, (approximately 300 µL),
was removed and discarded using a pipette. The MicroBead solution was added (300 µL) to
each extraction tube following disaggregation of the pellet. The entire contents of each tube
were transferred to a MicroBead tube and 50 µL of solution MD1 was added to each MicroBead
tube. The tubes were placed on the Mo Bio vortex adapter for 5 min at maximum speed,
removed from the vortex and incubated in a 65ºC water bath for 10 min. After incubation the
MicroBead tubes, they were placed on the vortex adapter and vortexed at maximum speed for an
additional 5 min. The manufacturer’s methods were applied from this point forward. The DNA
containing solution was transferred to a clean sterile Eppendorf® 1.5 mL Safe Lock Tube for
storage and to prevent evaporation. DNA was verified by gel electrophoresis prior to PCR
amplification of 16S rRNA genes.
POLYMERASE CHAIN REACTION (PCR) AND PCR PURIFICATION
The 16S rRNA genes from the isolates were amplified by PCR from 20 to 200 ng of
DNA in reaction mixtures, totaling 50 μl, containing (as final concentrations): 1X PCR Buffer
(Applied Biosystems), 50 mM each deoxynucleoside triphosphate, 200 nM each forward and
reverse primer, and 33.3 μl sterile distilled water. Reaction mixtures for universal primers were
incubated in a BioRad iCycler® at 98°C for six min. (for initial denaturation). After the
temperature was reduced to 90°C, 0.2 µL of Taq polymerase (Roche) was added; followed by 28
cycles at 52°C for 30 sec., 54°C for one minute, and 94°C for 30 sec. A final extension period of
19
7 min. at 72°C was done. 16S rRNA genes were amplified with the forward primer 27F (specific
for Bacteria) (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-
GGTTACCTTGTTACGACTT-3’). PCR products were confirmed by gel electrophoresis prior
to PCR purification. PCR fragments were purified with the Invitek Invisorb® Spin PCRapid Kit,
using manufacturer’s guidelines.
SEQUENCING, SEQUENCE PURIFICATION, AND DATA ANALYSIS
16S rRNA gene PCR products were sequenced using the BigDye Terminator reagent
(Applied Biosystems) and the 27F primer. Total reaction volumes were 10 μl and comprised: 0.5
μl 16S rRNA cleaned gene PCR product, 2.5 μl 27F primer (25 ng/μl), 2.5 μl BigDye Terminator
reagent and 4.5 μl sterile distilled water. Reaction mixtures were incubated in a BioRad
iCycler® at 94°C for 30 sec., followed by 25 cycles at 96°C for 10 sec., 50°C for 5 sec, and 60°C
for 4 min.
The sequence reactions were purified by ethanol precipitation after completion of the
sequencing cycles. Sequence reaction tubes were removed from a BioRad iCycler® and 10 µL
of sterile distilled water were added to each sequence reaction tube, that was transferred to a
clean sterile 0.6 mL tube containing three µL of sodium acetate (3M pH 5.0) and 50 µL of 200
proof absolute ethanol. Tubes were placed on ice for 10 min. The tubes were centrifuged for 30
min. at 16,100 x g. Approximately 75 µL of the supernatant was removed by pipette from each
tube and discarded. Next, 250 µL of 70% (v/v) ethanol was added to each tube and centrifuged
for 10 min. at 16,100 x g. The supernatant was removed and discarded. The tubes were than
placed in a drying oven set at 60ºC for roughly 20 min. or until tubes were completely dry.
Purified DNA was stored at -20ºC until ready for resuspension. The purified DNA was
20
resuspended in HiDi Formamide (Applied Biosystems) and analyzed using an ABI Prism 3100
Genetic Analyzer.
The sequences were compiled using the Bioedit™ program version 7.0.0. The sequences
were subjected to BLAST searches in GenBank®, the National Institutes of Health genetic
sequence database, and the closest phylogenetic relative was determined. The BLAST program
was described by Altschul et al. (1990). Appendix B lists the isolate name, colony description,
medium type, source dilution, depth, BLAST result, sequence length, similarity, and accession
number for each identified isolate.
ISOLATE DATABASE
The following data were recorded in the database: isolate numbers, dilutions from which
isolates were recovered, media from which isolates were recovered, depths of sample from
which isolates were recovered, description of colonies isolated, lengths of sequences (bp)
imported into the BLAST interface of GenBank®, GenBank® BLAST results, nucleotide
identity similarities, sample sites from which isolates were recovered, year sites were sampled,
phylogenetic groups of closest relatives as identified by BLAST results, latitudes and longitudes
of sample sites, closest relatives as identified by BLAST results, growth temperature of isolates,
and sequences imported into the BLAST interface of GenBank® have been placed in an online
database. The Atacama Desert isolate database, which was designed by Abhishek
Satyendranath, is accessible at:
http://www.biology.lsu.edu/webfac/frainey/raineylab/databases/ATISOL/searchdata.php
DIRECT COUNTS USING FLUORESCENCE MICROSCOPY
Fluorescence microscopy was applied to three surface soils from the Atacama Desert
(AT04-165, AT04-166, and AT04-170) for direct counts of microbial cells within each sample.
21
DAPI, 4'-6-Diamidino-2-phenylindole, forms a fluorescent complex with DNA and can be used
as a vital tool when quantifying cells. A combined modified version of the total cell counts
DAPI protocol from Carman (1993), Janssen et al. (2002), was Glavin et al. (2004), was applied
to this study. For each sample, 2 g of soil were fixed in 2 mL of cold filter sterilized (FS)
phosphate-buffered saline (PBS) (0.13 M NaCl, 7 mM Na2HP04, 3 mM NaH2PO4; pH 7.2)
containing 4% (w/v) paraformaldehyde for at least 3 hours at 4ºC in a sterile 15 mL centrifuge
tube. The soil and fixing solution were agitated using a Sonifier® ultrasonic cell disruptor
(Branson Sonic Power Co). The sonication was carried out on ice with the probe tip at two
thirds of the liquid depth for 1 min. at 50% power (pulsing in 30 sec. intervals). The samples
were centrifuged at 2,350 x g for 10 min. and the supernatant (fixing solution) was removed and
discarded. Soil pellets were washed with 2 mL of FS PBS and gently vortexed. Samples were
centrifuged for 10 min at 2,350 x g and the supernatants removed and discarded. An additional 2
mL of PBS was added to the soil pellets and gently vortexed. Tubes were centrifuged again at
650 x g for 1 min to spin down larger soil particulates, but to allow the cells to remain suspended
in the supernatant (Carman 1993). The supernatant from each tube was split into 15 mL
centrifuge tubes containing 1 mL, 500 µL, 250 µL, 100 µL, and 50 µL. For each tube, a final
DAPI concentration of 5 µg/mL (stock concentration of 1000 µg/mL) was added and incubated
for at least 3 hours in the dark. The extra 100 µL of supernatant (not stained with DAPI) from
each sample was used as a control for autofluorescing particulates and treated in the same
manner as the DAPI stained samples. The liquid extract in each 15 mL sample tube was
increased to 5 mL with the addition of FS PBS. Each 5 mL solution was filtered onto a Millipore
Isopore™ Membrane Filter (0.2 µm 25 mm GTBP). A 5 mL aliquot of FS PBS was passed
through each filter to ensure affixing of all cells to the filter. The filters were mounted on clean
22
glass slides with Citifluor™, an anti-fade solution, and coverslips (22 by 22 mm) were placed on
each mounted filter. The preparations were examined under UV illumination (excitation filter
BP 365nm) at 800x magnification with a Nikon Microphot-FXA microscope.
At least 20 fields, evenly distributed over the area beneath the coverslip, were examined
for each of the samples and autofluorescing controls. Fluorescing cells in each of the 20 fields
were counted and an average number of cells per field determined. Calculations were made
using the area of the filter (490.65 mm2) and microscopic field measurements at 800x
magnification (88 µm x 117 µm). It was determined that at this magnification there are 47,652
fields per polycarbonate filter. The average was multiplied by the number of fields per filter and
then again to determine the number of cells per 1 g of soil. To account for the amount of soil the
supernatant was extracted from, the 1 mL extract calculations were multiplied by one, the 500
µL extract calculations were multiplied by two, the 250 µL extract calculations were multiplied
by four, the 100 µL extract calculations were multiplied by ten, and the 50 µL extract
calculations were multiplied by 20.
DETERMINING LIMIT OF DIRECT COUNTS ON ATACAMA DESERT SOILS
The Atacama Desert soil isolate strain AT03-37-10 grown on Nutrient Agar, had the
closest relative being Blastococcus aggregatus (AJ430193). In a sterile 15 mL tube, a loop full
of bacterial growth was transferred to 9 mL of Nutrient Broth. Differential Interference Contrast
(DIC) and fluorescence photomicrographs of the strain AT03-37-10 cells were captured in order
to observe the cell size and shape. Scanning electron microscope (SEM) photomicrographs were
also taken of strain AT03-36-10 (preparations given below), whose closest relative is also
Blastococcus aggregatus (AJ430193), to observe the cell size and shape. A 1/10 dilution of the
cell suspension was prepared (500 µL into 4.5 mL FS PBS) and filtered onto a Millipore
23
Isopore™ Membrane Filter (0.2 µm 25 mm GTBP). A 5 mL aliquot of FS PBS was passed
through each filter to ensure affixing of all cells to the filter. The filters were mounted on clean
glass slides with Citifluor™, an anti-fade solution, and coverslips (22 by 22 mm) were placed on
each mounted filter. The preparations were examined under UV illumination (excitation filter
BP 365nm) at 800x magnification with a Nikon Microphot-FXA microscope for the DAPI
fluorescence microscopy and DIC images were captured at 1000x magnification with a Nikon
Microphot-FXA microscope. DIC and fluorescence photomicrographs were acquired from the
same filter and can be viewed in Figure 3.5.
Preparations for the SEM photomicrographs were done by Cindy Henk of the Socolofsky
Microscopy Center, Department of Biological Sciences. The Atacama Desert isolate strain
AT03-36-10 was grown on 1/10 strength PCA. In a sterile 15 mL tube, a loop full of bacterial
growth was transferred to 5 mL of 1/10 strength PCB. The cell culture was fixed in an equal
volume of 4% (v/v) glutaraldehyde, 0.1 M sodium cacodylate buffer (pH 7.0), and half strength
culture medium for 30 min. then collected on a 0.22 µm polycarbonate filter. The filter was
transferred to 4% (v/v) glutaraldehyde in 0.1 M buffer for 5 min. and rinsed three times in 0.1 M
buffer. The filter was dehydrated in ethanol, critical-point dried, mounted on an aluminum
specimen stub, and coated with gold:palladium 60:40 in an Edwards S150 sputter coater. The
filter was viewed using a Cambridge S-260 SEM. A representative SEM photomicrograph can
be viewed in Figure 3.5.
The strain AT03-37-10 cell suspension was serially diluted to 1/1000 in Nutrient Broth.
The optical densities of each of the cell suspensions were determined using SmartSpec™ Plus
Spectrophotometer (BioRad). The cell suspension dilution series was plated to determine the
number of viable cells in the stock culture. Using these data a standard curve plotting turbidity
24
versus total viable cell number was created. Either 1 mL of the stock culture, each dilution, or
the control (Nutrient Broth) was added to 1 g of soil from site AT04-170, a soil previously found
to have zero CFUs on the five culture media, and spread evenly over the base of an empty Petri
dish. Soils were air dried overnight. The soils were transferred to a sterile 15 mL tube and fixed
at 4ºC for at least 3 hours in 2 mL of cold FS PBS containing 4% (w/v) paraformaldehyde. The
tubes were then centrifuged at 2,350 x g for 10 min. and the supernatant (fixing solution) was
discarded. Two milliliters of FS PBS was added to the soil pellets, gently vortexed, and
centrifuged at 2,350 x g for 10 min. The supernatants were discarded and 2 mL of FS PBS was
added to the soil pellets. The tubes were gently vortexed and centrifuged for 1 min. at 650 x g to
spin down larger soil particulates, but keep bacterial cells suspended in the supernatants (Carman
1993). The 2 mL supernatants were split in half; 1 mL was transferred to two clean sterile 1.5
mL tubes. In one of the tubes for each sample 5 µL of FS DAPI (stock concentration of 1000
µg/mL and final concentration of 5 µg/mL) was added and the tubes were incubated for at least 3
hours at room temperature in the dark. The tube for each sample without DAPI was used as a
control for autofluorescing particulates and treated in the same exact manner as the DAPI stained
samples. From each DAPI treated sample (and each autofluorescing control) 1 mL was
transferred to 4 mL of FS PBS. Each 5 mL solution was filtered onto a Millipore Isopore™
Membrane Filter 0.2 µm, 25 mm GTBP. Five milliliters of FS PBS was passed through each
filter to ensure affixing of all bacterial cells to the filters. The filters were mounted on clean
glass slides with Citifluor™, an anti-fade solution, and coverslips (22 by 22 mm) were placed on
each mounted filter. The preparations were examined under at a magnification of 400x with a
Nikon Microphot-FXA microscope under UV illumination (excitation filter BP 365nm). At least
20 fields, evenly distributed over the area beneath the coverslip, were examined for each sample,
25
and the blue fluorescing cells were counted. Focusing at different planes was done for each
field; this was especially necessary when counting cells in small clumps. Calculations of cell
numbers were made using the area of the filter (490.65 mm2) and microscopic field
measurements at 400x magnification (176 µm x 235 µm). At the magnification used, each filter
contains 11,913.61 fields, thus the average cell number per field was multiplied by 11,913.61.
The method used only accounts for 0.5 g of soil, thus the value was multiplied by two to convert
the value to cells per gram of soil. The computed results determine the quantity of cells
recovered from those added to 1 g of AT04-170 soil and consecutively serve to determine the
detection limit per gram of soil for the DAPI direct count procedure on the Atacama Desert soils
examined in this study.
The procedure above was repeated, but instead of allowing the AT04-170 soils to dry
overnight after the cell suspension or control (Nutrient Broth) was added, the soils were allowed
to dry for a month in an empty sterile Parafilmed® Petri dish on the countertop. The purpose of
this trial was to determine the effects of the Atacama Desert soil and extended desiccation on the
added cultured microbial cells and what differences were noticed (if any) in the ability for DAPI
to bind to these cells, consequently possibly changing the established detection limit.
LAKE WATER SAMPLE PREPARATIONS FOR DAPI STAINING CONTROL
Approximately 30 mL of water was collected from a Louisiana State University lake with
a clean sterile 50 mL centrifuge tube. Immediately after sample collection, the lake water was
brought back to the lab and 5 mL was transferred to a clean sterile 15 mL centrifuge tube with a
10 mL serological pipette in a clean bench environment. A 1/1 ratio of FS PBS containing 4%
(w/v) paraformaldehyde (5 mL) was transferred to the 5 mL of lake water within the 15 mL
centrifuge tube for at least 3 hours at 4ºC. Fifty microliters of FS DAPI (stock concentration of
26
1000 µg/mL and final concentration of 5 µg/mL) was added to the total 10 mL of cold filter FS
PBS containing 4% (w/v) paraformaldehyde and lake water. The solution was allowed to DAPI
stain for at least 3 hours at 4ºC. In three new clean sterile 15 mL tubes, the solution was split
into 7 mL, 2 mL, and 1 mL, which equated to roughly 3.5 mL of lake water, 1 mL of lake water,
and 0.5 mL of lake water. The solutions in each of the three 15 mL sample tubes were increased
to 5 mL with the addition of FS PBS. Each 5 mL solution was filtered onto a Millipore
Isopore™ Membrane Filter 0.2 µm, 25 mm GTBP. Five milliliters of FS PBS was passed
through each filter to ensure affixing of all bacterial cells to the filters. The filters were mounted
on clean glass slides with Citifluor™, an anti-fade solution, and coverslips (22 by 22 mm) were
placed on each mounted filter. The mounted filters were examined under at a magnification of
800x with a Nikon Microphot-FXA microscope under UV illumination (excitation filter BP
365nm).
DAPI STAINING PREPARATIONS FOR CONTROL SOIL SAMPLE
A soil sample was collected from a cornfield in close proximity to the Louisiana State
University campus. A Global Positioning System (GPS) device was used to record the
coordinates of the sample site. The soil sample was retrieved using a sterile plastic scoop and
sterile Nasco Whirl-Pak® bag. One gram of soil was fixed in 1 mL of cold FS PBS containing
4% (w/v) paraformaldehyde for at least 3 hours at 4ºC in a sterile 15 mL centrifuge tube. The
samples were centrifuged at 2,350 x g for 10 min. and the supernatant (fixing solution) removed
and discarded. The soil pellet was washed with 1 mL of FS PBS and vortexed gently. The
sample was centrifuged for 10 min. at 2,350 x g and the supernatant was removed and discarded.
An additional one mL of PBS was added to the soil pellet and vortexed gently. The tube was
centrifuged again at 650 x g for 1 min. to spin down larger soil particulates, but allow the cells to
27
remain suspended in the supernatant (Carman 1993). A volume of 250 µL of the supernatant
was transferred to a 15 mL centrifuge tube and 1.25 µL of DAPI (stock concentration of 1000
µg/mL and final concentration of 5 µg/mL) was added and allowed to incubate for at least 3
hours in the dark. The liquid extract in the 15 mL sample tube was increased to 5 mL with the
addition of 4.75 mL of FS PBS. The 5 mL solution was filtered onto a Millipore Isopore™
Membrane Filter (0.2 µm 25 mm GTBP). A 5 mL aliquot of FS PBS was passed through each
filter to ensure affixing of all bacterial cells to the filter. The filter was mounted on clean glass
slides with Citifluor™, an anti-fade solution, and a coverslip (22 by 22 mm) was placed on the
mounted filter. The mounted filter was examined under UV illumination (excitation filter BP
365nm) at 800x magnification with a Nikon Microphot-FXA microscope.
DETERMINATION OF SALINITY IN ATACAMA DESERT SOIL SAMPLES
All Atacama Desert samples and the control cornfield soil sample were tested for soil
salinity, which is a measure of the total amount of soluble salt in soil. One gram of soil for each
site was weighed into a clean sterile 50 mL centrifuge tube and 20 mL of water from a Millipore
Milli-Q® Ultrapure Water Purification System were added to each tube containing soil with a
sterile 10 mL serological pipette. Tubes were shaken for an hour at room temperature at
approximately 180 rpms. The 20 mL of water from each sample tube was transferred to a
sample solution cup and the salinity was measured with an Extech EC400 Waterproof ExStik
Conductivity/TDS/Salinity Meter. The salinity, TDS (total dissolved solids), and conductivity
for all soil samples were recorded and are displayed in Appendix D.
PH DETERMINATION
One gram of each soil sample was weighed into a clean sterile 15 mL centrifuge tube and
1 mL of water from a Millipore Milli-Q® Ultrapure Water Purification System was added to
28
each tube, and then shaken for 10 min. An IQ Scientific Instruments model 170 isfet pH probe
(non-glass & temp corrected) was placed into the centrifuge tube to obtain a pH reading for each
soil sample. Duplicate readings were acquired for samples AT03-34, AT03-36, AT03-44, AT04-
169, AT04-150 10 cm, AT04-150 70 cm, AT04-152 20 cm, AT04-152 70 cm, AT04-152 80 cm,
AT04-153 10 cm, AT04-153 30 cm, AT04-153 40 cm, AT04-153 80 cm, AT04-159 surface, and
AT04-159 40 cm to determine if there were significant differences between each reading. After
24 hours, pH readings for samples AT04-163, AT04-166, AT04-150 80 cm, and AT04-152 80
cm were taken again to determine if there was a significant difference between readings taken
the previous day. Also, to detect any variance between pH readings taken immediately after
addition of distilled water and readings taken after samples (including distilled water) had shaken
for 10 min., pH readings for samples AT04-163, AT04-166, and AT04-150 80 cm were acquired
as distilled water was added.
DETECTION OF CARBONATE PRESENCE IN SELECTED SAMPLES
Approximately five drops of dilute hydrochloric acid (HCl) (approximately a 1/10
dilution) were added to approximately 0.5 g of dry samples AT04-167, AT04-152 70 cm, AT04-
153 30 cm to detect carbonation (bubbles) indicating presence of carbonates.
ELEMENTAL ASSAY ON ATACAMA DESERT SOILS
Dr. Robert Gambrell and associates of his laboratory completed metal analyses for every
surface and subsurface soil sample of Al, As, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, Si,
and Z (mg/L). The protocol used was on approximately 1 g of soil material that was weighed
into a 100 mL Pyrex test tube with aggregates broken up with a glass rod, if necessary. Five
milliliters of concentrated, trace metals grade nitric acid was added. A small glass funnel was
placed in the top of each tube to facilitate reflux action of the acid during the heating step to keep
29
the acid from evaporating. The tubes were put on a block digester at approximately 120º C for
about 8 hours. The next day, the glass funnels were removed and the block temperature
increased to 120º C again to evaporate the acid down to an approximate volume of 1.5 to 2 mL.
At this point, the tubes were removed from the block, allowed to cool, then the volume brought
to 50 mL with de-ionized water and the tubes were capped with parafilm. The contents
(acidified water and extracted soil solids) were mixed vigorously, then allowed to settle
overnight. The solids settled to the bottom and a clear supernatant appeared above the settled
solids. Part of the clear supernatant was poured into approximately 15 mL plastic tubes and
placed on the autosampler for a Varian MPX Inductively Coupled Plasma (ICP) Emission
Spectrometer. Also analyzed were two reagent blanks (diluted acid) and replicate extractions of
approximately 20% of the samples to demonstrate reproducibility. For metals present in
concentrations greater than the linear range of the instrument, dilutions were made and the
samples reanalyzed. The results were reported in mg/g, and were determined by multiplying the
extract concentration by 50 mL and by any applicable dilution factor, then dividing by the initial
soil sample weight. Table 6.1 displays the concentrations (mg/L) of the elemental components
for each soil sample.
INORGANIC ANION ANALYSIS
Sarah Jones at the Institute for Ecological Infrastructure Engineering and Water Quality
Laboratory at the College of Engineering at Louisiana State University performed anion analysis
on all Atacama Desert and cornfield soil samples. Samples were given to Sarah Jones in the
manner of 1 g of soil weighed into a clean sterile 50 mL centrifuge tube and 50 mL of water
from a Millipore Milli-Q® Ultrapure Water Purification System added to each tube, then shaken
overnight at room temperature at approximately 180 rpms. A Dionex IC25 Ion Chromatograph
30
and LC20 Chromatography Enclosure with an AS14A column (these two instruments make up
the DX 320 IC process analyzer) were used for the analysis. An AS14A eluent (purchased from
Dionex) was prepared with water filtered through a 0.2 µm membrane according to the directions
on the concentrate bottle: 8 mM sodium carbonate, 1 mM sodium bicarbonate, and 10 mL
Dionex AS14A. Samples were added to Dionex 5 mL autosampler vials, inserted into the filter
caps, and placed in the autosampler cassette. The eluent along with 25 µL of each sample was
run for 18 min. The latest five point standard curve for each anion (PO4, F, Br, Cl, NO3, SO4,
and NO2) was used to calculate the anion concentration. The anions ion chromatography
procedure is based on the EPA Method 300.0 (Determination of Inorganic Anions by Ion
Chromatography). Table 6.2 (chloride, fluoride, nitrate, and sulfate) and Appendix C
(phosphate, nitrite, and bromide) display the concentrations (mg/L) of analyzed anions present in
each soil sample.
31
CHAPTER 3 QUANTIFYING AND IDENTIFYING CULTURABLE HETEROTROPHIC BACTERIA
IN THE SURFACE SOILS OF THE ATACAMA DESERT
It was shown in a previous study of a limited number of samples that the surface soils
within this hyper-arid region of the Atacama Desert contained low numbers of or no culturable
heterotrophic bacteria (Narvarro-González et al. 2003). In order to examine the extent of these
soils containing low numbers of culturable heterotrophic bacteria, 33 additional samples in the
Yungay region were studied. It was hypothesized that the distribution, in terms of abundance
and diversity, of culturable bacteria in the surface soils would not be uniform and that patchiness
would be observed, similar to what has been observed for plant species in desert soils that
support plant growth (Rietkerk et al. 2004).
RESULTS
Colony Forming Units
Thirty-three accessible surface sites within the Yungay region were studied (Table 3.1).
Using the culture-dependent technique of dilution plating, a wide range of CFU/g values were
observed for the samples. Figure 3.1 shows the distribution of surface sites and the CFU values
determined for each site.
The surface site CFU data (Table 3.2 and Figure 3.2) showed that for all media, the
values from range 0 to 7.4 x105 CFUs/g of soil. Of the 33 sites, 22 showed no CFUs/g for at
least one of the media. Four sites showed no CFUs/g on all five media. Twenty of the 33 sites
had values for all five media of < 1 x 103 CFUs/g. Seven of the 33 sites had CFU/g values of > 1
x 104 and one of these (AT04-166) had CFU/g values of >105 (Table 3.3). The largest number of
samples showing no detectable CFUs/g were those plated on PCA (16 samples), while the lowest
number of samples with no detectable CFUs/g were those plated on 1/100 PCA (10 samples).
32
Samples plated on PCA consistently had lower CFU values (Table 3.3). For 1/100 PCA, higher
CFU values were found overall, with less samples showing CFU/g of 0 to 103 than those plated
on other culture media (Table 3.3). The highest CFU/g value of all samples that of site AT04-166
and was obtained on 1/10 PCA (Table 3.3).
Soil samples AT03-35 and AT04-170 were chosen for the experimental plating on media
differing in salt concentrations. AT03-35 represented sites with very low counts (i.e., below the
detection limit) and AT04-170 represented sites with 0 CFUs/g on all five principal media. After
35 days of incubation, AT04-170 showed 0 CFUs/g of soil on all media varying in marine salt
concentrations. After 35 days of incubation, AT03-35 had no CFUs/gram of soil on MAIO, 3.17
x 103 CFUs/gram of soil on 1X IO, 44.4 CFUs/gram of soil on 2X IO, and no CFUs/gram of soil
on 3X IO. More cells in sample AT03-35 were capable of being cultured on media prepared
with 1X IO than any other of the five key media and all other media containing varying salt
concentrations. The highest CFU value for AT03-35 on the five primary media occurred on the
culture medium MA with 4.44 x 102 CFUs/gram of soil. Colonies that appeared on the culture
media with 1X IO were all very similar in morphology to colonies observed on MA.
Soils from sites AT03-35 and AT04-170 were also plated onto the five media used in this
study (MA, NA, PCA, 1/10 PCA, 1/100 PCA) for a second time to determine if additional
colonies would grow after 20 days of incubation, i.e. to recover any slow-growing culturable
heterotrophic prokaryotes. Plates were allowed to incubate for 25 days beyond the original
dilution plates that were only allowed to incubate for a total of 20 days before colonies were
isolated. After 45 days of incubation, no further colonies had grown past 20 days.
Soils from sites AT04-163, AT04-158, and AT04-164 were plated on MA and 1/10
strength PCA media at pH 5.0 to determine whether a more acidic culture media would improve
33
Table 3.1: Degree minute decimal coordinates for surface sites within the core arid region of Yungay
Surface Sample Site West (minutes) South (minutes)
AT03-33 69º W 51.840′ 24º S 4.112′ AT03-34 69º W 51.806′ 24º S 4.103′ AT03-35 69º W 51.828′ 24º S 4.008′ AT03-36 69º W 51.854′ 24º S 3.836′ AT03-37 69º W 51.891′ 24º S 3.734′ AT03-38 69º W 52.088′ 24º S 3.645′ AT03-39 69º W 52.187′ 24º S 3.548′ AT03-40 69º W 52.451′ 24º S 3.637′ AT03-41 69º W 53.091′ 24º S 3.666′ AT03-42 69º W 54.649′ 24º S 3.445′ AT03-43 69º W 54.637′ 24º S 3.445′ AT03-44 69º W 54.496′ 24º S 3.695′ AT03-45 69º W 51.854′ 24º S 4.151′ AT03-46 69º W 51.897′ 24º S 4.151′ AT03-48 69º W 52.194′ 24º S 4.875′ AT03-49 69º W 52.632′ 24º S 4.545′ AT03-50 69º W 52.926′ 24º S 4.452′ AT04-151 69º W 51.858′ 24º S 3.720′ AT04-154 69º W 51.415′ 24º S 3.613′ AT04-155 69º W 51.445′ 24º S 3.728′ AT04-156 69º W 51.438′ 24º S 3.655′ AT04-157 69º W 51.495′ 24º S 3.548′ AT04-158 69º W 51.453′ 24º S 3.415′ AT04-161 69º W 54.065′ 24º S 4.440′ AT04-162 69º W 53.465′ 24º S 4.200′ AT04-163 69º W 52.925′ 24º S 3.900′ AT04-164 69º W 52.560′ 24º S 3.660′ AT04-165 69º W 52.680′ 24º S 4.140′ AT04-166 69º W 52.260′ 24º S 4.200′ AT04-167 69º W 51.480′ 24º S 4.800′ AT04-168 69º W 51.660′ 24º S 4.500′ AT04-169 69º W 53.580′ 24º S 3.900′ AT04-170 69º W 54.300′ 24º S 4.320′
34
AT03
-48
AT03
-33
AT03
-34*
AT03
-35
AT03
-36*
AT03
-37*
AT03
-38*
AT03
-39*
AT03
-40
AT03
-41
AT03
-42*
AT03
-43*
AT03
-44*
AT03
-45
AT03
-46
AT03
-49*
AT03
-50
AT04
-151
*
AT04
-154
*
AT04
-155
*
AT04
-156
*
AT04
-157
*
AT04
-158
*
AT04
-161
AT04
-162
*
AT04
-163
AT04
-164 AT
04-1
65 AT04
-166
AT04
-167
*
AT04
-168
AT04
-169
AT04
-170
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
50.0
50.5
51.0
51.5
52.0
52.5
53.0
53.5
54.0
54.5
55.0
Long
itiud
e - 69
º W (m
in.)
Latit
ude -
24
º S (m
in.)
Figu
re 3
.1:
Scat
ter p
lot i
llust
ratin
g pr
oxim
ity o
f sur
face
site
s in
core
arid
regi
on a
nd d
istri
butio
n of
CFU
s.
The
red
circ
les r
epre
sent
site
s with
no
(zer
o C
FUs/
g on
all
med
ia) h
eter
otro
phic
cul
tura
ble
bact
eria
l gro
wth
. Th
e pi
nk sq
uare
s re
pres
ent t
he si
tes w
ith lo
w c
ount
s bel
ow th
e de
tect
ion
limit
(CFU
s/g ≤
102 on
all
med
ia),
and
blue
squa
res r
epre
sent
site
s with
co
lony
num
bers
nea
r or s
light
ly a
bove
the
dete
ctio
n lim
it (C
FUs/
g be
twee
n 10
2 and
104
on a
t lea
st o
ne m
ediu
m).
The
gre
en
trian
gles
repr
esen
t the
site
s with
hig
h ba
cter
ial C
FUs (
CFU
s/g
of 1
04 or g
reat
er, o
n at
leas
t one
med
ium
).
* si
tes h
avin
g ze
ro c
olon
ies o
n at
leas
t one
med
ium
.
35
Table 3.2: Surface sample pH values and CFUs/g of soil on five media after 20 days incubation * average of duplicate pH
Media Surface Sites pH MA 1/10 PCA 1/100 PCA PCA NA
AT03-33 7.30 1.39 x 104 1.17 x 104 2.45 x 103 2.22 x 102 1.06 x 104 AT03-34 6.76* 67 0 22 1.11 x 102 0 AT03-35 7.22 4.44 x 102 3.78 x 102 2.22 x 102 67 89 AT03-36 6.48* 44 3.33 x 102 22 0 67 AT03-37 7.22 22 0 1.56 x 102 0 67 AT03-38 7.11 0 0 1.78 x 102 44 0 AT03-39 7.61 5.78 x 102 3.20 x 103 7.78 x 102 0 67 AT03-40 7.24 6.49 x 104 1.97 x 104 4.47 x 104 67 1.09 x 104 AT03-41 7.91 1.19 x 104 3.27 x 103 8.73 x 103 2.67 x 102 5.53 x 103 AT03-42 7.08 22 0 0 22 0 AT03-43 7.54 6.67 x 102 2.00 x 102 3.33 x 102 0 2.00 x 102 AT03-44 7.01* 0 22 0 0 0 AT03-45 7.35 2.47 x 103 2.89 x 102 7.78 x 102 22 9.33 x 102 AT03-46 7.09 1.11 x 104 4.85 x 103 1.43 x 104 8.87 x 103 7.40 x 103 AT03-48 7.56 1.76 x 103 1.40 x 103 1.04 x 103 0 1.33 x 102 AT03-49 7.11 0 0 0 22 0 AT03-50 7.42 3.11 x 102 1.56 x 102 67 1.11 x 102 44 AT04-151 7.16 3.56 x 102 67 3.11 x 102 0 0 AT04-154 7.58 0 0 1.62 x 103 0 0 AT04-155 7.58 0 0 8.44 x 102 22 0 AT04-156 7.22 22 0 7.33 x 102 0 0 AT04-157 7.79 0 22 0 0 22 AT04-158 7.49 67 67 67 0 22 AT04-161 7.97 1.77 x 104 1.70 x 104 1.78 x 104 1.49 x 103 5.15 x 103 AT04-162 8.46 3.91 x 103 2.66 x 102 0 22 2.67 x 102 AT04-163 8.21 0 0 0 0 0 AT04-164 7.55 1.22 x 103 2.18 x 103 8.44 x 102 3.33 x 102 2.02 x 103 AT04-165 8.02 6.49 x 104 8.20 x 104 5.07 x 104 5.82 x 103 4.20 x 104 AT04-166 7.38 6.80 x 105 7.40 x 105 4.67 x 105 1.91 x 104 6.65 x 105 AT04-167 8.23 0 0 0 0 22 AT04-168 7.37 0 0 0 0 0 AT04-169 8.15* 0 0 0 0 0 AT04-170 8.21 0 0 0 0 0
36
1.E+0
1
1.E+0
2
1.E+0
3
1.E+0
4
1.E+0
5
CFUs
/Gr
am of
Soil
AT03-33AT03-34AT03-35AT03-36AT03-37AT03-38AT03-39AT03-40AT03-41AT03-42AT03-43AT03-44AT03-45AT03-46AT03-48AT03-49AT03-50AT04-151AT04-154AT04-155AT04-156AT04-157AT04-158AT04-161AT04-162AT04-163AT04-164AT04-165AT04-166AT04-167AT04-168AT04-169AT04-170
Samp
le Sit
es
PCA
1/10 P
CA
1/100
PCA
MA
NA
Figu
re 3
.2:
CFU
s/g
of so
il fo
r eac
h of
the
33 su
rfac
e sa
mpl
es o
n fiv
e m
edia
37
Table 3.3: Values of CFU/g recovered on various culture media for the 33 sites sampled
Culture Media CFU/g of soil / number of sites MA 1/10 PCA 1/100 PCA PCA NA
0 11 13 10 16 13 <102 17 17 14 24 21 <103 22 23 24 29 25 <104 26 28 28 32 29 <105 32 32 32 33 32 >105 1 1 1 0 1
38
organism cultivation because Richard Quinn at the NASA Ames Research Center reported that
Yungay soils have an acidic pH 5. AT04-163 represented sites with zero CFUs on all principal
media, AT04-158 represented sites with very low counts, below the detection limit (CFUs below
103 on all five major media), AT04-164 represented sites with CFUs near or slightly above the
detection limit (CFUs between 103 and 104 on at least one medium). After 20 days of incubation,
all three representative surface sample sites showed zero CFUs/g of soil on the acidic media.
Regarding the results of this experiment, which suggest that organisms from the Yungay region
soils prefer conditions having a more neutral pH, I chose to determine the pH of all surface and
soil pit samples (soil pit pH readings in Table 4.1). The pH range for the surface samples was
found to be from 6.30 to 8.46, with a mean pH 7.5. Therefore, the Yungay surface soils were
found to have a more neutral pH than originally reported (Table 3.2). Selected sample pH
readings were obtained in duplicates to confirm accuracy of pH readings: AT03-34 (6.65 and
6.87), AT03-36 (6.30 and 6.66), AT03-44 (6.90 and 7.12), AT04-169 (8.15 and 8.15). The pH
for selected samples was determined again 24 hours after the first reading. The sample AT04-
163 had pH 8.21, and 24 hours later, the pH reading was 8.22. Sample AT04-166 had pH 7.38,
and 24 hours later, the pH reading was 7.45. Thus, there was no significant difference in the pH
readings obtained over a 24 hour period. Also, pH values as distilled water was added to the
chosen soil samples were recorded. The sample AT04-163 had pH between 8.25 and 8.17 at the
time water was added; the sample AT04-166 had pH between 7.65 and 7.26 at the time water
was added. Hence, the pH readings for the 33 surface samples were concluded to be consistent
despite if a reading was obtained as water was added to soil, 10 min. after water was added, or 24
hours after water was added (pH readings for soil pit samples were also consistent regardless if a
39
reading was obtained as water was added to soil, 10 min. after water was added, or 24 hours after
water was added).
Diversity of Culturable Bacteria
From each of the 33 sites, isolates were recovered and identified to examine the diversity
of culturable heterotrophic bacteria in the soils. The identity of the isolates was based on
comparison of partial 16S rRNA gene sequences (400 to 1100 nucleotide positions) with the
GenBank database using the BLAST tool. A total of 859 isolates were recovered from the 33
soil samples studied (Figure 3.3). The majority of the isolates (833) recovered from the 33
surface soils belong to the Actinobacteria phylum. The remaining 26 isolates were divided
between the two phyla, Proteobacteria (5 isolates) and Firmicutes (19 isolates). The total number
of isolates recovered from each soil sample and identified to the genus/species level was related
to the CFU count of that sample, as well as the successful subculturing of the initial isolate.
Therefore, in some cases only a single isolate was recovered and maintained for some of the soil
samples that had very low CFU values. The list of isolates, colony characteristics and 16S rRNA
gene sequence based identity are provided in Appendix B. The summary of the identities (Figure
3.4) is based on closest relatives provided by the BLAST search and are for the most part
identities at the genus level although in many cases the closest species identity is provided.
Figure 3.4 only includes isolates whose closest relatives were identified to at least genus level
according to BLAST result from GenBank® database.
The majority of the isolates recovered from the surface soils are strains of the genus
Blastococcus. These 623 isolates identified as members of the genus Blastococcus came from 24
of the 28 soils analyzed. In addition to Blastococcus spp., 11 of the 33 soil samples contained
species of the genera Geodermatophilus and Modestobacter, which are taxonomically close
40
483
81
122
161
84
112
159
198
11
13012689
211
213
15311914010
376
227
152
31
0%20
%40
%60
%80
%10
0%
Perc
entag
e
AT03
-33AT
03-34
AT03
-35AT
03-36
AT03
-37AT
03-38
AT03
-39AT
03-40
AT03
-41AT
03-42
AT03
-43AT
03-44
AT03
-45AT
03-46
AT03
-48AT
03-49
AT03
-50AT
04-15
1AT
04-15
4AT
04-15
5AT
04-15
6AT
04-15
7AT
04-15
8AT
04-16
1AT
04-16
2AT
04-16
3AT
04-16
4AT
04-16
5AT
04-16
6AT
04-16
7AT
04-16
8AT
04-16
9AT
04-17
0
Surfa
ce
Samp
le Si
te
Actin
obac
teria
Firmi
cutes
Prote
obac
teria
Figu
re 3
.3:
Div
ersi
ty a
t the
phy
la le
vel w
ithin
surf
ace
sam
ples
Id
entit
y of
the
clos
est r
elat
ives
is b
ased
on
com
paris
on o
f par
tial 1
6S rR
NA
gen
e se
quen
ces (
400
to 1
100
nucl
eotid
e po
sitio
ns)
with
the
Gen
Ban
k da
taba
se u
sing
the
BLA
ST to
ol.
The
num
bers
with
in e
ach
bar r
epre
sent
the
num
ber o
f iso
late
s sel
ecte
d an
d id
entif
ied
to p
hylu
m le
vel f
rom
that
par
ticul
ar si
te.
41
371
21
15
21
13
12
12
71
213
21
25
11
11
121
545
177
93
111
31
11
122
22
120
13
822
13
119
21
11
94
15
11
112
41
120
21
81
411
82
342
21
13
11
214
11
751
912
1141
123
1611
1
0%20
%40
%60
%80
%10
0%
Perc
enta
ge
AT03
-33AT
03-34
AT03
-35AT
03-36
AT03
-37AT
03-38
AT03
-39AT
03-40
AT03
-41AT
03-42
AT03
-43AT
03-44
AT03
-45AT
03-46
AT03
-48AT
03-49
AT03
-50AT
04-15
1AT
04-15
4AT
04-15
5AT
04-15
6AT
04-15
7AT
04-15
8AT
04-16
1AT
04-16
2AT
04-16
3AT
04-16
4AT
04-16
5AT
04-16
6AT
04-16
7AT
04-16
8AT
04-16
9AT
04-17
0
Surfa
ceSa
mple
Site
s
Blas
toco
ccus
sp.
Bacil
lus s
p.No
card
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es sp
.M
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acter
ium
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erma
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ilus s
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r sp.
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s Sp
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42
relatives of Blastococcus. The remaining actinobacterial species were representatives of a
number of taxa including species of the genera Arthrobacter, Cellulomonas, Frankia, Georgenia,
Kocuria, Micrococcus, Mycobacterium, Nocardioides, Promicromonospora, Rothia,
Streptomyces, and Williamsia. The 21 species of the low G+C Gram positive phylum (the
Firmicutes) represent a range of genera that fall within the order Bacilliales, including species of
the genera Bacillus, Brevibacillus, Exigubacterium, Halobacillus, Ornithinicoccus,
Paenibacillus, and Staphylococcus. Representatives of only four genera within the
Proteobacteria were isolated from the surface soil samples, namely Acinetobacter, Oxalobacter,
Pantoea, and Sphingomonas represented a total of five isolates (Figure 3.4).
DISCUSSION
Patchiness in the distribution of CFUs/g is clearly shown in Figure 3.1. There is no
obvious explanation for the observed patchiness throughout the surface of the Yungay region
with respect to the proximity of one site to another. Microbes obtain nutrients from the
environment in which they inhabit; thus, it can be supposed that microbial populations are
located in the core arid region directly as a result of the availability of resources throughout the
soils. It has been reported that the crucial requirement for self-organized patchiness to develop is
that organisms are positively correlated with resource abundance at short spatial range, but
negatively correlated at long spatial range (Rietkerk et al. 2004). This idea supports the short
range patchiness seen in the 15.3 km2 of the core arid region of the Atacama Desert.
Wind currents are known it move across the Atacama Desert from the Pacific Ocean. It
can be considered that marine salts could be carried by these winds and deposited in the inland
desert region. The two samples that were plated onto media containing different salt
concentrations were AT03-35, representing sites with low counts below the detection limit
43
(CFUs <103 on all five major media), and AT04-170, representing sites with zero CFUs on all
five principal media. The sample AT04-170 showed zero colonies on all media including media
varying in marine salt concentrations. The sample AT03-35 showed a higher CFU value on
media containing 1X Instant Ocean® than on any other culture media. The colonies viewed on
the 1X IO medium had the same morphology as colonies observed on all other media; the only
apparent difference was the number of colonies. These finding may indicate that recoverable
heterotrophic organisms that exist in Yungay soils prefer to mature in an environment having
slightly high marine salt concentrations due to in situ conditions of rare marine salt moisture
events.
In order to recover any slow-growing culturable heterotrophic bacteria two surface
samples (AT03-35 and AT04-170) were plated and incubated for 45 days. No additional
colonies were observed after 20 days of incubation indicating that the culturable heterotrophic
bacteria from the collected Yungay surface samples are capable of becoming active and
replicating within 20 days on the five culture media selected for this study.
According to Richard Quinn of the NASA Ames Research Center in Moffett Field,
California, the Atacama Desert soil within the core arid region had a pH value of 5.0. Three
surface soils were plated onto 1/10 PCA (a dilute nutrient medium) and MA (a nutrient rich
marine medium) at a pH 5.0 in order to consider whether or not culturable bacteria from this soil
would grow better at acidic pH. Regarding the findings, organisms existing within the hyper-
arid soils grow better under neutral pH conditions. In order to provide substantiating evidence
for the results, I performed pH analyses for all soil samples (surface and subsurface). The results
are consistent for the surface and soil pit samples; they contradict the initial pH report and
44
indicate that the hyper-arid soils have a neutral pH. Also, the results suggest that the
heterotrophic culturable bacteria inhabiting the soils are neutrophilic organisms.
For each of the 33 sites, isolates were recovered and identified to compare every site’s
bacterial diversity (Figures 3.3 and 3.4). The number of isolates from each sample in the phyla
and closest relative charts may not match exactly due to the fact that the BLAST results do not
always give a closest relative down to genus, the results are sometimes only defined to phylum
or family. In the closest relative charts diagramed in this study, only isolates that are identified
particular to genera are included.
There is low diversity among the culturable heterotrophic bacteria recovered from surface
soils (Figures 3.3 and 3.4). The majority of the culturable bacteria from surface samples
belonged to the genus Blastococcus, a member of the Actinobateria phylum. This group is the
most prevalent of the recoverable microorganisms from the arid regions of the Atacama Desert
using the dilution plating method. Blastococcus is a member of the family
Geodermatophilaceae, with the other genera Geodermatophilus and Modestobacter, which were
also recovered Yungay surface samples (Geodermatophilus was also found in Yungay
subsurface samples). Blastococcus is a Gram-positive organism whose name in Greek means
“sprouting berry”. Strains showing the highest similarity to Blastococcus aggregatus was found
in nine surface samples, along with strains related to Blastococcus sp. strains BC448, which is
the most abundant strain observed throughout this study, BC412, BC521, and BC512. Colonies
of the genus Blastococcus are various shades of pink or orange, are round, vary in size depending
on the media, and are smooth to rough in surface texture. To determine cell morphology,
photomicrographs of Blastococcus sp. strains AT03-36-10 and AT03-37-10 were taken using
DIC microscopy, DAPI epifluorescence microscopy, and SEM (Figure 3.5). Cells occurred
45
Figure 3.5: Photomicrographs depicting cell morphology of Blastococcus aggregatus DAPI-stained B. aggregatus (A), DIC photomicrograph of B. aggregatus (B), and scanning electron micrographs of B. aggregatus (C and D).
B
A
budding cell
46
Figure 3.5: (Continued)
C
D dividing cell
detached bud
47
singly, in pairs, tetrads, and often formed aggregates (Figure 3.5). Cells ranged from
approximately 0.5 to 2.0 µm in diameter, which is very close to the diameter range for the genus
Blastococcus reported in Urzì et al. (2004) of 0.5-1.7 µm. Under the conditions used, bud
formation was observed, motile spores were produced, elongated cells formed, and germ tubes
and long filaments were produced. Urzì et al. (2004) describe that strains BC412 and BC521
also bud, form elongated cells, and produce germ tubes and long filaments. Blastococcus
aggregatus was first discovered by Ahrens and Moll (1970) in a sample recovered from the
Baltic Sea. Members of this genus have mostly been isolated from arid environments.
The increase in the numbers of culturable bacteria among sites did not always correlate to
an increase in diversity. The sample site AT04-166, which had the highest CFU value and from
which the highest number of isolates could be recovered, shows more diversity than any of the
other sites, but about 50% of the culturable heterotrophic bacteria recovered from AT04-166 are
members of the genus Blastococcus. Sites with higher CFU values did not have more isolates
identified, as isolates were recovered based on the different colony morphologies. If a site had
elevated CFU values, but all colonies were identical in morphology, then fewer colonies were
selected for isolation and identification.
Surface samples studied in the Yungay region showed overall low numbers of
recoverable prokaryotes, yet patchiness in value for CFUs/g of soil was observed. Low diversity
among the culturable heterotrophic bacteria was detected. Also, the abundance of recoverable
cells occurred erratically throughout the surface samples. These observations give new insights
to what could have occurred or may currently take place in Martian soils, thus Mars’ surface
explorations should sample a vast area at as many possible sites before any conclusions are made
regarding the microbiology past or present of the surface layers of soils on Mars.
48
CHAPTER 4 HETEROTROPHIC BACTERIA BENEATH THE SURFACE
NASA intends to send several missions to Mars in the upcoming years. At least two of
these missions will focus on subsurface Martian soils; hence, it is also important to explore the
subsurface soils of environments analogous to Mars. For this reason, the subsurface soils in the
hyper-arid core region, the Yungay region, of the Atacama Desert were investigated. Four soil
pits were excavated with depths of 40 cm to 90 cm to examine the subsurface microbial
communities. Three of these soil pits were excavated in the hyper-arid region of the Atacama
Desert. An additional soil pit was constructed in a more southern region of the Atacama Desert,
known as Altamira, as a comparison with the pits in the Yungay region. Samples were collected
at the surface and at 10 cm intervals from the surface. All samples were examined using the
previously described culture-dependent techniques. The identity of pure-culture isolates,
selected on the basis of their availability or novel colony morphology, was determined by 16S
rRNA gene sequence determination.
RESULTS
Colony Forming Units
Three soil pits (AT04-150, AT04-152, and AT04-153) were excavated in the Yungay
region of the Atacama Desert. Soil pit AT04-150 was located at 69º W 51.847′ and 24º S 3.672′
to a total depth of 90 cm. Soil pit AT04-152 was located at 69º W 51.858′ and 24º S 3.745′ to a
total depth of 80 cm. The soil pit designated AT04-153 at 69º W 51.423′ and 24º S 3.585′ to a
total depth of 80 cm. The fourth soil pit, which was constructed at a southern site with sparse
vegetation, was constructed at 70º W 11.758′ and 25º S 45.543′ to a depth of 40 cm.
49
The hyper-arid region subsurface CFU/g data are shown in Table 4.1 and Figure 4.1. The
CFU/g values for all media range from 0 to 8.07 x 103. Of the 28 layers from the three hyper-
arid pits, 27 showed no CFUs/g on at least one of the media used for detection of culturable
bacteria. There were seven layers that showed no CFUs/g on all five media (Table 4.1).
Twenty-six layers from the three Yungay pits had CFU/g values for all five culture media of < 1
x 103/g of soil and none of the 28 layers had CFU/g values of > 1 x 104. The largest number of
layers showing no detectable CFU/g were those plated on 1/100 PCA (25 layers) while the
lowest number of layers showing no detectable CFU/g were those plated on MA (11 samples).
Most CFU values were below the detection limit of the dilution plating method, but two high
values were observed (Table 4.1). The soil pit AT04-150 had 8 x 103 CFUs/g occurring at a
depth of 40 cm, while the soil pit AT04-152 has a slightly higher culturable population at 4 x 103
CFU/g at 10 cm below the surface; both of these were observed on MA culture medium (Table
4.1).
The CFUs/g of subsurface layers in the southern pit AT04-159, range from 6.4 x 103 to
1.86 x 106 for all media (Table 4.1 and Figure 4.1). Of the five layers of pit AT04-159, only the
surface layer displayed < 1 x 104 CFU/g on the culture medium PCA. The depths of 30 cm and
40 cm have CFU/g values of > 1 x 106 per gram of soil on at least one of the five culture media
used for detecting culturable bacteria. On MA culture medium, two layers (surface and 10 cm)
show CFU/g of soil > 1 x 104 and < 1 x 105, one layer (20 cm) shows CFU/g of soil > 1 x 105 and
< 1 x 106, and two layers (30 cm and 40 cm) show CFU/g of soil > 1 x 106 and < 1 x 107. On NA
culture medium, three layers (surface, 10 cm, and 20 cm) show CFU/g of soil > 1 x 104 and < 1 x
105, one layer (30 cm) shows CFU/g of soil > 1 x 105 and < 1 x 106, and one layer (40 cm) shows
CFU/g of soil > 1 x 106 and < 1 x 107. On PCA culture medium, one layer (surface) shows
50
Table 4.1: CFUs/g of soil for all four soil pits sampled on five media * average of duplicate pH
Media Soil Pit Depth (cm) pH PCA 1/10 PCA 1/100 PCA MA NA
AT04-150 0 7.25 0 0 0 0 0 10 7.76* 0 0 0 22 44 20 7.71 0 0 0 89 0 30 7.84 0 0 0 1.78 x 102 0 40 7.91 44 0 0 8.07 x 103 0 50 7.96 0 0 0 1.56 x 102 0 60 8.02 0 0 0 0 0 70 7.95* 1.56 x 102 0 0 0 0 80 8.08 89 0 0 0 0 90 8.20 0 0 0 0 0
AT04-152 0 7.16 22 0 0 0 22 10 7.80 1.18 x 103 4.44 x 102 6.00 x 102 3.96 x 103 4.22 x 102 20 7.86* 0 0 22 1.78 x 102 22 30 7.67 22 0 0 3.78 x 102 0 40 8.01 0 1.11 x 102 0 67 0 50 7.93 0 0 0 0 0 60 8.03 0 0 0 44 0 70 7.87* 0 0 0 22 0 80 7.77* 0 0 0 0 0
AT04-153 0 7.52 0 3.11 x 102 1.11 x 102 5.11 x 102 67 10 7.94* 0 0 0 44 0 20 7.88 1.56 x 102 0 0 22 0 30 7.79* 0 0 0 67 0 40 8.27* 0 0 0 0 0 50 8.09 22 0 0 0 0 60 8.14 0 0 0 0 0 70 8.02 0 22 0 67 22 80 7.86* 22 22 0 22 0
AT04-159 0 7.99* 6.40 x 103 5.51 x 104 1.08 x 105 6.80 x 104 4.09 x 104 10 7.60 6.47 x 104 1.04 x 105 1.50 x 105 8.95 x 104 8.98 x 104 20 7.54 9.96 x 104 1.53 x 105 1.16 x 105 4.33 x 105 9.96 x 104 30 7.70 4.87 x 105 1.53 x 106 5.56 x 105 1.60 x 106 3.91 x 105 40 7.85* 1.09 x 106 1.53 x 106 1.80 x 106 1.54 x 106 1.29 x 106
51
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
CFUs/gram of soil
0 10 20 30 40 50 60 70 80 90
Depth below surface (cm)
Soil Pit AT04-150 CFUs/g
PCA1/10 PCA1/100 PCAMANA
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
CFUs/gram of soil
0 10 20 30 40 50 60 70 80
Depth below surface (cm)
Soil Pit AT04-152 CFUs/g
PCA1/10 PCA1/100 PCAMANA
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
CFUs/gram of soil
0 10 20 30 40 50 60 70 80
Depth below surface (cm)
Soil Pit AT04-153 CFUs/g
PCA1/10 PCA1/100 PCAMANA
1.00E+001.00E+011.00E+02
1.00E+031.00E+041.00E+051.00E+061.00E+07
CFUs/gram of soil
0 10 20 30 40
Depth below surface (cm)
Soil Pit AT04-159 (Altamira Soil Pit) CFUs/g
PCA1/10 PCA1/100 PCAMANA
Figure 4.1: CFUs/g of soil for layers within each soil pit on five media
52
CFUS/g of soil > 1 x103 and < 1 x 104, two layers (10 cm and 20 cm) show CFU/g of soil > 1 x
104 and < 1 x 105, one layer (30 cm) shows CFU/g of soil > 1 x 105 and < 1 x 106, and one layer
(40 cm) shows CFU/g of soil > 1 x 106 and < 1 x 107. On 1/10 PCA culture medium, one layer
(surface) shows CFU/g of soil > 1 x 104 and < 1 x 105, three layers (10 cm, 20 cm, and 30 cm)
show CFU/g of soil > 1 x 105 and < 1 x 106, and one layer (40 cm) shows CFU/g of soil > 1 x 106
and < 1 x 107. On 1/100 PCA culture medium, four layers (surface, 10 cm, 20 cm, and 30 cm)
show CFU/g of soil > 1 x 105 and < 1 x 106 and one layer (40 cm) shows CFU/g of soil > 1 x 106
and < 1 x 107. This outlined data is also exhibited in Table 4.2.
Diversity of Culturable Heterotrophic Bacteria Isolated from the Soil Pits
Isolates were selected based on the criterion of their availability and colony morphology
uniqueness. Isolates were identified using the 16S rRNA gene sequence. Of the three Yungay
pits, 3 Firmicutes, 3 Proteobacteria, and 25 Actinobacteria were recovered from AT04-150; 2
Firmicutes, 4 Proteobacteria, and 98 Actinobacteria were recovered from AT04-152; and 1
Firmicutes, 1 Proteobacteria, and 27 Actinobacteria were recovered from AT04-153 (Figures 4.2,
4.3, and 4.4). Based on these data, there was not variation in the diversity among individual
layers. The most commonly observed taxa recovered from the Yungay pits belonged to the
genera Actinospora, Amycolatopsis, Arthrobacter, Bacillus, Blastococcus, Brevibacillus,
Cellulomonas, Chelatococcus, Curtobacterium, Frankia, Geodermatophilus, Micrococcus,
Mycobacterium, Nocardioides, Pseudomonas, Pseudonocardia, Sphingomonas, and
Streptomyces. Even in the two layers that have elevated CFUs, more of the same kinds of
organisms are found.
The most dominant cultured organism from the hyper-arid soil pit isolates based on its
closest relative is Pseudonocardia saturnea. P. saturnea is the closest relative of isolates
53
Table 4.2: Values of CFU/g recovered on various culture media for the AT04-159 southern soil pit
Culture Media CFU/g of soil / number for AT04-159 layers MA NA PCA 1/10 PCA 1/100 PCA
<104 0 0 1 0 0 >104 and <105 2 3 2 1 0 >105 and <106 1 1 1 3 4
>106 2 1 1 1 1
54
2
1 2 1
3
7 2
8
2
2 1
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
90 cm80 cm70 cm60 cm50 cm40 cm30 cm20 cm10 cm
surface
Depth
Soil Pit AT04-150 - Diversity at the Phlya Level
ActinobacteriaFirmicutesProteobacteria
2
1 2 1
3
6 2 1
2 2 1 1 1
21 1 1
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
90 cm80 cm70 cm60 cm50 cm40 cm30 cm20 cm10 cm
surface
Depth
Soil Pit AT04-150 - Diversity at the Closest Relative Level
Blastococcus sp.Bacillus sp.Pseudonocardia saturneaChelatococcus asaccharovoransNocardioides sp.Mycobacterium sp.Nostocoida aromativoraGeodermatophilus sp.Micrococcus sedentariusStaphylococcus sp.Methylobacterium mesophilicum Curtobacterium flaccumfaciensMicrococcus luteus
4 2
8 13
1 1
4 3 1
4
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
90 cm80 cm70 cm60 cm50 cm40 cm30 cm20 cm10 cm
surface
Depth
Soil Pit AT04-150B Enrichment - Diversity at the Phlya Level
ActinobacteriaFirmicutesProteobacteria
1 2 1 2
6 1 1
31
2 1 1 32 1 1
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
90 cm80 cm70 cm60 cm50 cm40 cm30 cm20 cm10 cm
surface
Depth
Soil Pit AT04-150B Enrichment - Diversity at the Closest Relative Level
Blastococcus sp.Pseudonocardia saturneaStreptomyces sp.Brevibacillus agriHerbaspirillum seropedicaeStreptomyces californicus
Figure 4.2: Diversity at the phyla and closest relative levels in soil pit AT04-150 and AT04-150B enrichment cultures
55
1
2
2 4
4 1
8
80
1 1
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
80 cm
70 cm
60 cm50 cm
40 cm
30 cm
20 cm
10 cmsurface
Depth
Soil Pit AT04-152 - Diversity at the Phlya Level
ActinobacteriaFirmicutesProteobacteria
1
1 1
3 1 1 1
4 1
5 3
2 12 12 3 1
1
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
80 cm
70 cm
60 cm
50 cm
40 cm
30 cm
20 cm
10 cm
surface
Depth
Soil Pit AT04-152 - Diversity at the Closest Relative LevelBlastococcus sp.Pseudonocardia saturneaNocardioides sp.Mycobacterium sp.Streptomyces sp.Pseudonocardia petroleophilaStaphylococcus epidermidis Actinobispora xinjiangensisSphingomonas sp.Amycolatopsis sp.Micrococcus sp.Pseudomonas stutzeri
1
2
1
2 1
5
6 3
11 1
7
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
80 cm
70 cm
60 cm
50 cm
40 cm
30 cm
20 cm
10 cm
surface
Depth
Soil Pit AT04-152B Enrichment - Diversity at the Phlya Level
ActinobacteriaFirmicutes
1
1 1
1
1 1 1
3 1
2 2 1 1 1
3 4 1 1
5 2
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
80 cm70 cm60 cm50 cm40 cm30 cm20 cm10 cm
surface
Depth
Soil Pit AT04-152B Enrichment - Diversity at the Closest Relative Level
Blastococcus sp.Pseudonocardia saturneaStreptomyces sp.Brevibacillus agriStreptomyces californicusBlastococcus aggregatusBacillus cereusAmycolatopsis sp.Streptomyces neyagawaensis
Figure 4.3: Diversity at the phyla and closest relative levels in soil pit AT04-152 and AT04-152B enrichment cultures
56
3
4
1
4 1
1 1
14
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
80 cm
70 cm
60 cm
50 cm
40 cm
30 cm
20 cm
10 cm
surface
Depth
Soil Pit AT04-153 - Diversity at the Phlya Level
ActinobacteriaFirmicutesProteobacteria
2 1
1 1 1 1
1
1 1 1
1
12 2
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
80 cm
70 cm
60 cm50 cm
40 cm
30 cm
20 cm
10 cmsurface
Depth
Soil Pit AT04-153 - Diversity at the Closest Relative Level
Blastococcus sp.Curtobacterium flaccumfaciensBlastococcus aggregatusBrevibacillus agriGeodermatophilus obscurusMicrococcus luteusCellulomonas cartaeFrankia sp.
7
11
6
1
7
6
3
4
9 6
0% 20% 40% 60% 80% 100%
Percentage
80 cm
70 cm60 cm
50 cm40 cm
30 cm20 cm
10 cmsurface
Depth
Soil Pit AT04-153B Enrichment - Diversity at the Phlya Level
ActinobacteriaFirmicutes
7
6 2 3
5
1
4 1 2
5 1
2
2 2
8 1 5 1
0% 20% 40% 60% 80% 100%
Percentage
80 cm70 cm
60 cm
50 cm40 cm
30 cm20 cm
10 cm
surface
Depth
Soil Pit AT04-153B Enrichment - Diversity at the Closest Relative Level
Blastococcus sp.Pseudonocardia saturneaStreptomyces sp.Brevibacillus agriStreptomyces californicusBlastococcus aggregatusBacillus cereusMicrococcus luteusGeodermatophilus obscurus
Figure 4.4: Diversity at the phyla and closest relative levels in soil pit AT04-153 and AT04-153B enrichment cultures
57
recovered from soil pit AT04-150 in layers 10 cm (1 isolate out of 3), 20 cm (2 isolates out of 2),
30 cm (2 isolates out of 7), 40 cm (6 isolates out of 9), and 50 cm (3 isolates out of 3); this
organism was also recovered from soil pit AT04-152 in sample layers 10 cm (12 isolates out of
30), 20 cm (5 isolates out of 8), and 30 cm (4 isolates out of 5). However none of the isolates
from pit AT04-153 were identified as a closest relative to P. saturnea. The other dominant
organisms isolated from soil pit AT04-152 have closest relatives in species of the genus
Streptomyces.
Isolates whose closest relatives are members of the genus Streptomyces were recovered in
pit AT04-152 in sample layers 10 cm (12 isolates recovered out of 30) and 20 cm (3 isolates
recovered out of 8); as with P. saturnea, members of the genus Streptomyces were not detected
in the dilution plating method in soil pit AT04-153. In contrast, the isolates from soil pit AT04-
153 are dominated by organisms most closely related to species of the genus Blastococcus,
similar to what was found in the surface samples. Members of the genus Blastococcus were the
closest relatives of isolates recovered from soil pit AT04-153 on the surface layer (12 isolates out
of 14), 30 cm below the surface (1 isolate out of 1), 70 cm below the surface (2 isolates out of 4),
and 80 cm below the surface (2 isolates out of 3).
Isolates were selected and identified from the southern soil pit at Altamira, AT04-159, on
the basis of their availability and novel morphologies. All isolates were identified based on 16S
rRNA gene sequence determination. A few morphology types (approximately six) undeniably
dominated the solid culture media (both nutrient-rich and nutrient-dilute) (Figure 4.5).
Increased diversity in AT04-159 was observed when compared with isolate diversity from the
three core arid region pits (Figure 4.5). In the southern-most soil pit, about 42 different bacterial
taxa were recovered. The most commonly observed isolates recovered from the Altamira soil pit
58
8 1 5
12 1
22 9 6
16 6 1
76 11 3
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage
40 cm
30 cm
20 cm
10 cm
surface
Depth
Soil Pit AT04-159 (Altimira Soil Pit) - Diversity at the Phyla Level
ActinobacteriaFirmicutesProteobacteriaBacteroidetes
8 1
9 1 1
5 2 6 3 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1
5 1 3 1 1 1 1 1 2 1 1 1
10 1 4 7 21 18 10 3 21 3 8 21 3 111
0% 20% 40% 60% 80% 100%
Percentage
40 cm
30 cm
20 cm
10 cm
surface
Depth
Soil Pit AT04-159 (Altimira Soil Pit) - Diversity at the Closest Relative LevelBlastococcus sp. Bacillus sp.Chelatococcus asaccharovorans Nocardioides sp.Geodermatophilus sp. Arthrobacter sp.Streptomyces sp. Pseudonocardia petroleophilaActinobispora xinjiangensis Sphingomonas sp.Blastococcus aggregatus Geodermatophilus obscurusHymenobacter sp Actinobispora alaniniphilaArthrobacter agilis Cellulomonas cellasea Modestobacter sp. Modestobacter multiseptatus Paenibacillus sp. Cellulosimicrobium funkei Saccharothrix sp. Cellulomonas septicaCellulomonas uda Hongia sp. Streptomyces albidochromogenes Actinobispora yunnanensisBacillus niacini Subtercola pratensisPseudonocardia zijingensis Mesorhizobium sp.Streptomyces plemorphus Streptomyces carpinensisPromicromonospora aerolata Mycobacterium celatum Cellulomonas sp. Actinomadura spBacillus litoralis Rhizobium spOxalophagus oxalicus Streptomyces rutgersensis Bacillus krulwichiae
Figure 4.5: Diversity at the phyla and closest relative levels in soil pit AT04-159 (the Altamira soil pit)
59
have closest relatives in the genera Arthrobacter, Bacillus, Blastococcus, Geodermatophilus,
Modestobacter, and Streptomyces. Although the CFUs/g increased with depth in the Altamira
pit, the diversity decreased with depth. A greater number of the Altamira soil pit isolates were
recovered from layers at or near surface because most of the colonies growing on the dilution
plates from these depths were morphologically unique; the bacterial colonies from deeper layers
were more numerous, but less diverse both in their morphology and phylogenetic diversity.
Enrichment cultures were set up for the three pits constructed within the Yungay region
of the Atacama Desert. Isolates from the enrichment cultures were recovered just as the dilution
plate isolates were selected on the basis of their availability and morphological uniqueness and
identified based on their 16S rRNA gene sequence. Of the enrichment isolates recovered from
soil pit AT04-150 and sequenced, 6 belonged to the Firmicutes, 2 to the Proteobacteria, and 24 to
the Actinobacteria; from pit AT04-152, 5 isolates belonged to the Firmicutes and 35 to the
Actinobacteria; from pit AT04-153, 6 isolates belonged to the Firmicutes, and 54 Actinobacteria
strains were recovered (Figures 4.2, 4.3, and 4.4 (the third chart in each figure)). The enrichment
approach did not result in the recovery of addition bacterial diversity from the soil pits of hyper-
arid region and the closest relatives to these isolates were species of the genera Amycolatopsis,
Bacillus, Blastococcus, Brevibacillus, Geodermatophilus, Herbaspirillum, Micrococcus,
Pseudonocardia, and Streptomyces (Figures 4.2, 4.3, and 4.4 (the fourth chart in each figure)).
The most dominant isolate among the Yungay enrichment isolates has P. saturnea as their
closest relative. P. saturnea was the closest relative of enrichment isolates recovered in pit
AT04-150 in the surface layer (1 isolate out of 4), the layer at 10 cm below surface (1 isolate out
of 7), 20 cm below the surface (1 isolate out of 1), 30 cm below the surface (3 isolates out of 3),
40 cm below the surface (6 isolates out of 8), and 50 cm below the surface (2 isolates out of 6).
60
This organism was also found at AT04-152 on the surface (2 isolates out of 7), 10 cm below the
surface (3 isolates out of 9), 20 cm below the surface (2 isolates out of 7), 30 cm below the
surface (3 isolates out of 4), 70 cm below the surface (1 isolate out of 2), and 80 cm below the
surface (1 isolate out of 1). From pit AT04-153, only isolates from the layer at 40 cm below the
surface (1 isolate out of 7) and 70 cm below the surface (2 isolates out of 11) were identified as
closest relatives to P. saturnea.
Other dominant organisms were detected in the Yungay pits enrichment cultures were
those whose closest relatives were related to the genus Streptomyces and these were recovered
from enrichment cultures of soil pit AT04-150. From the surface layer (1 isolate out of 4), 10 cm
below the surface (1 isolate out of 7), 40 cm below the surface (1 isolate out of 8), and 50 cm
below the surface (1 isolates out of 6). Enrichment isolates identified by partial 16S rRNA gene
sequences as Streptomyces spp. were recovered from soil pit AT04-152 in the layers 10 cm
below the surface (5 isolates out of 9), 20 cm below the surface (1 isolate out of 7), and 30 cm
below the surface (1 isolate out of 4). Also, enrichment culture isolates from soil pit AT04-153
were found to be similar to members of the genus Streptomyces at 10 cm below the surface (2
isolates out of 4) and at 30 cm below the surface (1 isolate out of 6).
Another dominant species among the Yungay soil pit enrichment isolates are
Blastococcus spp. This organism is much more dominant in pit AT04-153; 43 out of the 58
enrichment isolates recovered from this pit were identified as close relatives of Blastococcus spp.
Enrichment isolates with relatives most similar to those of the genus Blastococcus were
recovered from soil pit AT04-150 in the surface layer (2 isolates out of 4), 10 cm below surface
(2 isolates out of 7), and 50 cm below the surface (1 isolate out of 6); in pit AT04-152 the
surface layer (5 isolates out of 7), 20 cm below the surface (2 isolates out of 7), 40 cm below the
61
surface (1 isolate out of 3), and 70 cm below the surface (1 isolate out of 2). Also, most of the
enrichment isolates recovered from soil pit AT04-153 were identified as members of the genus
Blastococcus. Members of this genus were detected in every layer of soil pit AT04-153; surface
(8 isolates out of 15), 10 cm below the surface (2 isolates out of 4), 20 cm below the surface (2
isolates out of 2), 30 cm below the surface (5 isolates out of 6), 40 cm below the surface (4
isolates out of 7), 50 cm below the surface (1 isolate out of 1), 60 cm below the surface (5
isolates out of 5), 70 cm below the surface (9 isolates out of 11), and 80 cm below the surface (7
isolates out of 7).
The genus Brevibacillus is also uniformly distributed throughout the enrichment isolates
recovered from layers within all three of the hyper-arid region soil pits. In soil pit AT04-150,
isolates whose closest relatives were Brevibacillus spp. were observed at 10 cm below the
surface (3 isolates out of 7) and 50 cm below the surface (2 isolates out of 6); in soil pit AT04-
152, isolates whose closest relatives were Brevibacillus spp. were recovered at 10 cm below the
surface (1 isolate out of 9) and 20 cm below the surface (1 isolate out of 7); also in soil pit AT04-
153 at the surface layer (1 isolate out of 15).
DISCUSSION
The three Yungay pits show very low CFU values overall throughout the surface and
subsurface layers sampled and investigated. These findings further contribute to the idea that the
hyper-arid region of the Atacama Desert is an analog of Mars both on the surface and in the
subsurface. The majority of the subsurface layers sampled and investigated show zero CFUs on
all culture media utilized for this study. Even in the Atacama Desert at sites outside the core
hyper-arid region, such that at Altamira, the CFU/g of soil is as high 1.08 x 105 at surface layers
and 1.80 x 106 at subsurface depths on at least one of the five culture media employed for this
62
study. The two CFU spikes observed in pits AT04-150 and AT04-152 (core hyper-arid region
pits) are not comparable to the CFUs/g from the Altamira soil pit samples, nevertheless these
elevated CFUs/g are indicative of the need for multiple site surface and subsurface sampling on
Mars where similar microbial patchiness with depth may exist.
The fact that the largest number of colonies from the Yungay soil pit samples was
observed on the Marine Agar (MA) culture media suggests that recoverable heterotrophic
bacteria existing in hyper-arid Atacama soils may be of marine origin and brought there by
deposition from winds from the coastal region to the west.
The CFUs/g found for Altamira pit samples (especially near the surface) in this study
coincide with the Navarro-González et al. (2003) reports of surface CFU levels at the latitude 25º
S 45′ to be between 104 and 105 CFUs/g. The higher CFUs/g at this site are to be expected when
comparing this region to Yungay CFUs/g due to the increase of culturable bacterial numbers
along the north-to-south precipitation gradient (Navarro-González et al. 2003). The Altamira
site, a more southern site, accumulates more moisture throughout the year than the hyper-arid
Yungay region, thus the area is more accommodating to microbial life.
The diversity of culturable heterotrophic bacteria isolated from each of the three pits
within the hyper-arid core area is much less than that recovered in the Altamira pit. The isolates
from three Yungay pits fall within three phyla, whereas the Altamira pit was determined to
contain representatives of at least four phyla. The most prevalent of the isolates belong to the
phylum Actinobacteria, which is the phylum that such organisms as Arthrobacter spp.,
Blastococcus spp., Cellulomonas spp., Frankia spp., Geodermatophilus spp., Hymenobacter
spp., Kocuria spp., Micrococcus spp., Modestobacter spp., Nocardioides spp., Pseudonocardia
spp., and Streptomyces spp. belong. The isolates falling into the phylum Firmicutes were
63
Bacillus spp., Brevibacillus spp., and Paenibacillus spp. Finally, the isolates belonging to the
phylum Proteobacteria were Chelatococcus spp., Herbaspirillum spp., Pseudomonas spp., and
Sphingomonas spp.
There is not a great deal of diversity observed between individual layers among the three
hyper-arid region pits. Even in the two pits that exhibit elevated CFUs in two layers, we find
more of the same types of bacteria. This is consistent with the patchiness in the surface
diversity, and may indicate spatial patchiness vertically as well as horizontally. Isolates
recovered from the surface of each pit are in many cases identical to the bacteria found at lower
depths of the soil pit. When comparing isolates recovered from the surface samples (Chapter 3)
and Yungay pit samples, it is apparent that the diversity is slightly greater in the surface samples.
Nevertheless, many overlapping organisms were found in surface samples and in samples taken
at some depths. Because the diversity of the species found throughout the core region samples is
very low, this overlap relates surface soils to subsurface soils and may signify similar resource
abundance at short spatial range (Rietkerk et al. 2004).
An interesting finding revealed from the comparison of the Yungay surface and Yungay
pit samples was that the large number of Blastococcus sp. cultured from surface samples were
not found in the subsurface soil pit samples, especially at sites AT04-150 and AT04-152. Also,
Pseudonocardia spp. were recovered only from the soil pit samples and was not observed among
the organisms recovered from surface sites. Of the three core soil pits, AT04-150 and AT04-152
were constructed on the top of a hill with an altitude of about 1070 m (nearly 50 m above the
valley), whereas AT04-153 was dug in the valley adjacent to the hill. Most of the 33 surface
sites showing patchiness were also sampled in a valley. As shown in the surface study (Chapter
3) and in the soil pit located in the valley (AT04-153), the culturable bacteria that dominate were
64
members of the genus Blastococcus. In the two soil pits constructed on the hill (AT04-150 and
AT04-152), less than 2% of the culturable bacteria isolated were members of the genus
Blastococcus, instead the dominating genera were Pseudonocardia and Streptomyces. Of the
total culturable heterotrophic bacteria recovered from the soil pit AT04-150, about 30% were
members of the genus Pseudonocardia, and from soil pit AT04-152 about 20% were members of
the genus Pseudonocardia and 25% were of the genus Streptomyces. This suggests that the
difference in the bacterial populations may be attributed to the differences between the types of
soils found in low lying valleys and those found at elevated sites.
As anticipated, the soil pit AT04-159 in the less arid, more southern region has CFUs up
to four magnitudes greater than those in the core arid region. As formerly discussed, the
Altamira soil pit was found to contain about 42 different bacterial taxa, which supports the
theory that further south, outside of the hyper-arid region of the Atacama Desert, moisture levels
increase in soils, therefore they have a greater diversity of culturable heterotrophic bacteria and
much higher CFUs especially in the subsurface layers (Navarro-González 2003). While the CFU
values increase at greater depths in the Altamira pit, the diversity is reduced with depth. At
layers near the surface, fewer bacterial colonies were observed using the dilution plating method
than at deeper subsurface layers, yet a larger number of the Altamira pit isolates were recovered
from layers at nearer the surface because most colonies growing on the dilution plates at these
depths were morphologically different. Although bacterial colonies from deeper subsurface
layers within the Altamira pit were more numerous, they were less diverse in morphological
characteristics; therefore, less diversity of isolates were recovered. A detail apparent throughout
this study is that samples having elevated CFUs do not necessarily contain more recovered
isolates if indeed the high numbers of colonies all appear to be similar in colony morphology.
65
Enrichment cultures were prepared for each of the pits sampled in the core arid region.
Although they cannot be used for quantification purposes, they are useful in uncovering
organisms that grow rapidly in favorable nutrient conditions. A fog event, which is a rare
moisture occurrence in the hyper-arid region, could be one source of a natural enrichment
environment in which organisms generally found at low numbers can rapidly flourish.
For the three soil pits sampled in the Yungay region, the diversity in the enrichment
isolates is similar to that of the dilution plating isolates since both have low diversity of
organisms, and the organisms recovered during the enrichment are generally the same as the
isolates from the dilution plating experiment. The enrichment isolates from soil pits AT04-150
and AT04-152 like the dilution plating isolates, are dominated by the P. saturnea like strains.
These P. saturnea related strains are important to this study because they have been found to be
abundant in the Yungay soil pit samples (both from dilution plating and enrichment methods),
but were never recovered from surface samples. As previously mentioned, this may be attributed
to the fact that the soil pits from which this particular organism was recovered were located on
the elevated area in Yungay.
Strains of the genera Brevibacillus and Streptomyces were not only consistently identified
from the enrichment isolates that were recovered from the three Yungay pits, but they were also
identified from isolates recovered throughout this study within the hyper-arid region (both
surface and subsurface dilution plate isolates). These taxa along with other overlapping
microorganisms (mentioned earlier in this chapter), belong to the culturable heterotrophic
bacterial community surviving in the core arid region of the Atacama Desert.
Blastococcus spp. have also been found to be abundant among the enrichment isolates,
especially those from soil pit AT04-153. This is to be expected since the majority of the surface
66
study revealed the genus Blastococcus to be the principal recoverable microorganism inhabiting
the hyper-arid region. The fact that more enrichment isolates from soil pit AT04-153 (the
Yungay valley pit) were identified as closest relatives of Blastococcus sp. than enrichment
isolates from the two pits constructed at elevated area also implies that the difference in the
bacterial inhabitants may be accredited to distinctions between the soils found in low lying
valleys and those found at elevated sites. Since bacteria obtain nutrients from the environment in
which they inhabit, bacterial populations in the core arid region may be distributed directly as a
result of the availability of resources throughout the soils.
The studies described in this and the previous chapter, highlight the need for multiple site
selection for either surface or subsurface sampling of Martian soils where similar patterns of
patchiness as uncovered in the Atacama Desert might exist.
67
CHAPTER 5
DIRECT CELL COUNTS USING FLUORESCENCE MICROSCOPY
DAPI, 4'-6-Diamidino-2-phenylindole, is a fluorochrome that binds with DNA to form a
fluorescent complex that will fluoresce blue under ultraviolet exposure and is popularly used in
direct count protocols to quantify both viable and nonviable bacterial cells in environmental
samples. In order to investigate the actual bacterial cell concentrations in Yungay soil samples, a
direct count method was attempted using DAPI (4'-6-Diamidino-2-phenylindole) fluorescence
microscopy. However, this method, which is described in chapter 2, proved to be problematic in
conjunction with the Atacama Desert soils due to low bacterial cell numbers and non-specific
binding of DAPI with soil particles from the Yungay region. The complications that were
recognized by applying the DAPI direct count method for Atacama Desert hyper-arid soils lead
to an inquiry to determine the limit of detection of these particular soils in conjunction with the
DAPI direct count protocol applied.
RESULTS
DAPI Direct Count Detection Limit
Known cell concentrations (determined by dilution plating technique) of a suspension of
an Atacama Desert isolate whose closest relative was identified to be Blastococcus aggregatus
were added to AT04-170 soils (CFU value of zero as described in Chapter 3). This strain was
selected for the experiment given that the genus Blastococcus is the predominate organism found
in Yungay soils. The organism was added to the soil as a stock suspension and a 1/10 dilution of
the stock solution and allowed to dry overnight (another experiment discussed later allowed the
soil and cell suspension to dry for one month). The liquid extract from the AT04-170 soils along
with the added cells was stained with DAPI as described in Chapter 2 to determine the detection
68
limit for the DAPI method used in this study. The cell concentration of the Blastococcus strain
AT03-37-10 stock solution was determined to be 1.05 x 106 CFUs per mL. Figure 5.1 shows
three epifluorescence photomicrographs, two photomicrographs at varying bacterial cell
concentrations added to soil and dried overnight and one representing a control of DAPI stained
soil lacking additional cells, that represent the detection limit of the direct count method
performed on Yungay soils.
Twenty fields were counted and averaged from the filter with the stock cell suspension
added to soil and from the filter with 1/10 diluted stock suspension added to soil. Table 5.1 gives
the results of counted cells per field, averages, and known versus calculated cells per gram of soil
for both the stock and 1/10 diluted cell suspension. Each field counted has an area of 41,184
µm2 at the magnification used (400x), and since the filters that the cells were captured on have
an area of 490.625 mm2, there are 11,913 fields per filter.
The number of cells per field range from 20 to 101 for the filter containing the stock cell
suspension and AT04-170 soil, and the average cell number is 45.5 Blastococcus spp. strain
AT03-37-10 cells per field. Fields 3 and 16 have unusually high cell numbers (101 and 92) due
to cells aggregating amid clusters of soil particles; Figure 5.1 (A) demonstrates this arrangement
in the upper left-hand portion of the image. The average (45.5) was multiplied by 11,913.61 and
then again by 2 to account for the dilution within this procedure. The final cell concentration
was calculated to be 1.08 x 106 cells per gram (or cells per mL since 1 mL of cell suspension was
added to 1 gram of soil), which is almost exactly the known concentration added to the soil.
Therefore, it can be assumed that for a Yungay soil with a cell concentration above 1 x 106 cells
per gram the numbers of cells present can accurately be detected using the DAPI staining
69
procedure used in this study. Figure 5.1 (A) shows a photomicrograph of field 12 from the filter
with the stock suspension (1.05 x 106 cells per mL) added to AT04-170 soil.
A 1/10 dilution of the cell suspension (1.05 x 105 cells per mL) was prepared and handled
exactly as the stock solution. The number of cells per field range from 0 to 4. Fields 3, 8, 10,
and 11 zero fluorescing bacterial cells were observed, for fields 5, 9, 12, 14, and 17 one cell was
observed, for fields 1, 4, 6, 7, 13, 18, and 19 two cells were observed, for fields 2, 15, and 20
three cells were observed, and for field 16 four cells were observed. The average number of
cells observed from the 20 evenly distributed fields is 1.6. Calculations were performed in the
same manner as above and the final cell concentration was determined to be 3.81 x 104 cells per
gram of Yungay soil. Figure 5.1 (B) shows field 4 from the polycarbonate filter prepared with 1
x 105 cells per mL added to AT04-170 soil. The known cell concentration and direct count cell
concentration (3.81 x 104 cells per gram of soil) contradict one another. Therefore, the detection
limit of the DAPI staining method used on Yungay soils is concluded to exist at some point
between 1 x 105 and 1 x 106 cells per gram of soil.
Figure 5.1 (C) shows the control epifluorescence photomicrograph of the soil AT04-170
without the addition of a cultured cell suspension. This figure demonstrates the non-specific
binding of DAPI to the Atacama Desert soil (the green fluorescing particles).
The experiment above was repeated, but the soil was allowed to dry for one month after
the cells (stock cell suspension and 1/10 dilution) were added to the AT04-170 soils instead of
overnight in order to compare the appearance of added cells after an extended time period in
AT04-170 soil. The filters were viewed under a higher magnification (800x); therefore, the
calculations to convert average cells per field to total cells per gram of soil vary from the above
experiment. Twenty fields were counted and averaged. Table 5.1 gives the counted cell
70
Figure 5.1: Photomicrographs of AT04-170 spiked with cultured bacterial cells and dried overnight to determine DAPI direct count detection limit Stock + AT04-170 soil: truly detected (gold arrow shows aggregate cells where cells are distinguishable; red arrow shows cells difficult to tell apart since amongst fluorescing soil) (A), 1/10 dilution + soil: below detection limit (arrows show two bacterial cells) (B).
A
B
71
Figure 5.1: (Continued) AT04-170 soil stained with DAPI without Blastococcus strain AT03-37-10 cells added (C).
C
72
numbers, averages, and known versus calculated cells per gram of soil for the one month drying
experiment and the overnight drying experiment for both the stock and 1/10 diluted cell
suspension. Each field counted has an area of 10,296 µm2 at the magnification used (800x), and
since the filter that the cells were captured on has area of 490.625 mm2, there are 47,652 fields
per slide. The average cell number was multiplied by 47,652 and then again by 2 to account for
the dilution within this procedure.
Figure 5.2 shows two representative epifluorescence photomicrographs at varying
concentrations of the bacterial cell stock added to soil and dried for one month. As Figure 5.2
indicates, the cells appeared less bright blue than the cells that dried on AT04-170 soil overnight.
Also the cells somewhat lost the characteristic coccid shape that was apparent in Figure 5.1.
Regarding the one month drying experiment, the number of cells per field range from 3 to 23
and the average cell number on the stock cell suspension plus AT04-170 soil filter is 10.9 cells
per field. After the calculations mentioned in the previous paragraph were performed, the final
cell concentration was calculated to be 1.04 x 106 cells per gram of soil (or per mL since one mL
was added to one gram of soil), which is practically the known concentration that was added to
the soil just as the experiment where the cell suspension and soil were dried overnight.
Therefore, it can be still be assumed that a Yungay soil with a cell concentration above 1 x 106
cells per gram can accurately be detected using the DAPI staining procedure used in this study.
Figure 5.2 (A) shows a photomicrograph taken of field 1 from filter containing 1.05 x 106 cells
per mL added to one gram of AT04-170 soil and dried for one month. The number of cells
observed per field from the 1/10 dilution filter (1.05 x 105 cells per mL) range from zero to three.
For fields 2, 3, 8, 12, 13, 14, 15, 16, 17, and 19 zero fluorescing bacterial cells were observed,
for fields 5, 18, and 20 one cell was observed, and for fields 1, 4, 6, 7, 10, and 11 two cells were
73
Table 5.1: Direct count detection limit trials displaying cell numbers per field, average cells per field, and known versus calculated cells/g for stock and 1/10 diluted cell suspension after overnight and one month drying experiment
Number of Cells per Field in DAPI Detection Limit Experiment
Stock 400x (overnight)
1/10 dilution 400x (overnight)
Stock 800x (one month)
1/10 dilution 800x (one month)
Field Number
Field 1 34 2 12 2 Field 2 36 3 8 0 Field 3 101 0 9 0 Field 4 49 2 15 2 Field 5 25 1 10 1 Field 6 66 2 7 2 Field 7 26 2 5 2 Field 8 72 0 11 0 Field 9 40 1 9 3 Field 10 39 0 6 2 Field 11 36 0 14 2 Field 12 30 1 23 0 Field 13 42 2 8 0 Field 14 20 1 16 0 Field 15 28 3 3 0 Field 16 92 4 10 0 Field 17 24 1 5 0 Field 18 51 2 19 1 Field 19 29 2 13 0 Field 20 70 3 15 1 Average Cells/Field 45.5 1.6 10.9 0.9 Known Cells/Gram 1.05 x 106 1.05 x 105 1.05 x 106 1.05 x 105 Calculated Cells/Gram 1.08 x 106 3.81 x 104 1.04 x 106 8.58 x 104
74
Figure 5.2: Detection limit photomicrographs of AT04-170 spiked with cultured bacterial cells and dried for one month 1.05 x 106 cell/mL plus AT04-170 soil: accurately detected (arrows indicate counted fluorescing cells (A), 1.05 x 105 cell/mL plus soil: detection limit for this study (arrow indicates one counted fluorescing bacterial cell) (B).
A
B
75
observed. The average number of cells observed was 0.9; the average was multiplied by 47,652
and then again by 2 for a final cell concentration of 8.58 x 104 cells per gram of Yungay soil.
Figure 5.2 (B) shows a photomicrograph taken of field 5 from the filter containing 1 x 105 cells
per mL added to AT04-170 soil. The known cell concentration (1 x 105 cells per mL) and direct
count cell concentration (8.58 x 104 cells per gram of soil) disagree with one another just as the
experiment where the cell suspension and soil were dried overnight. Therefore, the detection
limit of the DAPI staining method used on Yungay soils can still be assumed to exist somewhere
between 1 x 105 and 1 x 106 cells per gram of soil.
DAPI Direct Cell Count Determination for AT04-166
On the basis the detection limit stated above, only the soil sample having the highest CFU
value of 7.4 x 105 per gram of soil on 1/10 PCA culture medium, AT04-166, was able to be
counted using the fluorescence direct count procedure described in Chapter 2. Extraction fluid
from five different amounts of soil (1.0 gram, 0.5 grams, 0.25 grams, 0.1 grams, and 0.05 grams)
was stained with DAPI and used to determine a soil amount that would achieve countable
bacteria under an epifluorescence microscope without serious interference from fluorescing soil
particles. The enumeration of total bacterial cells was accomplished by counting and averaging
bacterial cells in 20 countable fields evenly distributed over the area beneath the coverslip.
Figure 5.3 shows representative epifluorescence photomicrographs of the DAPI direct count
method performed on the extraction supernatants from all five amounts of AT04-166 soil.
Regarding the AT04-166 soil, the filter containing extraction fluids that account for 0.25
grams of soil attested to be the countable filter; the number of cells counted per field, the average
cell number per field, and calculated total cells per gram of soil is displayed in Table 5.2. The
number of cells per field for sample AT04-166 range from 17 to 51 and the average cell number
76
Figure 5.3: Photomicrographs from the fluorescence direct count method employing DAPI in surface site AT04-166 Photomicrograph represents 1.0 g of soil: overly concentrated with fluorescing cells and soil particles (A), represents 0.5 g of soil: still too concentrated with fluorescing cells and soil particles (B).
A
B
77
Figure 5.3: (Continued) Photomicrograph represents 0.25 g of soil: countable (arrows indicate all fluorescing cells counted in this field) (C), represents 0.1 g of soil: too few cells; not countable (arrows indicate all fluorescing cells) (D).
C
D
78
Figure 5.3: (Continued) Photomicrograph represents 0.05 g of soil: no fluorescing cells in this field (E).
E
79
per 20 evenly distributed fields on the countable filter is 34.2. Each field counted has an area of
10,296 µm2, and since the filter that the cells were captured on has an area of 490.625 mm2, there
are 47,652 fields per slide. The average cell count per field (34.2) was multiplied by 47,652
fields. Since the method used for this experiment accounts for 0.25 grams of soil, the value was
then multiplied by four to convert the value to cells per gram of soil. It was resolved that the
total number of bacterial cells (viable and nonviable) per gram of AT04-166 soil is 6.52 x 106.
The concluded direct count value is above the determined detection limit for the utilized method
and soils, thus the total cell count for AT04-166 can be assumed to be an accurate enumeration.
On the filter containing extraction fluids that account for 0.1 grams of AT04-166 soil less
than or equal to five cells in eight fields were observed, but the remaining fields contained zero
countable cells, therefore, was not selected as the soil concentration containing a countable total
bacterial cell number. Figure 5.3 (D) shows one of the eight fields (out of 20) on the filter
containing extracts from 0.1 grams of soil in which fluorescing bacterial cells could be observed;
for fields 9, 14, and 19 one fluorescing bacterial cell was observed, for fields 2, 8, and 11 two
cells were observed, for field 5 three cells were observed, and for field 17 (image shown in
Figure 5.3 (D)) five cells. Liquid extract from 1.0 and 0.5 grams of soil contained high
concentrations of fluorescing soil particles which prevented the ability to view any bacterial
cells. The filter prepared with 0.05 grams of soil showed zero fluorescing bacterial cells in all 20
evenly distributed fields.
With respect to the appearance of the cells in Figure 5.3, it was demonstrated in the one
month drying experiment that fluorescing cells became dimmer and lost characteristic bacterial
forms after extended time periods in the Yungay desert soil. This is also true for bacteria that
permanently inhabit environmental hyper-arid soils from within the Yungay region; thus, the
80
fluorescing bacterial cells viewed in AT04-166 soils were difficult to count. The fluorescing
cells were also difficult to differentiate from fluorescing soil debris, a problem encountered
throughout this experiment because DAPI non-specifically binds to Atacama Desert soil
particles.
Sample Sites Having Cell Totals below DAPI Direct Count Detection Limit
AT04-165, a sample having the second highest CFUs/g (8.20 x 104) on 1/10 PCA culture
medium, and AT04-170, a sample having a 0 CFUs/g on all five primary culture media, were
prepared and observed using the DAPI direct count method to determine if the remaining
Yungay samples are adequate candidates for DAPI direct count methods based on the detection
limit. These two surface samples were not able to be accurately quantified due to the constraints
of the determined detection limit per gram of Yungay region soil. Extraction supernatant from
five soil amounts (1.0 g, 0.5 g, 0.25 g, 0.1 g, and 0.05 g) was stained with DAPI from both
AT04-165 and AT04-170 soils and filtered onto a black polycarbonate filter to determine a soil
amount that would generate countable bacteria using epifluorescence microscopy without
significant obstruction from fluorescing soil particles. Figure 5.4 shows one representative
photomicrograph from each of the five AT04-165 soil sample amount trials. The filter prepared
using 0.5 g of soil is the only amount at which fluorescing cells can be viewed because the
extract from 1.0 g contains a high concentration of fluorescing soil particles which obstruct the
view of any bacterial cells, and the other three filters that were prepared with smaller amounts of
soil (0.25 g, 0.1 g, and 0.05 g) show a decrease in fluorescing soil, but zero fluorescing bacterial
cells are observed in all 20 fields. All 20 evenly distributed countable fields for sample AT04-
165 are presented in Table 5.2 as number of fluorescing cells/field, average cells per field, and
calculated total cells per gram of soil. The average cells per field on the filter prepared from 0.5
81
Figure 5.4: Photomicrographs of AT04-165 soils unable to be truly quantified by DAPI direct counts Photomicrograph represents 1.0 g of soil: overly concentrated with fluorescing soil particles to count (A), photomicrograph represents 0.5 g of soil (arrows indicate counted fluorescing cells) (B).
B
A
82
Figure 5.4: (Continued) Photomicrograph represents 0.25 g of soil: no fluorescing cells (C), represents 0.1 g of soil: no fluorescing cells (D).
C
D
83
Figure 5.4: (Continued) Photomicrograph represents 0.05 g of soil: no fluorescing cells in this field (E).
E
84
Table 5.2: Direct count trials displaying cell numbers per field, average cells per field, and calculated cells/g for each tested sample site
Number of Cells per Field in Soil Sample AT04-165 (800x) AT04-166 (800x) AT04-170 (800x) Field Number
Field 1 2 44 0 Field 2 4 23 0 Field 3 0 17 0 Field 4 2 27 0 Field 5 0 36 0 Field 6 0 40 0 Field 7 1 47 0 Field 8 1 37 0 Field 9 0 34 0 Field 10 2 28 0 Field 11 0 21 0 Field 12 0 36 0 Field 13 6 44 0 Field 14 5 41 0 Field 15 0 30 0 Field 16 0 29 0 Field 17 0 26 0 Field 18 1 33 0 Field 19 3 51 0 Field 20 0 40 0 Average Cells/Field 1.35 34.2 0 Calculated Cells/Gram 1.29 x 105 6.52 x 106 0 < or > Detection Limit < detection limit > detection limit < detection limit
85
grams of AT04-165 soil was found to be 1.35. The representative photomicrograph labeled B in
Figure 5.4 portrays field 14 (5 fluorescing cells). For sample AT04-165, fields 3, 5, 6, 9, 11, 12,
15, 16, 17, and 20 contain zero fluorescing bacterial cells and fields 7, 8, and 18 contain one
fluorescing cell. Also, fields 1, 4, and 10 have two fluorescing bacterial cells, field 19 has three
cells, field 2 has four cells, and field 13 has six cells. The average (1.35) was multiplied by
47,652 fields per filter and then again by two to convert the value to cells per gram of soil. The
total number of bacterial cells (viable and nonviable) per gram of AT04-165 soil was found to be
1.29 x 105, but because this number falls below the determined detection limit, it cannot be
presumed an accurate total bacterial cell count.
Sample AT04-170 was prepared in the same manner as AT04-165 and AT04-166. Figure
5.5 shows one representative photomicrograph from each of the five soil amount trials from
sample AT04-170. All 20 evenly dispersed fields for all five soil amounts from sample AT04-
170 have zero fluorescing microbial cells; thus, the average number of fluorescing cells per field
is zero. Because zero cells were detected at all attempted soil concentrations, it is understood
that sample AT04-170 contains no viable or non-viable bacterial cells.
DISCUSSION
The widely used polycarbonate filters contain thousands of fields (depending on the
magnification) capable of quantification purposes. Therefore, evenly distributed sample fields
are counted and averaged to compute a representative number of cells per field. For this reason,
DAPI direct counts should only be performed on filters consisting of microbial cells uniformly
distributed over the entire surface of the filter. When preparing filters from soil samples, it is
important to prepare an appropriate soil dilution in order to obtain a countable microbial cell
concentration, and to find a dilution that is not so overly concentrated with soil that cells cannot
86
Figure 5.5: Photomicrographs of AT04-170 soils unable to be quantified by DAPI direct counts Photomicrograph represents 1.0 g of soil: overly concentrated with fluorescing soil particles (A), photomicrograph represents 0.5 g of soil: no apparent fluorescing bacterial cells (B).
A
B
87
Figure 5.5: (Continued) Photomicrograph represents 0.25 g of soil: no apparent fluorescing bacterial cells (C), photomicrograph represents 0.1 g of soil: no apparent fluorescing bacterial cells (D).
C
D
88
Figure 5.5: (Continued) Photomicrograph represents 0.05 g of soil: no apparent fluorescing bacterial cells (E).
E
89
be distinguished from fluorescing soil particles. Also, too many cells per field could result in
cell clumping resulting in inefficient cell totals. Conversely, cells at extremely low
concentrations can result in inaccurate counts because every cell at low concentrations is
imperative for reliable findings. Given that the Yungay samples have such low microbial cell
numbers, more soil must be used for direct count methods to increase cell concentrations in order
to enumerate cells more accurately. Unfortunately increasing soil volumes to concentrate
microbial cell volumes creates a downside for DAPI direct count procedures because DAPI
shows evidence of nonspecific binding to Atacama Desert soil. The nonspecific binding of
DAPI to mineral particles makes it difficult to distinguish microbial cells from mineral particles
although the mineral particles fluoresce green instead of blue.
Another disadvantage of direct count procedures occurs when microbial cells are grouped
together, making it challenging to distinguish single cells from one another. This is particularly
troublesome in this study because the genus Blastococcus dominates the culture-dependent
studies in all surface samples from the core arid region.
In spite of this problem, I attempted to count blue fluorescing cells ranging from
diameters 0.2 µm and 5.0 µm (the typical bacterial cell size range). Only one soil sample, AT04-
166, was able to be counted, although number of cells in sample AT04-166 was still difficult to
quantify for the reasons stated above. Cells that have survived in the extreme environment of the
Yungay region in the Atacama Desert appear differently under DAPI epifluorescence
microscopy than cells that have been thriving in a nutrient rich environment such as culture
media. This is apparent when comparing the differences between the appearance of cells dried
overnight within Yungay soil (Figure 5.1) and cells dried over a month within Yungay soil
90
(Figure 5.2). This is yet another disadvantage to quantifying cells in Atacama Desert hyper-arid
soils even when cells are able to be observed via DAPI staining, such as in sample AT04-166.
Quantifying microbial cells with DAPI staining in samples with low CFU/g, except
AT04-166, proved impossible with the DAPI direct count method. This dilemma, along with the
obstacles mentioned above, lead to an investigation of the detection limit of Yungay soil in
conjunction with the DAPI direct count protocol applied. Even samples that have obvious
fluorescing cells in certain fields, such as sample AT04-165, may not be adequate for
quantification purposes if the final calculated total cells per gram of soil is below the determined
detection limit for the method and soils used.
Wagner et al. (2003) states that the detection limit of fluorescence in situ hybridization
(FISH) is about 1 x 103 cells/mL. Another study done by the Ocean Drilling Program reports at
that the detection limit of the DAPI direct count procedure used by microbiologists researching
ocean drilling samples is approximately 1 × 105 cells/cm3 (Asada et al. 2002). Considering these
reported detection limits, I explored the detection limit for the DAPI method used for this study.
Known cell concentrations (determined by dilution plating) of microbial cells, particularly
Blastococcus sp. strain AT03-37-10, were added to soil AT04-170 (CFU value and detected total
cell number per gram of soil is zero), stained with DAPI, and counted to find the detection limit
for the DAPI technique used in this study. The detection limit was found to exist at someplace
between 1 x 105 and 1 x 106 cells/g.
AT04-166 is the only sample in this study (except the control pit) that had CFU values
for culturable organisms greater than 1 x 105 cells/g of soil. AT04-166 was also the only Yungay
tested soil to have total cell counts above the determined detection limit. The CFU value per
gram of soil for AT04-166 was as high as 7.40 x 105 on 1/10 PCA medium and the total cell
91
count is found to be 6.52 x 106 cells/g of soil. The sample site having the next highest CFU
value per gram of soil (8.20 x 104) on 1/10 PCA medium is AT04-165, but the calculated total
cell count (1.29 x 105) is below the detection limit; therefore, all other samples fall below the
detection limit of the direct count method of this study and cannot be accurately calculated.
For AT04-166, the total cell count per gram of soil is higher than the CFU value found
using the dilution plating technique and is to be expected because the dilution plating technique
only includes viable culturable heterotrophic bacteria. In non-extreme environments the
difference would be greater; the culturable cell concentrations would be much lower than actual
cell numbers since culturable bacteria only account for 0.001-15% of the total bacterial cell
numbers in most environmental samples (Amann et al. 1995). The extent to how inaccurate the
culturable portion of microorganisms is, when compared to total microbial cell numbers, depends
greatly on the soil under study. The Atacama Desert soils in the Yungay region is known to be
unique in their hyper-aridity and as this study indicates the culturable versus actual microbial cell
concentrations in Yungay region desert soils may not vary significantly. A study by Hartmann et
al. (1997) states that the culturable fraction of prokaryotes underestimates the inhabiting
population of active and dormant microbes by at least one to two orders of magnitude. With
regards to this statement, this thesis focuses on the least amount of variance (one order of
magnitude) between the culturable and total cells. Culturable cell concentrations and total cell
concentration for sample AT04-166 differ only by one order of magnitude. The results from the
direct counts on AT04-165 soils cannot be declared completely valid due to the detection limit,
but the difference between the values of CFUs per gram of soil and total cells per gram of soil is
less than one order of magnitude indicating that some Yungay soils may be exceptions to the “at
least one to two magnitudes” (Hartmann et al. 1997). Organisms cultured from certain Yungay
92
samples may in fact only underestimate the total resident population by less than one order of
magnitude due to low nutrient availability and low diversity among the inhabiting organisms.
For this reason, samples researched in this thesis, other than AT04-166, all contain total cell
counts below the detection limit of this study; thus, DAPI direct count procedures are not
suitable for quantifying most Atacama Desert soils from within the hyper-arid region of Yungay.
Control samples of lake water and soil were also prepared using DAPI epifluorescence
microscopy. A water sample from a Louisiana State University campus lake was fixed, DAPI
stained (a final concentration of 5 µg/µL), and filtered onto a black polycarbonate filter. Also, a
soil sample was retrieved from a cornfield near the Louisiana State University campus and
prepared in a similar manner to the other soil samples tested in this study. The only difference in
this sample preparation was that the cornfield sample was not sonicated due to the likely delicate
nature of some cells thriving in this fertile environment.
The cornfield soil is extremely organic in comparison to the Yungay soils researched in
this study. The purpose of the cornfield experiment was not to quantify, but to determine
whether the problems of non-specific DAPI binding to Yungay soil particles is a drawback when
handling other soils, in particular, soils from non-extreme environments. Figure 5.6 shows four
photomicrographs that were captured from a filter prepared with an extract from 0.25 grams of
cornfield soil. These images reveal that non-specific binding of DAPI to the cornfield soil
particles also is a setback in soils other than Atacama Desert soils.
The water sample was collected in order to compare necessary DAPI direct count
methods on soil samples with necessary methods on water samples. Excessive preparations that
are required for soil samples were not necessary for the water sample; the water was simply
fixed, stained, and filtered. When handling water, the DAPI direct count problems that are
93
obvious with soil samples (discussed throughout this chapter) are no longer concerns. All
volumes showed numerous various types of microorganisms in every field; the number of
fluorescing cells decreased consecutively from the 3.5 mL volume to the 0.5 mL volume. Figure
5.7 shows four images of each water volume filtered. Compared to all other figures throughout
this chapter (including the cornfield figures), Figure 5.7 indicates the substantial variation
between soil and water samples. This control study indicates the simplicity of obtaining direct
counts from water samples by DAPI epifluorescence and makes the case that DAPI direct count
methods are more efficient when employed on water environmental samples as opposed to soil
samples.
94
Figure 5.6: Comparison photomicrographs of cornfield soil sample Four photomicrographs represent 0.25 g of cornfield soil sample.
95
Figure 5.7: Comparison photomicrographs of LSU lake water sample Four photomicrographs represent 3.5 mL of lake water (A), four photomicrographs represent 1.0 mL of lake water (B).
A
B
96
Figure 5.7: (Continued) Four photomicrographs represent 0.5 mL of lake water (C).
C
97
CHAPTER 6 ASSOCIATION BETWEEN VALUES OF CFUS AND
SOIL CHEMICAL COMPOSITION
To examine the potential influence of the chemical composition of the soils studied on
heterotrophs in the Atacama Desert soils, major cation and anion concentrations were measured.
Also, a simple check for carbonate (CO32-) was done using dilute HCl in selected hyper-arid
soils.
RESULTS
Elemental Cation Analysis of Soil Samples
Table 6.1 displays all cation concentrations for each Atacama Desert soil sample studied.
There appears to be no relationship between metal concentrations and CFUs/g (Figures 6.1 and
6.2), except possibly for Na concentrations for surface and subsurface sites sampled within the
Yungay area. Specifically, three of the four sites that had no CFUs/g of soil on all media (AT04-
168, AT04-169, and AT04-170) and another site (AT04-167) that had < 102 (below detection
limit) CFUs/g on one culture medium and 0 CFUs/g on the other four media all had increased Na
levels (> 400 mMol/L) compared to the other soil samples. Conversely, some samples that
contained between 102 and 106 CFUs/g on at least one culture medium had lower Na
concentrations (< 50 mMol/L) (Figure 6.1). For some soils (AT03-42, AT03-44, and AT03-49)
with CFU/g values of < 102 on all five culture media, however, there were also low Na
concentrations measured. For these soils, however, Al concentrations were elevated (Table 6.1
and Figure 6.1).
The concentration of most of the major cations did not vary significantly with depth for
the hyper-arid and southern soil pits (Figure 6.2). However, Na concentrations did vary,
generally increasing with depth, and Ca concentrations decreased in two of the four pits. The
98
Table 6.1: Cation concentrations (mg/L) for Atacama Desert soils All values are reported in mg/L. ND refers to no data obtained.
Sample Site Al As Ca Cd Cr Cu Fe K
AT03-33 17311.48 12.63 18796.51 1.79 15.33 43.80 21767.50 3224.65AT03-34 20831.21 17.38 37856.49 2.51 18.15 75.49 26597.38 3992.88AT03-35 21778.58 11.98 25810.72 2.66 21.20 60.78 29910.39 4324.80AT03-36 14781.87 12.30 21006.71 1.65 13.73 53.78 19443.31 1907.50AT03-37 23982.85 15.45 27847.81 2.49 18.12 73.48 27517.94 4611.27AT03-38 21206.19 18.16 19903.36 2.01 14.33 63.18 22234.04 3866.73AT03-39 15033.08 9.07 17961.55 1.88 16.24 46.47 23865.70 2484.88AT03-40 11544.42 12.96 24908.00 1.35 11.89 93.79 17621.40 2177.02AT03-41 11469.02 9.72 19190.00 1.63 15.23 46.20 21389.48 2101.73AT03-42 24362.06 17.17 8771.11 2.22 16.28 57.84 25040.13 4627.96AT03-43 19016.96 14.09 9033.38 1.85 15.03 50.60 22018.89 3691.51AT03-44 22450.92 15.76 25668.55 2.45 18.39 66.09 24850.20 4582.33AT03-45 18788.49 14.57 17164.40 1.90 15.54 57.64 22298.79 3531.13AT03-46 18530.35 13.31 18892.83 1.85 15.76 63.51 21065.35 3255.45AT03-48 23838.18 17.30 36127.54 2.51 20.59 81.81 26571.73 4953.20AT03-49 16699.24 16.23 26468.40 1.70 13.63 64.10 18928.56 4041.59AT03-50 14903.95 11.60 13745.10 1.35 12.16 43.92 17857.14 3133.42AT04-151 19418.26 14.96 20170.94 1.91 17.00 55.29 23324.81 3502.57AT04-154 15400.95 10.79 19441.67 1.59 13.84 35.68 21227.53 2371.29AT04-155 15301.79 11.07 15913.07 2.11 19.26 42.93 27682.66 2337.01AT04-156 17863.81 11.62 15235.26 1.98 17.37 47.98 25970.93 2726.07AT04-157 16820.66 8.47 7203.69 1.85 17.45 37.96 24700.99 2747.49AT04-158 23654.97 14.93 14402.50 1.90 15.96 59.30 23308.34 3844.11AT04-161 15948.41 8.32 31796.12 2.24 20.37 43.92 26812.20 3829.01AT04-162 8465.73 5.24 34724.80 1.12 13.55 20.24 16933.68 2029.26AT04-163 12136.24 5.97 25315.87 2.04 26.05 35.91 29227.86 2053.89AT04-164 11350.73 3.25 16284.94 1.03 8.36 156.10 15750.05 1723.10AT04-165 8656.57 8.02 28230.61 0.97 9.25 32.65 12966.62 2065.61AT04-166 23181.98 14.78 17149.21 2.41 20.32 50.35 27383.14 5208.40AT04-167 9777.78 7.72 30791.76 0.96 10.44 20.77 12811.04 4225.21AT04-168 12996.20 10.55 23162.22 1.13 15.43 30.79 13211.95 5588.61AT04-169 18988.05 8.09 23170.60 1.56 16.19 42.85 22211.38 2833.86AT04-170 11404.57 6.57 32332.01 1.86 18.85 25.27 24219.15 2330.26
99
Table 6.1: (Continued) Sample Site Mg Mn Na Ni P Pb Si Zn
AT03-33 6897.46 327.19 1006.57 12.09 455.76 10.95 97.12 49.67 AT03-34 8587.15 382.83 1064.93 15.31 510.79 21.89 230.20 73.73 AT03-35 8672.53 364.64 1125.51 16.00 475.01 17.24 117.89 77.28 AT03-36 6430.00 312.57 876.24 10.63 379.56 13.86 203.19 41.71 AT03-37 10376.88 412.76 1153.73 18.94 599.39 17.50 81.79 84.21 AT03-38 8553.22 361.74 1037.73 14.41 537.68 16.01 124.26 65.47 AT03-39 5010.76 277.73 1284.55 9.90 368.22 10.42 153.49 42.41 AT03-40 5234.75 238.02 1120.22 7.66 321.26 9.91 330.25 35.62 AT03-41 4591.66 239.08 1057.89 8.34 399.84 9.76 207.77 34.51 AT03-42 11462.47 430.11 993.53 17.89 662.94 12.67 235.48 72.50 AT03-43 9394.18 369.04 731.49 15.25 576.18 10.59 154.85 57.12 AT03-44 9562.77 415.47 1118.10 16.79 590.74 15.02 148.79 82.48 AT03-45 8441.62 349.24 999.27 14.16 539.01 13.40 65.94 58.48 AT03-46 9122.64 357.49 935.30 14.71 508.72 15.61 93.88 60.66 AT03-48 13063.53 458.89 1086.36 18.49 473.19 21.32 133.52 79.91 AT03-49 8061.01 332.13 1240.64 11.52 524.83 16.03 88.55 57.57 AT03-50 7378.50 312.85 774.80 9.20 416.95 10.32 191.42 44.62 AT04-151 7559.75 344.09 1252.54 13.87 510.04 12.86 91.92 57.30 AT04-154 5160.14 316.71 1150.89 10.02 426.76 9.75 123.56 39.12 AT04-155 5263.43 308.24 1204.55 11.74 466.24 11.66 57.05 46.62 AT04-156 5588.04 334.33 1283.15 11.79 457.57 12.27 108.68 47.61 AT04-157 5596.40 309.68 2402.64 11.44 450.47 8.82 170.25 43.69 AT04-158 9729.95 379.21 2628.14 15.69 584.57 12.27 77.61 60.15 AT04-161 7885.17 301.09 1524.06 13.69 401.85 13.21 104.64 49.97 AT04-162 5027.07 195.83 3573.49 6.07 216.12 5.25 341.22 26.07 AT04-163 4149.75 205.72 3645.65 8.58 359.29 6.76 282.56 33.01 AT04-164 5199.71 264.96 794.13 5.78 264.44 6.60 42.65 28.24 AT04-165 4534.41 221.09 3064.48 5.43 314.84 8.92 183.23 27.75 AT04-166 11247.46 405.25 1110.29 17.67 534.48 12.72 125.31 63.05 AT04-167 4020.94 235.51 41635.13 10.26 291.11 7.61 201.92 42.68 AT04-168 8781.30 212.86 65840.75 7.70 272.90 9.15 179.91 41.88 AT04-169 6366.58 232.46 13682.44 9.50 352.46 6.22 158.59 33.35 AT04-170 5043.64 180.27 9935.88 7.97 291.31 7.41 160.93 32.27
100
Table 6.1: (Continued) Sample Site Al As Ca Cd Cr Cu Fe K
AT04-150 0cm 18015.36 14.48 18130.15 2.33 21.23 50.01 27841.65 3136.59 AT04-150 10cm 10777.60 10.17 46252.91 1.18 11.89 12.01 15969.52 2135.61 AT04-150 20cm 11984.70 8.81 37054.24 1.35 11.83 14.27 17948.12 2611.72 AT04-150 30cm 13227.53 17.66 55166.27 1.36 14.06 15.11 17762.63 3452.11 AT04-150 40cm 11925.56 6.89 39526.98 1.84 17.23 15.14 23497.39 2480.83 AT04-150 50cm 13695.33 18.92 43350.73 1.62 14.76 16.89 20446.43 3502.28 AT04-150 60cm 12298.46 10.18 27273.39 1.05 11.11 14.82 14142.91 3202.53 AT04-150 70cm 10852.27 19.11 37620.61 0.98 11.08 12.97 13463.13 3500.42 AT04-150 80cm 10451.96 24.27 31122.99 0.61 9.82 14.15 8238.36 5406.99 AT04-150 90cm 10699.02 15.56 23732.78 0.90 11.03 15.69 12617.48 3848.15 AT04-152 0cm 21575.38 14.42 19384.57 2.11 17.04 53.87 24068.80 3795.26 AT04-152 10cm 7448.39 5.96 71929.80 0.71 7.00 9.85 9700.99 2005.43 AT04-152 20cm 11665.19 8.16 58427.04 1.20 11.97 14.90 15630.08 3054.93 AT04-152 30cm 13148.50 9.14 59981.19 1.12 11.59 17.55 15371.47 3814.85 AT04-152 40cm 12164.52 8.36 40271.38 1.15 10.44 16.72 14962.68 3339.63 AT04-152 50cm 12409.81 9.41 35310.10 1.14 12.50 15.51 15650.16 3400.24 AT04-152 60cm 13855.61 10.33 16353.51 1.07 10.58 16.67 15210.93 2813.29 AT04-152 70cm 13151.05 12.18 29201.90 1.05 12.20 16.41 14569.32 4307.67 AT04-152 80cm 12268.11 12.81 27100.99 1.07 12.06 15.36 15257.03 3778.53 AT04-153 0cm 16145.22 8.93 7877.56 1.57 15.25 26.47 19832.51 2270.56 AT04-153 10cm 8245.30 8.55 78082.17 0.60 6.54 10.30 8578.90 2936.92 AT04-153 20cm 10649.00 8.55 55835.20 0.94 8.82 12.46 12712.52 2629.64 AT04-153 30cm 9377.53 8.36 49228.73 0.86 7.75 12.49 11807.43 2540.22 AT04-153 40cm 11484.18 14.10 53403.54 1.07 8.32 14.44 11790.34 3514.62 AT04-153 50cm 14194.64 23.23 35192.94 1.41 11.68 18.09 15734.38 3836.49 AT04-153 60cm 10968.92 12.46 23399.33 0.55 9.41 14.34 7780.04 3722.38 AT04-153 70cm 12502.35 14.08 24909.71 0.89 9.08 16.98 10845.91 4533.28 AT04-153 80cm 14160.91 14.99 21563.66 0.90 11.06 18.51 11960.97 4841.45 AT04-159 0cm 14322.63 4.29 15925.79 1.97 11.67 43.48 25592.00 2184.88 AT04-159 10cm 11340.49 3.30 16261.76 1.12 6.34 25.37 15854.48 1669.81 AT04-159 20cm 9500.60 4.64 43184.61 0.94 6.45 20.80 13820.81 1964.10 AT04-159 30cm 15935.83 6.68 17007.05 1.69 12.31 33.12 24147.68 3067.44 AT04-159 40cm 17978.00 9.71 16585.50 1.58 12.62 36.30 22778.00 3979.00
Cornfield ND ND ND ND ND ND ND ND
101
Table 6.1: (Continued) Sample Site Mg Mn Na Ni P Pb Si Zn
AT04-150 0cm 6725.49 338.18 1096.98 12.77 459.40 12.25 107.46 52.18 AT04-150 10cm 4936.55 204.68 998.52 4.84 256.77 3.97 19.18 21.55 AT04-150 20cm 5952.84 203.80 959.19 6.92 282.12 3.57 22.54 26.38 AT04-150 30cm 6894.31 224.61 1133.69 6.12 310.69 3.97 37.29 27.49 AT04-150 40cm 5394.75 231.96 1138.73 8.02 313.76 4.62 63.42 30.83 AT04-150 50cm 7140.27 252.41 2642.80 7.48 328.18 5.49 42.67 29.72 AT04-150 60cm 6212.97 246.34 22664.09 6.98 78.75 4.12 33.75 27.11 AT04-150 70cm 6453.35 192.58 22531.65 5.23 205.93 3.22 63.54 21.66 AT04-150 80cm 7107.77 172.75 48175.61 4.92 118.09 2.57 33.80 20.30 AT04-150 90cm 6076.92 226.44 24619.80 5.98 126.61 3.97 51.41 23.98 AT04-152 0cm 9336.82 383.56 1302.48 15.18 509.83 11.74 68.67 58.70 AT04-152 10cm 3955.04 127.36 524.71 3.45 189.43 2.20 34.81 16.83 AT04-152 20cm 6231.26 198.67 997.70 6.99 291.94 3.68 46.03 27.09 AT04-152 30cm 6812.13 215.69 951.29 7.38 305.07 4.11 41.54 30.31 AT04-152 40cm 6484.66 225.01 3228.97 6.82 309.84 4.44 32.11 27.20 AT04-152 50cm 6124.77 224.14 6262.57 6.71 287.34 4.48 47.65 26.84 AT04-152 60cm 6574.17 289.55 4337.63 7.46 304.64 5.33 30.64 25.98 AT04-152 70cm 8773.74 214.75 3319.58 6.30 260.65 3.88 44.90 27.76 AT04-152 80cm 8412.82 217.85 3322.14 5.81 255.59 3.96 32.38 26.55 AT04-153 0cm 4916.17 296.94 1273.21 9.68 399.84 7.61 254.59 35.01 AT04-153 10cm 5224.92 116.24 750.52 3.28 183.24 2.44 30.63 16.97 AT04-153 20cm 4618.08 176.24 1073.49 4.41 260.12 3.68 33.85 20.46 AT04-153 30cm 4877.63 164.02 925.76 4.52 259.33 3.36 23.39 19.72 AT04-153 40cm 6313.82 182.28 3142.76 5.05 270.87 3.70 72.32 22.16 AT04-153 50cm 7589.97 224.15 4860.07 7.29 328.61 5.05 67.93 28.04 AT04-153 60cm 6074.91 167.49 48792.51 5.63 122.79 3.01 25.28 22.05 AT04-153 70cm 9480.13 197.90 28408.63 6.53 218.70 3.86 24.05 26.70 AT04-153 80cm 10301.81 206.39 22670.00 7.07 207.42 4.03 33.98 29.15 AT04-159 0cm 7375.20 380.27 580.96 8.12 279.74 8.36 29.81 42.69 AT04-159 10cm 4029.76 253.83 322.15 4.66 202.97 3.32 32.53 26.38 AT04-159 20cm 3754.48 201.95 292.16 3.68 169.51 2.50 26.82 20.97 AT04-159 30cm 6450.76 315.16 536.71 6.69 226.82 5.08 80.96 36.76 AT04-159 40cm 9064.00 363.50 659.00 7.13 274.00 5.98 44.15 40.10
Cornfield ND ND ND ND ND ND ND ND
102
010
020
030
040
050
060
070
080
090
010
0011
0012
0013
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0015
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00
Sam
ple
Site
s
mMol/L
110100
1000
1000
0
1000
00
1000
000 CFU
Na
(mM
ol/L
)K
(mM
ol/L
)C
a (m
Mol
/L)
Mg
(mM
ol/L
)Fe
(mM
ol/L
)M
n (m
Mol
/L)
Pb (m
Mol
/L)
Zn (m
Mol
/L)
As (
mM
ol/L
)C
d (m
Mol
/L)
Cr (
mM
ol/L
)C
u (m
Mol
/L)
Ni (
mM
ol/L
)A
l (m
Mol
/L)
Si (m
Mol
/L)
P (m
Mol
/L)
CFU
AT04-170 AT04-169 AT04-168 AT04-167 AT04-166 AT04-165 AT04-164 AT04-163 AT04-162 AT04-161 AT04-158 AT04-157 AT04-156 AT04-155 AT04-154 AT04-151 AT03-50 AT03-49 AT03-48 AT03-46 AT03-45 AT03-44 AT03-43 AT03-42 AT03-41 AT03-40 AT03-39 AT03-38 AT03-37 AT03-36 AT03-35 AT03-34 AT03-33
Figu
re 6
.1:
CFU
/g v
alue
s and
cat
ion
conc
entra
tions
for Y
unga
y su
rfac
e so
ils
103
Figu
re 6
.2:
Cha
rts d
ispl
ayin
g re
latio
nshi
p be
twee
n C
FU/g
val
ues a
nd c
atio
n co
ncen
tratio
ns fo
r Ata
cam
a D
eser
t soi
l pits
AT
04-1
59 c
atio
ns
010
020
030
040
050
060
070
080
090
010
0011
0012
0013
0014
0015
0016
0017
0018
0019
0020
0021
0022
00
010
2030
40
Dep
th (c
m)
mMol/L
110100
1000
1000
0
1000
00
1000
000
1000
0000 CFU
Na
(mM
ol/L
)K
(mM
ol/L
)C
a (m
Mol
/L)
Mg
(mM
ol/L
)Fe
(mM
ol/L
)M
n (m
Mol
/L)
Pb (m
Mol
/L)
Zn (m
Mol
/L)
As (
mM
ol/L
)C
d (m
Mol
/L)
Cr (
mM
ol/L
)C
u (m
Mol
/L)
Ni (
mM
ol/L
)A
l (m
Mol
/L)
Si (m
Mol
/L)
P (m
Mol
/L)
CFU
AT
04-1
53 c
atio
ns
010
020
030
040
050
060
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090
010
0011
0012
0013
0014
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00
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2030
4050
6070
80
Dep
th (c
m)
mMol/L
110100
1000
1000
0 CFU
Na
(mM
ol/L
)K
(mM
ol/L
)C
a (m
Mol
/L)
Mg
(mM
ol/L
)Fe
(mM
ol/L
)M
n (m
Mol
/L)
Pb (m
Mol
/L)
Zn (m
Mol
/L)
As (
mM
ol/L
)C
d (m
Mol
/L)
Cr (
mM
ol/L
)C
u (m
Mol
/L)
Ni (
mM
ol/L
)A
l (m
Mol
/L)
Si (m
Mol
/L)
P (m
Mol
/L)
CFU
AT
04-1
52 c
atio
ns
010
020
030
040
050
060
070
080
090
010
0011
0012
0013
0014
0015
0016
0017
0018
0019
0020
0021
0022
00
010
2030
4050
6070
80
Dep
th (c
m)
mMol/L
110100
1000
1000
0 CFU
Na
(mM
ol/L
)K
(mM
ol/L
)C
a (m
Mol
/L)
Mg
(mM
ol/L
)Fe
(mM
ol/L
)M
n (m
Mol
/L)
Pb (m
Mol
/L)
Zn (m
Mol
/L)
As (
mM
ol/L
)C
d (m
Mol
/L)
Cr (
mM
ol/L
)C
u (m
Mol
/L)
Ni (
mM
ol/L
)A
l (m
Mol
/L)
Si (m
Mol
/L)
P (m
Mol
/L)
CFU
AT
04-1
50 c
atio
ns
010
020
030
040
050
060
070
080
090
010
0011
0012
0013
0014
0015
0016
0017
0018
0019
0020
0021
0022
00
010
2030
4050
6070
8090
Dep
th (c
m)
mMol/L
110100
1000
1000
0 CFU
Na
(mM
ol/L
)K
(mM
ol/L
)C
a (m
Mol
/L)
Mg
(mM
ol/L
)Fe
(mM
ol/L
)M
n (m
Mol
/L)
Pb (m
Mol
/L)
Zn (m
Mol
/L)
As (
mM
ol/L
)C
d (m
Mol
/L)
Cr (
mM
ol/L
)C
u (m
Mol
/L)
Ni (
mM
ol/L
)A
l (m
Mol
/L)
Si (m
Mol
/L)
P (m
Mol
/L)
CFU
104
three soil pits sampled from within the Yungay hyper-arid region had no CFUs/g, other than the
two CFU/g peaks at 10 cm in soil pit AT04-152 and at 40 cm in soil pit AT04-150; therefore, it
was difficult to determine if there was any correlation among the CFU/g values and cation
concentrations (Figure 6.2). But, some layers from the three core soil pits with the highest Na
concentrations did have 0 or < 102 CFUs/g on all five culture media (Figure 6.2). For soil pit
AT04-152, CFU/g values decreased with depth. Ca concentrations also decreased with depth, as
Na concentrations. At 10 cm, consistently high CFU/g values between 102 and 104 were
measured on all five culture media, and this layer also had a low Na concentration. However, at
50 cm depth, the Na concentration was the highest in the pit and there was 0 CFUs/g measured
on all five culture media. Although this Na concentration was not higher than Na concentrations
for other soil samples (Table 6.1), it was higher than Na concentrations measured at sites having
higher culturable heterotrophic bacteria abundances (Figure 6.1). Because Ca concentrations
also decreased with depth, there could be a correlation with CFU/g. For the southern soil pit
(AT04-159), all of the five layers had > 105 CFUs/g on at least one of the five culture media.
This site, as discussed in Chapter 4, was considered a control soil pit because the region is less
arid than the Yungay region; the increased CFUs/g may reflect the increased amounts of
precipitation and a larger organic component in this soil. The concentration of Na was low
throughout the soil pit, with values much lower than the Yungay soils (Table 6.1 and Figure 6.2).
Inorganic Anion Analysis of Soil Samples
Table 6.2 lists major anion concentrations for each surface and subsurface sample along
with their CFU/g range. Of the 33 Yungay surface sites, only seven sites had Cl anion
concentrations > 1.0 mMol/L (Figure 6.3). Of these seven soils, samples AT04-162 and AT04-
165 had CFUs/g of soil > 103 on at least one culture medium, while the remaining soils had 0 or
105
Table 6.2: Major anion concentrations (mg/L) and CFU/g values for Atacama Desert soils NA represents the anion value was below detection (< 0.01 mg/L). ND refers to no data was obtained.
Sample Site Cl F NO3 SO4 CFUs/g AT03-33 0.88 0.60 0.51 1657.97 < 105 AT03-34 0.53 NA 0.51 2013.20 < 103 AT03-35 0.78 0.51 0.72 1397.27 < 103 AT03-36 0.46 0.84 0.55 2126.90 < 103 AT03-37 0.73 0.48 0.63 2059.77 < 103 AT03-38 0.66 0.27 0.65 1747.73 < 103 AT03-39 0.42 NA 0.32 979.20 < 104 AT03-40 0.81 0.47 0.41 2358.29 < 105 AT03-41 0.84 0.52 0.46 1485.75 < 105 AT03-42 1.12 0.28 0.71 93.22 < 102 AT03-43 0.66 0.48 0.59 158.78 < 103 AT03-44 1.01 0.48 0.91 2362.98 < 102 AT03-45 0.44 0.44 0.30 1344.27 < 104 AT03-46 NA NA 0.48 875.42 < 105 AT03-48 0.61 NA 0.47 1923.91 < 104 AT03-49 0.52 NA 0.61 1940.37 < 102 AT03-50 0.32 0.88 0.36 633.87 < 103 AT04-151 1.11 NA 1.33 2279.62 < 103 AT04-154 0.89 NA 0.54 1827.93 < 104 AT04-155 0.57 0.50 0.53 754.53 < 103 AT04-156 0.56 0.41 0.57 989.19 < 103 AT04-157 21.1 NA 12.54 27.78 < 102 AT04-158 16.00 NA 5.36 71.87 < 102 AT04-161 1.38 NA 0.64 1554.82 < 105 AT04-162 124.62 NA 4.27 712.50 < 104 AT04-163 53.38 0.61 4.61 2843.06 0 AT04-164 4.07 0.92 0.29 1788.43 < 104 AT04-165 40.75 0.60 7.92 2671.57 < 105 AT04-166 0.35 NA 0.21 514.02 < 106 AT04-167 65.11 1.75 253.30 1877.13 < 102 AT04-168 46.63 3.75 99.43 3141.63 0 AT04-169 207.61 1.29 8.30 1217.13 0 AT04-170 580.14 4.71 2.67 1851.42 0
106
Table 6.2: (Continued)
Sample Site Cl F NO3 SO4 CFUs/g AT04-150 0cm 0.39 NA 0.52 1659.12 0 AT04-150 10cm 0.42 NA NA 2077.94 < 102 AT04-150 20cm 1.26 0.21 0.14 2305.36 < 102 AT04-150 30cm 0.73 0.33 0.08 2318.13 < 103 AT04-150 40cm 1.05 0.27 0.09 2322.96 < 104 AT04-150 50cm 11.95 0.78 10.65 2780.12 < 103 AT04-150 60cm 64.59 1.14 3.41 3391.57 0 AT04-150 70cm 52.72 1.46 9.83 3189.31 < 103 AT04-150 80cm 295.55 4.36 32.07 4969.68 < 102 AT04-150 90cm 71.08 3.40 21.19 3527.04 0 AT04-152 0cm 0.69 0.30 0.41 1483.39 < 102 AT04-152 10cm 0.40 NA 0.02 2289.31 < 104 AT04-152 20cm 0.58 NA 0.06 2316.73 < 103 AT04-152 30cm 0.68 0.32 0.07 2304.91 < 103 AT04-152 40cm 27.74 NA 1.21 2703.80 < 103 AT04-152 50cm 77.62 NA 10.24 2559.13 0 AT04-152 60cm 45.89 0.55 12.51 1834.08 < 102 AT04-152 70cm 53.63 1.45 24.80 2831.09 < 102 AT04-152 80cm NA NA 21.99 2791.28 0 AT04-153 0cm 0.19 0.12 0.16 31.26 < 103 AT04-153 10cm 0.24 NA NA 2319.46 < 102 AT04-153 20cm 0.32 0.28 NA 2326.12 < 103 AT04-153 30cm 2.98 0.2118 0.66 2299.37 < 102 AT04-153 40cm 51.23 1.29 3.07 2652.08 0 AT04-153 50cm 83.24 2.72 3.20 2883.70 < 102 AT04-153 60cm 72.90 1.01 11.86 4873.15 0 AT04-153 70cm 138.90 1.82 15.82 3279.26 < 102 AT04-153 80cm 653.09 1.58 13.56 2822.31 < 102 AT04-159 0cm 0.38 NA 0.03 9.34 < 106 AT04-159 10cm 0.60 NA NA 1406.09 < 106 AT04-159 20cm 2.47 NA NA 2323.30 < 106 AT04-159 30cm 3.21 NA NA 1209.20 > 106 AT04-159 40cm 2.76 NA NA 1380.97 > 106
Cornfield 0.34 NA 0.03 0.62 ND
107
0246810121416182022242628303234
Sam
ple
Site
s
mMol/L
110100
1000
1000
0
1000
00
1000
000 CFU
Cl (
mM
ol/L
)
F (m
Mol
/L)
SO4
(mM
ol/L
)
NO
3 (m
Mol
/L)
CFU
AT04-170 AT04-169 AT04-168 AT04-167 AT04-166 AT04-165 AT04-164 AT04-163 AT04-162 AT04-161 AT04-158 AT04-157 AT04-156 AT04-155 AT04-154 AT04-151 AT03-50 AT03-49 AT03-48 AT03-46 AT03-45 AT03-44 AT03-43 AT03-42 AT03-41 AT03-40 AT03-39 AT03-38 AT03-37 AT03-36 AT03-35 AT03-34 AT03-33
Figu
re 6
.3: C
FU/g
val
ues a
nd a
nion
con
cent
ratio
ns fo
r Yun
gay
surf
ace
soils
108
Figu
re 6
.4:
CFU
/g v
alue
s and
ani
on c
once
ntra
tions
at d
epth
for A
taca
ma
Des
ert s
oil p
its
AT
04-1
53 A
nion
s
0510152025303540455055
010
2030
4050
6070
80
Dep
th (c
m)
mMol/L
110100
1000
CFU
Cl (
mM
ol/L
)F
(mM
ol/L
)SO
4 (m
Mol
/L)
NO
3 (m
Mol
/L)
CFU
AT
04-1
59 A
nion
s
0510152025303540455055
010
2030
40
Dep
th (c
m)
mMol/L
110100
1000
1000
0
1000
00
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000
1000
0000 CFU
Cl (
mM
ol/L
)F
(mM
ol/L
)SO
4 (m
Mol
/L)
NO
3 (m
Mol
/L)
CFU
AT
04-1
52 A
nion
s
0510152025303540455055
010
2030
4050
6070
80
Dep
th (c
m)
mMol/L
110100
1000
1000
0 CFUC
l (m
Mol
/L)
F (m
Mol
/L)
SO4
(mM
ol/L
)N
O3
(mM
ol/L
)C
FU
AT
04-1
50 A
nion
s
0510152025303540455055
010
2030
4050
6070
8090
Dep
th (c
m)
mMol/L
110100
1000
1000
0 CFU
Cl (
mM
ol/L
)F
(mM
ol/L
)SO
4 (m
Mol
/L)
NO
3 (m
Mol
/L)
CFU
109
near 0 CFUs/g on all five culture media. Moreover, soil from AT04-169 and AT04-170 both
contained 0 CFUs/g and had the two highest Cl concentrations of all the analyses. Therefore, the
elevated Cl values may be correlated to no recoverable bacteria per gram of soil and soils having
elevated CFU/g values also had relatively low Cl concentrations (Figure 6.3).
Compared to Cl concentration, there was no apparent relationship with the other anion
concentrations and CFU/g for the surface soils (Table 6.2 and Figure 6.3). For instance, of all 33
surface samples from within the Yungay region, only two (AT04-167 and AT04-168) had NO3
levels > 1.0 mMol/L, with most of the NO3 concentrations < 0.2 mMol/L (Table 6.2 and Figure
6.3). Both of the soils with high NO3 concentrations had low CFUs/g on all five culture media
(Figure 6.3). Sixteen soils with NO3 concentrations between 0.003 mMol/L and 0.01 mMol/L
had > 102 CFUs/g on at least one medium. The concentration of SO4 in all of the Yungay
surface soils were very high, and yet there was no correlation to high or low CFUs/g values .
For the soil pits, Cl and SO4 concentrations increased with depth, which did not
necessarily correlate to CFU/g values (Figure 6.4). The other anions did not seem to relate to
CFU/g values, either. As with the surface soils, although it appeared that low CFU/g values
were associated with high Cl levels, the 80 cm layer from soil pit AT04-152 had 0 CFUs/g and
yet it had a low Cl concentration. SO4 levels were just as elevated as were measured in surface
soil samples and SO4 concentrations did not correlate to the CFU/g levels in the soil pits.
Detection of Carbonates in Atacama Desert Soils
Carbonate anions (e.g., HCO3-, CO3
2-) were not measured with the other anions, but to
check if carbonate was present in the soils, dilute HCl was added to dry soil. Sample AT04-167,
representing the surface samples from the Yungay region, and sample AT04-153 at 30 cm,
representing a subsurface layer from the hyper-arid region, both tested positive for carbonates.
110
Soil from AT04-152 at 70 cm, representing Yungay region subsurface soils at deep layers, had
negative results for carbonates.
DISCUSSION
The chemical composition of the soils was determined in this study to establish if there
were any possible correlations between soil chemical composition and CFU/g values in Atacama
Desert soils. The results of the chemical analyses described provide some insight into the results
previously discussed in Chapters 3 and 4. The similarities observed between the ion analyses
and the CFU/g values relate to the idea that a requisite for self-organized patchiness at short
spatial range is a positive connection between organisms and nutrient availability (Rietkerk et al.
2004). Therefore, excess soluble salts in soils can shape bacterial distributions because elevated
salinity concentrations can cause nutrient discrepancies that will result in the accumulation of
elements that might be harmful to prokaryotic cells (Kotuby-Amacher et al. 1997).
“Chilean nitrate” is the trade name for the naturally occurring mined inorganic mineral
salt that is found in high abundance within the soils of the Atacama Desert because of a lack of
precipitation (NOSB TAP 2002). The substance quickly ionizes into Na and NO3 in water, but it
can also contain Cl, NO3, and SO4 (NOSB TAP 2002). As a result, these anions may build up in
the soil and could account for the correlations observed between soils with high concentrations
of anions and little to no CFUs/g of soil. Specifically, Na is locally persistent in soil systems in
that it is relatively immobile. Soil has a net negative charge; thus, it binds Na strongly and Na
tends to accumulate in arid environments (NOSB TAP 2002). As indicated by Kotuby-Amacher
et al. (1997), high soil salinity can cause nutrient imbalances that result in a buildup of elements,
which can be harmful to cells; this reduces water infiltration if the amount of the salt element,
Na, is elevated. For these reasons, it was expected that soils containing high concentrations of
111
Na, namely soils from the studied hyper-arid region, would not result in high values of
heterotroph CFUs/g.
The results from the elemental analyses of the Atacama Desert soils support the
suggestion that elevated concentrations for some elements and compounds (e.g., Na, Cl, NO3)
may contribute to decreased abundances of bacteria (Figures 6.1 and 6.3). For instance, higher
Na levels in the majority of the soils correlated to low CFU/g values. Regarding the depth at
which the increased levels were observed, Na may accumulate over time more efficiently in
subsurface layers because of its persistent nature (Figure 6.2). By comparison, the control soil
pit at Altamira contained considerably higher amounts of culturable heterotrophic bacteria and
exhibited Na concentrations less than most of the Yungay soil samples. Elevated SO4
concentrations were ubiquitous throughout Atacama Desert samples (Yungay and Altamira soil
pit samples). Although there was no direct correlation between levels of culturable heterotrophic
bacteria within individual samples and SO4 concentrations, the relatively low levels of CFUs/g
observed throughout the Atacama Desert samples, in comparison with soils from non-arid
environments, may have been influenced by the accumulation of SO4 in the soils. Carbonate
detected in the Yungay region of the Atacama Desert at surface and near surface layers, but not
in more subterranean layers, could be in the mineral form of CaCO3 in the soils. The absence of
precipitation probably results in the accumulation of CaCO3 in the surface soils of the hyper-arid
region; this mineral can have a neutralizing effect on pH and may be one of the reasons why the
soil pH for most of the desert soils was pH 7 or above.
112
CHAPTER 7 CONCLUSIONS
The purpose of this thesis was to study the microbiology of the hyper-arid core region of
the Atacama Desert, Chile. The research on these hyper-arid soils involved the use of culture-
dependent methods, epifluorescence direct count techniques, and chemical composition analyses.
The findings confirm the Mars like nature of the soils within the hyper-arid region and
demonstrate the need for extensive and comprehensive sampling strategies on any future Mars
exploration missions that involve surface or subsurface sampling for life detection.
Chapter 2 presents a thorough description of the techniques applied to Atacama Desert
soil samples throughout this research to better understand microbiological aspects of the “Mars-
like” soils. The essential components of this study include culturable bacteria quantification and
identification methods which were employed on 33 surface soil sites as well as four soil pits
from which subsurface layers were sampled. Serial dilution plating procedures were applied to
each surface and subsurface sample using five culture media, three nutrient rich and two nutrient
low for the determination of CFUs/g of soil. Isolation methods of pure cultures, preservation
procedures, DNA extraction methods, PCR and PCR purification protocols, and sequencing
techniques are described within Chapter 2. Sequencing amplified 16S rRNA genes from pure
isolates permitted identification of the isolates to the closest phylogenetic relatives by using the
BLAST interface and the GenBank database. The methodologies for DAPI epifluorescence
studies are described in Chapter 2. Also, procedures to perform pH, salinity, elemental,
inorganic anion, and carbonate analyses are outlined.
Chapter 3 represents the culture-dependent findings for surface samples acquired from
the hyper-arid core region of the Atacama Desert. The chapter investigates the hypothesis that
113
the distribution of culturable heterotrophic bacteria in terms of quantity and diversity in surface
soils would not be homogeneous because organisms acquire nutrients from their environment
and are spread across desert regions according to resource accessibility throughout the soils
(Rietkerk et al. 2004). Patchiness was anticipated and later confirmed to exist throughout the
surface of the hyper-arid region. Evidence that at short spatial range, for self-arranged
patchiness to take place, organisms must be directly linked to resource abundance from Rietkerk
et al. 2004, supports the short range patchiness found in the 15.3 km2 core arid region of the
Atacama Desert, Yungay. The patchiness in terms of numbers is apparent in the CFUs
determined for the 33 surface sites since the range was found to be between 0 and 7.4 x105
CFUs/g of soil.
Isolates belonging to three phyla, Acintobacteria, Firmicutes, and Proteobacteria were
found within the culturable heterotrophic bacteria inhabiting the surface sites. Members of the
phylum Actinobacteria are prevalent throughout the sampled surface sites. One family included
in the phylum Actinobacteira, Geodermatophilaceae, was found to dominate the majority of the
surface sites, especially species of the genus Blastococcus. Chapter 3 also discusses
characteristics of species in the genus Blastococcus.
Chapter 4 describes the examination of the subsurface samples via culture-dependent
methods. Other than two CFU value peaks that barely exceed 1 x 103 CFUs/g of soil on at least
one of the five culture media used, the three soil pits in the core hyper arid region demonstrate
very low CFU values in all layers sampled. The southern site at Altamira, where a control pit
was excavated, had higher CFU values per gram of soil than soils samples from within the
hyper-arid region. The pit at Altamira, AT04-159, was found to have CFUs/g of soil of
approximately 1.0 x 105 on at least one culture media at layers near the surface, and > 1.0 x 106 at
114
the deepest subsurface layer on all culture media employed for this study. The diversity of
culturable heterotrophic bacteria retrieved from each of the three pits within the hyper-arid core
region is greatly reduced as compared to the diversity recovered in the Altamira pit. The isolates
from these three Yungay soil pits consist of members of the phyla, Acintobacteria, Firmicutes,
and Proteobacteria, while the Altamira pit was found to have representatives of at least four
phyla including the three listed for the core region soil pits and an additional phylum,
Bacteroidetes. The most abundant of the three phyla are representatives of the phylum
Actinobacteria, precisely as was found in the surface samples. The diversity recovered from the
individual layers of the three hyper-arid region soil pits is low. Even in the two pits that exhibit
spiked CFU levels, larger amounts of the same types of bacteria that were recovered from the
surface layers were detected. Bacteria able to be cultured from the surface of each of the three
pits are in many instances the same bacteria found at some depths. Comparative evaluations of
isolates recovered from the surface samples and isolates obtained from the Yungay soil pit layers
show that the diversity is slightly greater in the surface samples; although, many transverse
organisms were discovered.
The amount of Blastococcus sp. recovered from surface samples is greatly reduced in the
Yungay soil pit samples, specifically in soil pits AT04-150 and AT04-152. Pseudonocardia sp.
are found to be dominate the culturable isolates within pits AT04-150 and AT04-152 and are
only detected in Yungay pit samples. Of the three hyper-arid region soil pits, AT04-150 and
AT04-152 were excavated on the top of a hill, while soil pit AT04-153 was constructed in the
valley adjacent that hill. The majority of the surface sites showing spatial patchiness in terms of
CFU levels and diversity were also sampled in a valley at Yungay. The culturable bacteria that
dominate surface samples and the valley sampled soil pit, AT04-153, are members of the genus
115
Blastococcus. Soil pits AT04-150 and AT04-152, the two soil pits that were dug on the hilltop
have less than 2% of the recoverable bacteria identified as members of the genus Blastococcus,
and the dominate organisms are members of the genera Pseudonocardia and Streptomyces. This
suggests some differences in the nature of the soils found in Yungay low lying valley and those
soils found at elevated sites in the Yungay region that may result in the observed variation within
the bacterial communities. The findings reported and discussed in Chapters 3 and 4 suggest the
existence of spatial patchiness in terms of numbers of recoverable bacteria and diversity on a
vertical as well as on a horizontal plane.
The soil pit in the less arid, more southern region at Altamira has CFU/g values up to four
magnitudes greater than those in the hyper-arid region. The Altamira soil pit was found to
contain approximately 42 different prokaryotic taxa. Vegetation increases along a moisture
gradient in southern regions of the Atacama Desert (Navarro-González et al. 2003). For this
reason, it was projected that CFU levels and bacterial community diversity of organisms
recovered from the Altamira soil pit increase in comparison with the CFUs and diversity within
the hyper-arid region.
Enrichment cultures for each of the Yungay pits proved useful in uncovering organisms
that grow rapidly in favorable conditions; although, they cannot function as enumeration studies.
A fog event, which is a rare moisture occurrence in the hyper-arid region, could be one source of
a natural enrichment process wherein organisms normally found at small concentrations could
multiply quickly. In the three Yungay pits, the diversity among the recovered enrichment
isolates was found to be similar to that of the dilution plating isolates in terms that both contain
low diversity of organisms, and the organisms recovered were commonly the same.
116
Chapter 5 describes the direct count research carried out in an attempt to quantify
bacterial cells through DAPI epifluorescence microscopy. DAPI staining for enumeration
purposes in low CFU value samples, which includes all samples from the Yungay region besides
AT04-166, proved undoable with the DAPI direct count method used for this study. An
investigation of the detection limit for the Yungay soils with the applied DAPI direct count
procedure was carried out. Blastococcus sp. strain AT03-37-10 cells were added to soils
containing zero culturable bacteria, stained with DAPI, and counted to find the detection limit for
the DAPI technique used in this study. The detection limit was found to exist between 1 x 105
and 1 x 106 cells per gram of soil.
AT04-166 was the only sample in this study (besides the Altamira pit samples) that has
CFU values greater than 1 x 105 cells per gram of soil. AT04-166 was also the only Yungay soil
found to have total cell counts above the determined detection limit. The CFU value per gram of
soil for AT04-166 is as high as 7.40 x 105 on one of the culture media utilized and the total cell
count was found to be 6.52 x 106 cells per gram of soil. Direct count procedures performed on
the sample site, AT04-165, which has the second highest CFU/g value of 8.20 x 104, were
unsuccessful because the computed total cell count of 1.29 x 105 was beneath the determined
detection limit. Because other soil samples from the Yungay region were found to contain lower
CFU numbers than AT04-165, the results obtained from the direct cell count attempt on AT04-
165 suggest that other samples would show results that also fall below the detection limit of the
direct count method and therefore not be able to accurately be calculated by the method
employed in this study.
In chapter 6 elevated concentrations of the metal, Na, and inorganic anionic molecules
Cl, F, SO4, and NO3 within Yungay soils, was shown, somewhat, to correlate with low numbers
117
of culturable bacterial cells; the inverse was also observed. This parallel may be related to the
idea that claims a necessity for self-organized patchiness at short spatial range is an established
positive association linking organisms and nutrient availability (Rietkerk et al. 2004). Thus, an
abundance of soluble salts in soils can affect bacterial distributions as well since high soil
salinity can cause nutrient imbalances that result in an accrual of elements that is lethal to cells
(Kotuby-Amacher et al. 1997).
This thesis adds to previous reports by Navarro-González et al. 2003 characterizing this
region as having soils considered analogous to Martian soils. Discoveries made as a result of
this study not only are beneficial to future Atacama Desert research, but can also be applied to
preparations concerning upcoming Mars exploration. The increased CFU values at surface sites
and subsurface layers from the Yungay region signify the need for numerous site surface and
subsurface sampling on Mars where similar patchiness of life over the surface of the planet and
with depth at subsurface layers may exist.
118
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123
APPENDIX A MEDIA INSTRUCTIONS
The media recipes listed are for solid and liquid given that this study uses culture media in both agar and broth forms. Broth forms are identical to solid media except for they do not contain agar, which is the solidifying agent.
For the experiment involving acidic media (pH 5), the MA and MB and 1/10 PCA and 1/10 PCB were made exactly as stated here except that HCl was added to the media before being autoclaved in order to drop the pH to 5.
Plate Count Agar (PCA)
5.0 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 2.5 grams Bacto™ Yeast Extract 1.0 grams Dextrose GR from EM® Science 15.0 grams Bacto™ Agar Fill to 1 liter with distilled H20
Plate Count Broth (PCB)
5.0 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 2.5 grams Bacto™ Yeast Extract 1.0 grams Dextrose GR from EM® Science Fill to 1 liter with distilled H20
1/10 Plate Count Agar (1/10 PCA)
0.5 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 0.25 grams Bacto™ Yeast Extract 0.1 grams Dextrose GR from EM® Science 15.0 grams Bacto™ Agar Fill to 1 liter with distilled H20
1/10 Plate Count Broth (1/10 PCB)
0.5 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 0.25 grams Bacto™ Yeast Extract 0.1 grams Dextrose GR from EM® Science Fill to 1 liter with distilled H20
1/100 Plate Count Agar (1/100 PCA)
0.05 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 0.025 grams Bacto ™Yeast Extract 0.01 grams Dextrose GR from EM® Science 15.0 grams Bacto™ Agar Fill to 1 liter with distilled H20
1/100 Plate Count Broth (1/100 PCB)
124
0.05 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 0.025 grams Bacto ™Yeast Extract 0.01 grams Dextrose GR from EM® Science Fill to 1 liter with distilled H20
Nutrient Agar (NA) 3.0 grams Bacto™ Beef Extract 5.0 grams Bacto™ Peptone (Enzymatic Digest of Protein) 15.0 grams Bacto™ Agar Fill to 1 liter with distilled H20 Nutrient Broth (NB) 3.0 grams Bacto™ Beef Extract 5.0 grams Bacto™ Peptone (Enzymatic Digest of Protein) Fill to 1 liter with distilled H20
Marine Agar (MA)
55.1 grams Difco™ Marine Agar Fill to 1 liter with distilled H20 Marine Broth (MB)
37.4 grams Difco™ Marine Broth Fill to 1 liter with distilled H20 Marine Agar + Instant Ocean® (MA+IO) 55.1 grams Difco™ Marine Agar 35.79 grams Instant Ocean® Fill to 1 liter with distilled H20 pH to 8 Marine Broth + Instant Ocean® (MB+IO) 37.4 grams Difco™ Marine Broth 35.79 grams Instant Ocean® Fill to 1 liter with distilled H20 pH to 8 1 times Instant Ocean® (1xIO) 35.79 grams Instant Ocean®
5.0 grams of Bacto™ Tryptone (Pancreatic Digest of Casein)
125
1.0 gram of Dextrose GR from EM® Science Fill to 1 liter with distilled H20
pH to 8 (for agar - 15.0 grams Bacto™ Agar was added before H20) 2 times Instant Ocean® (2xIO)
71.58 grams Instant Ocean® 5.0 grams of Bacto™ Tryptone (Pancreatic Digest of Casein) 1.0 gram of Dextrose GR from EM® Science Fill to 1 liter with distilled H20
pH to 8 (for agar - 15.0 grams Bacto™ Agar was added before H20) 3 times Instant Ocean® (3xIO)
107.37 grams Instant Ocean® 5.0 grams of Bacto™ Tryptone (Pancreatic Digest of Casein) 1.0 gram of Dextrose GR from EM® Science Fill to 1 liter with distilled H20
pH to 8 (for agar - 15.0 grams Bacto™ Agar was added before H20)
126
Surf
ace
Isol
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sp. B
C44
8 0.
97
AJ3
1657
1 A
T03-
33-1
5 -2
N
A
surf
ace
oran
ge g
loss
y ~i
rreg
ular
con
vex
~opa
que
810
Blas
toco
ccus
sp. B
C44
8 0.
97
AJ3
1657
1 A
T03-
33-1
6 -2
1/
100
PCA
sur
face
pk
glos
sy c
onve
x ~t
rans
83
1 Bl
asto
cocc
us sp
. BC
448
0.98
A
J316
571
AT0
3-33
-17
-2
1/10
0 PC
A s
urfa
ce o
rang
e (d
arke
r cen
ter)
glo
ssy
conv
ex ~
trans
82
2 Bl
asto
cocc
us sp
. BC
448
0.99
A
J316
571
AT0
3-33
-18
-2
1/10
0 PC
A s
urfa
ce !
lt pk
glo
ssy
conv
ex ~
trans
81
1 Bl
asto
cocc
us sp
. BC
448
0.98
A
J316
571
AT0
3-33
-19
-2
1/10
0 PC
A s
urfa
ce o
rang
e ~t
rans
glo
ssy
conv
ex
810
Blas
toco
ccus
sp. B
C44
8 0.
98
AJ3
1657
1 A
T03-
33-2
0 -2
1/
100
PCA
sur
face
whi
te tr
ans ~
flat g
loss
y 10
34
Blas
toco
ccus
sp. B
C44
8 0.
98
AJ3
1657
1 A
T03-
33-2
1 -2
1/
100
PCA
sur
face
pk/
red
trans
glo
ssy
conv
ex
679
Blas
toco
ccus
sp. B
C44
8 0.
98
AJ3
1657
1 A
T03-
33-2
2 -1
1/
100
PCA
sur
face
whi
te/o
ffw
hite
flat
glo
ssy
~tra
ns fr
ayed
edg
es
804
Paen
ibac
illus
sp. T
RO
4 0.
95
AJ2
5119
3 A
T03-
33-2
3 -2
M
A
surf
ace
yello
w g
loss
y op
aque
con
vex
~irr
egul
ar
817
Prom
icro
mon
ospo
ra e
nter
ophi
la
0.94
X
8380
7 A
T03-
33-2
4 -2
M
A
surf
ace
lt pk
con
vex
~dul
l ~op
aque
~bu
mpy
surf
ace
912
Blas
toco
ccus
sp. B
C44
8 0.
99
AJ3
1657
1 A
T03-
33-2
5 -2
M
A
surf
ace
offw
hite
~irr
egul
ar ra
ised
~op
aque
glo
ssy
~bum
py su
rfac
e 86
2 Pr
omic
rom
onos
pora
ent
erop
hila
0.
95
X83
807
AT0
3-33
-27
-2
MA
su
rfac
e ho
t ora
nge/
hot p
each
um
bona
te o
paqu
e ~g
loss
y 85
6 Bl
asto
cocc
us sp
. BC
448
0.99
A
J316
571
AT0
3-33
-29
-1
1/10
0 PC
A s
urfa
ce t
an tr
ans !
glos
sy c
onve
x 93
3 Bl
asto
cocc
us sp
. BC
448
0.99
A
J316
571
AT0
3-33
-30
-1
1/10
0 PC
A s
urfa
ce l
t pk
~tra
ns g
loss
y co
nvex
83
8 Bl
asto
cocc
us sp
. BC
448
0.99
A
J316
571
AT0
3-33
-33
-1
1/10
0 PC
A s
urfa
ce t
an d
ull ~
opaq
ue fl
at
809
Mod
esto
bact
er m
ultis
epta
tus
0.98
A
J871
304
AT0
3-33
-34
-1
1/10
0 PC
A s
urfa
ce !
lt pk
~tra
ns g
loss
y co
nvex
85
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
33-3
5 -2
1/
10 P
CA
su
rfac
e or
ange
~tra
ns g
loss
y co
nvex
86
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
33-3
8 -2
1/
10 P
CA
su
rfac
e or
ange
/pea
ch ~
trans
glo
ssy
conv
ex
861
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-33
-39
-2
1/10
PC
A
surf
ace
pk/ta
n tra
ns g
loss
y co
nvex
65
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
33-4
0 -2
1/
10 P
CA
su
rfac
e of
fwhi
te ~
trans
glo
ssy
conv
ex
923
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-33
-41
-2
1/10
PC
A
surf
ace
hot o
rang
e/ho
t pk
~glo
ssy
opaq
ue c
onve
x 91
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
33-4
3 -2
1/
10 P
CA
su
rfac
e ho
t pk/
hot p
each
~du
ll co
nvex
opa
q 92
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
33-4
4 -2
1/
10 P
CA
su
rfac
e lt
pk tr
ans g
loss
y co
nvex
91
0 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
33-4
7 -2
1/
10 P
CA
su
rfac
e lt
pk d
ull ~
roug
h co
nvex
~op
aque
59
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
33-4
8 -1
1/
10 P
CA
su
rfac
e da
rker
ora
nge
glos
sy c
onve
x ~o
paqu
e 88
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
33-4
9 -1
1/
10 P
CA
su
rfac
e lt
oran
ge/ta
n gl
ossy
con
vex
opaq
ue
924
Mod
esto
bact
er m
ultis
epta
tus
0.99
A
J871
304
AT0
3-33
-50
-1
1/10
PC
A
surf
ace
crea
m/ta
n (d
arke
r in
cent
er) o
paqu
e gl
ossy
con
vex
811
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-33
-53
-1
NA
su
rfac
e of
fwhi
te/y
ello
w c
ream
y bi
g co
nvex
glo
ssy
opaq
ue
911
Mic
roco
ccus
sp. R
G-6
4
0.99
A
Y56
1623
APP
EN
DIX
B
TA
BL
E O
F SU
RFA
CE
AN
D S
OIL
PIT
ISO
LA
TE
S Th
e ta
ble
belo
w sh
ows i
sola
tes r
ecov
ered
from
surf
ace
and
pit s
ampl
es, t
he d
ilutio
n, m
edia
, and
dep
th th
at th
e is
olat
e w
as re
cove
red
from
, col
ony
mor
phol
ogy,
leng
th o
f th
e se
quen
ce (b
p) im
porte
d in
to th
e B
LAST
inte
rfac
e of
Gen
Ban
k®, c
lose
st re
lativ
e as
iden
tifie
d by
BLA
ST re
sult,
nuc
leot
ide
iden
tity
sim
ilarit
y, a
nd G
enB
ank®
ac
cess
ion
num
ber.
127
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
C
olon
y M
orph
olog
y
S
eque
nce
Len
gth
B
LA
ST R
esul
t
Si
mila
rity
Acc
essi
on n
o.
AT0
3-33
-54
-1
NA
su
rfac
e ta
nger
ine
big
~fla
t rou
gh b
umpy
ring
s ~op
aque
~gl
ossy
91
5 M
odes
toba
cter
mul
tisep
tatu
s 0.
99
AJ8
7130
4 A
T03-
33-5
6 -1
N
A
surf
ace
oran
ge p
ink
~opa
que
glos
sy c
onve
x m
ed
989
Baci
llus s
p. H
SCC
165
1
0.95
A
B04
5098
A
T03-
33-5
6A
-1
NA
su
rfac
e sm
all t
ange
rine
opaq
ue g
loss
y co
nvex
81
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
33-5
7 -1
N
A
surf
ace
trans
glo
ssy
conv
ex
980
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-33
-58
-1
NA
su
rfac
e pk
bum
py ir
regu
lar ~
trans
dul
l con
cave
cen
ter
751
Blas
toco
ccus
sp. B
C41
2
0.98
A
J316
574
AT0
3-33
-59
-1
NA
su
rfac
e ta
n/m
auve
glo
ssy
irreg
ular
~tra
ns ra
ised
86
1 Bl
asto
cocc
us sp
. BC
412
0.
99
AJ3
1657
1 A
T03-
33-6
0 -1
N
A
surf
ace
mau
ve g
loss
y co
nvex
~tra
ns
864
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-33
-61
-1
NA
su
rfac
e or
ange
rais
ed g
loss
y irr
egul
ar tr
ans d
arke
r spo
t in
cent
er
845
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-33
-63
-1
NA
su
rfac
e lt
pk o
paqu
e gl
ossy
con
vex
854
Mod
esto
bact
er m
ultis
epta
tus
0.98
A
J871
304
AT0
3-34
-1
-2
PCA
su
rfac
e ta
n no
n-gl
ossy
~co
nvex
(mor
e in
cen
ter)
opa
que
text
ured
(bum
py)
787
Baci
llus f
ortis
stra
in R
-651
4
0.97
A
Y44
3038
A
T03-
34-2
-3
PC
A
surf
ace
pk g
loss
y co
nvex
~tra
ns ~
irreg
ular
79
2 M
odes
toba
cter
mul
tisep
tatu
s 0.
98
AJ8
7130
4 A
T03-
34-3
-1
PC
A
surf
ace
hot p
k/or
ange
rigi
d bu
mpy
roug
h op
aque
irre
gula
r ~du
ll 92
7 W
illia
msi
a m
ural
is is
olat
e 50
0/04
0.
99
AY
9867
34
AT0
3-34
-4
-1
PCA
su
rfac
e ho
t pk
huge
irre
gula
r ~co
nvex
opa
que
glos
sy
833
Will
iam
sia
mur
alis
isol
ate
500/
04
0.99
A
Y98
6734
A
T03-
34-6
-1
PC
A
surf
ace
yello
w (m
usta
rd) o
paqu
e gl
ossy
con
vex
92
0 N
ocar
dioi
des s
p. V
4.M
E.19
0.
97
AJ2
4465
6 A
T03-
34-7
-1
M
A
surf
ace
hot p
k ~i
rreg
ular
opa
que
~glo
ssy
rais
ed b
umpy
smoo
th
925
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-34
-8
-1
MA
su
rfac
e ho
t ora
nge/
hot p
k ~i
rreg
ular
glo
ssy
~rou
gh ra
ised
92
5 M
odes
toba
cter
mul
tisep
tatu
s 0.
99
AJ8
7130
4 A
T03-
34-9
-1
M
A
surf
ace
pk ~
glos
sy c
onve
x op
aque
96
8 Bl
asto
cocc
us sp
. BC
412
0.
98
AJ3
1657
4 A
T03-
34-1
0 -1
M
A
surf
ace
dark
er p
k ~d
ull c
onve
x op
aque
89
7 Bl
asto
cocc
us sp
. BC
412
0.
98
AJ3
1657
4 A
T03-
35-1
-1
1/
10 P
CA
su
rfac
e or
ange
/pin
k gl
ossy
con
vex
opaq
ue
960
Blas
toco
ccus
sp. B
C41
2
0.97
A
J316
574
AT0
3-35
-2
-1
1/10
PC
A
surf
ace
lt ta
n gl
ossy
~fla
t with
con
vex
cent
er ~
trans
80
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
35-6
-1
1/
100
PCA
sur
face
lt t
an g
loss
y ~f
lat w
ith c
onve
x ce
nter
~tra
ns
711
Unc
ult e
arth
wor
m c
ast b
acte
rium
clo
ne c
250
0.96
A
Y15
4604
A
T03-
35-7
-1
1/
100
PCA
sur
face
lt t
an g
loss
y ~f
lat w
ith c
onve
x ce
nter
~tra
ns
575
Bac
teriu
m E
llin6
023
0.
98
AY
2346
75
AT0
3-35
-11
-1
MA
su
rfac
e pe
ach/
lt pk
~co
nvex
glo
ssy
~bum
py su
rfac
e op
aque
51
3 Bl
asto
cocc
us sp
. BC
448
0.
96
AJ3
1657
1 A
T03-
35-1
2 -1
M
A
surf
ace
lt pk
(lik
e br
ain)
ripp
led
glos
sy ra
ised
~irr
egul
ar m
argi
ns o
paqu
e 77
3 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
35-1
3 -1
M
A
surf
ace
oran
ge/p
k op
aque
con
vex
glos
sy
745
Blas
toco
ccus
sp. B
C41
2
0.97
A
J316
574
AT0
3-35
-18
-1
PCA
su
rfac
e lt
yello
w b
ig ir
regu
lar r
ough
with
bum
ps ri
gid
opaq
ue ~
dull
90
8 M
icro
cocc
us sp
. RG
-64
1.
00
AY
5616
23
AT0
3-35
-19
-2
1/10
0 PC
A s
urfa
ce l
t pk
glos
sy c
onve
x ~t
rans
82
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
35-2
0 -1
1/
100
PCA
sur
face
lt p
k gl
ossy
con
vex
~opa
que
908
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
3-35
-21
-1
1/10
0 PC
A s
urfa
ce t
an ~
trans
glo
ssy
conv
ex
718
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
3-35
-22
-2
1/10
0 PC
A s
urfa
ce p
k gl
ossy
con
vex
~tra
ns
831
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-36
-2
-1
1/10
0 PC
A s
urfa
ce l
t pk
~tra
ns ir
regu
lar f
lat g
loss
y 79
6 B
acte
rium
Elli
n602
3
0.98
A
Y23
4675
A
T03-
36-3
-1
PC
A
surf
ace
dark
ora
nge/
pink
irre
gula
r rai
sed
roug
h bi
g gl
ossy
but
dul
l 83
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
36-4
-1
1/
100
PCA
sur
face
lt p
ink
glos
sy ir
regu
lar ~
flat ~
trans
76
3 Ex
iguo
bact
eriu
m sp
.BTA
H1
0.98
A
Y20
5564
A
T03-
36-5
-1
1/
100
PCA
sur
face
lt p
ink/
lt pe
ach
~tra
ns ~
flat g
loss
y da
rker
in c
ente
r 66
4 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T03-
36-6
-1
1/
100
PCA
sur
face
lt p
ink
~tra
ns ~
flat g
loss
y da
rker
in c
ente
r 40
9 Bl
asto
cocc
us sp
. BC
521
0.
98
AJ3
1657
3 A
T03-
36-7
-1
1/
100
PCA
sur
face
lt p
ink
non-
glos
sy d
ull f
lat ~
trans
dar
ker i
n ce
nter
81
2 Bl
asto
cocc
us sp
. BC
521
0.
97
AJ3
1657
3 A
T03-
36-1
0 -1
1/
10 P
CA
su
rfac
e lt
oran
ge c
onve
x ~g
loss
y ~t
rans
mor
e op
aque
rais
ed d
ot in
cen
ter
961
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
3-36
-11
-1
1/10
PC
A
surf
ace
pk fl
at ~
opaq
ue ro
ugh
rigid
~du
ll 86
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
36-1
2 -1
1/
10 P
CA
su
rfac
e ta
n/cr
eam
~op
aque
glo
ssy
conv
ex
941
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-36
-13
-1
1/10
PC
A
surf
ace
pk c
onve
x op
aque
~du
ll 73
7 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
36-1
4 -1
1/
10 P
CA
su
rfac
e pk
con
vex
opaq
ue ~
dull
830
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-36
-15
-1
1/10
PC
A
surf
ace
hot o
rang
e gl
ossy
con
vex
~tra
ns
924
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-36
-16
-1
NA
su
rfac
e ta
n ~t
rans
glo
ssy
conv
ex
949
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-36
-17
-1
NA
su
rfac
e or
ange
/pk
~tra
ns g
loss
y co
nvex
92
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
36-1
8 -1
M
A
surf
ace
pk sm
all o
paqu
e ~d
ull c
onve
x 98
1 Bl
asto
cocc
us sp
. BC
412
0.
98
AJ3
1657
4 A
T03-
36-1
9 -1
M
A
surf
ace
lt pk
opa
que
~dul
l con
vex
965
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-36
-20
-1
1/10
0 PC
A s
urfa
ce p
k tra
ns g
loss
y co
nvex
85
0 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1
128
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
C
olon
y M
orph
olog
y
S
eque
nce
Len
gth
B
LA
ST R
esul
t
Si
mila
rity
Acc
essi
on n
o.
AT0
3-37
-1
-1
1/10
0 PC
A s
urfa
ce l
t pk/
lt pe
ach
~tra
ns g
loss
y co
nvex
66
2 B
acte
rium
Elli
n502
4
0.95
A
Y23
4441
A
T03-
37-2
-1
1/
100
PCA
sur
face
lt p
k ~t
rans
glo
ssy
conv
ex
839
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-37
-4
-1
1/10
0 PC
A s
urfa
ce l
t pk/
lt pe
ach
~tra
ns g
loss
y co
nvex
~irr
egul
ar
664
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-37
-6
-1
MA
su
rfac
e pk
~bu
mpy
surf
ace
rais
ed o
paqu
e gl
ossy
86
6 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T03-
37-7
-1
M
A
surf
ace
pk ~
bum
py su
rfac
e ra
ised
opa
que
glos
sy
700
Blas
toco
ccus
agg
rega
tus
0.96
A
J430
193
AT0
3-37
-8
-1
MA
su
rfac
e da
rker
pk
~opa
que
~glo
ssy
rais
ed e
dges
~irr
egul
ar su
rfac
e 82
8 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T03-
37-9
-2
M
A
surf
ace
~lt p
k op
aque
~irr
egul
ar &
~bu
mpy
surf
ace
glos
sy ra
ised
! 63
0 Bl
asto
cocc
us a
ggre
gatu
s 0.
96
AJ4
3019
3 A
T03-
37-1
0 -1
N
A
surf
ace
pk ~
opaq
ue d
ull c
onve
x 10
03
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
3-38
-1
-3
PCA
su
rfac
e ta
nger
ine
oran
ge v
ery
conv
ex g
loss
y op
aque
82
2 B
acte
rium
Elli
n500
4
0.94
A
Y23
4421
A
T03-
38-2
-2
1/
10 P
CA
su
rfac
e w
hite
smal
l fla
t irr
egul
ar o
paqu
e 81
7 Ro
thia
sp. C
CU
G 2
5688
0.
99
AJ1
3112
2 A
T03-
38-4
-2
1/
10 P
CA
su
rfac
e sm
all w
hite
glo
ssy
conv
ex ~
trans
82
7 M
ycob
acte
rium
sp. E
SD
0.98
A
F284
430
AT0
3-38
-5
-2
1/10
PC
A
surf
ace
trans
whi
te ir
regu
lar m
ore
opaq
ue a
roun
d ed
ge w
hite
spec
s tra
ns in
cen
ter
835
Stap
hylo
cocc
us a
ureu
s 0.
99
BX
5718
57
AT0
3-38
-8
-2
1/10
0 PC
A s
urfa
ce p
k !~
trans
glo
ssy
conv
ex w
ith b
row
n sp
ec in
cen
ter
577
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-39
-1
-2
1/10
PC
A
surf
ace
smal
l whi
te ~
trans
glo
ssy
conv
ex
808
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-39
-2
-2
1/10
PC
A
surf
ace
whi
te fu
ngi l
ooki
ng b
ut n
ot a
hai
ry su
rfac
e fla
t glo
ssy
grow
th o
n su
rfac
e 64
7 Ba
cillu
s fir
mus
0.
99
D16
268
AT0
3-39
-3
-1
1/10
PC
A
surf
ace
smal
l lt p
urpl
e gl
ossy
con
vex
light
er a
roun
d ed
ge ~
trans
76
7 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
39-4
-1
1/
10 P
CA
su
rfac
e sm
all l
t pur
ple
glos
sy c
onve
x lig
hter
aro
und
edge
~tra
ns
784
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-39
-5
-1
1/10
PC
A
surf
ace
lt pi
nk sm
all g
loss
y co
nvex
~tra
ns
838
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-39
-6
-1
1/10
0 PC
A s
urfa
ce l
t pin
k sm
all g
loss
y co
nvex
~tra
ns
704
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-39
-7
-1
1/10
0 PC
A s
urfa
ce w
hite
/lt p
ink
smal
l glo
ssy
conv
ex
770
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-39
-11
-1
1/10
PC
A
surf
ace
smal
l lt p
urpl
e gl
ossy
con
vex
light
er a
roun
d ed
ge ~
trans
80
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
39-1
2 -1
1/
10 P
CA
su
rfac
e lt
pink
smal
l glo
ssy
conv
ex ~
trans
84
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
39-1
3 -1
1/
10 P
CA
su
rfac
e lt
purp
le sm
all g
loss
y co
nvex
ligh
ter a
roun
d ed
ge ~
trans
70
7 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
39-1
4 -1
1/
10 P
CA
su
rfac
e lt
pink
~tra
ns sm
all g
loss
y co
nvex
83
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
39-1
5 -2
1/
100
PCA
sur
face
lt p
ink
trans
glo
ssy
~fla
t sm
all
800
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-39
-16
-1
NA
su
rfac
e ta
nger
ine/
peac
h ~t
rans
glo
ssy
conv
ex
842
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-40
-1
-3
1/10
PC
A
surf
ace
lt pe
ach/
pink
mor
e da
rk in
cen
ter g
loss
y co
nvex
77
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-2
-3
1/
10 P
CA
su
rfac
e lt
peac
h fla
t glo
ssy
781
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-3
-3
1/10
PC
A
surf
ace
lt pe
ach/
pink
irre
gula
r glo
ssy
flat
830
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-4
-3
1/10
0 PC
A s
urfa
ce !
lt pi
nk f
lat i
rreg
ular
glo
ssy
smal
l col
ony
arou
nd b
lob
741
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-5
-2
1/10
0 PC
A s
urfa
ce !
lt pi
nk (a
lmos
t whi
te) ~
trans
flat
glo
ssy
smal
l 87
0 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
40-6
-2
1/
100
PCA
sur
face
!lt
pink
(alm
ost w
hite
) ~tra
ns fl
at g
loss
y sm
all
932
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-7
-1
1/10
PC
A
surf
ace
smal
l irr
egul
ar lt
pin
k/pe
ach
conv
ex ~
trans
83
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
40-8
-1
1/
10 P
CA
su
rfac
e lt
pink
smal
l con
vex
~tra
ns g
loss
y 78
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-1
1 -1
PC
A
surf
ace
dark
er o
rang
e co
nvex
big
glo
ssy
~tra
ns
458
Blas
toco
ccus
sp. B
C52
1
0.99
A
J316
573
AT0
3-40
-12
-1
PCA
su
rfac
e lt
pink
/lt o
rang
e co
nvex
~gl
ossy
tran
s bum
p at
cen
ter t
op
737
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-13
-1
PCA
su
rfac
e hu
ge d
arke
r. pi
nk ri
gid
~tra
ns n
on-g
loss
y irr
egul
ar
747
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-40
-14
-1
PCA
su
rfac
e lt
pink
con
vex
non-
glos
sy ~
opaq
ue
785
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-15
-1
PCA
su
rfac
e bi
g lt
pink
/lt ta
n gl
ossy
con
vex
~tra
ns
805
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-17
-1
PCA
su
rfac
e lt
pink
tran
s irr
egul
ar n
on-g
loss
y ra
ised
roug
h 77
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-1
9 -2
1/
100
PCA
sur
face
tra
nslu
cent
~fla
t glo
ssy
rais
ed sp
ot in
cen
ter
950
Orn
ithin
icoc
cus h
orte
nsis
0.
94
Y17
869
AT0
3-40
-20
-2
1/10
0 PC
A s
urfa
ce v
ery
lt pi
nk (a
lmos
t whi
te) g
loss
y co
nvex
~tra
ns
947
Orn
ithin
icoc
cus h
orte
nsis
0.
94
Y17
869
AT0
3-40
-21
-2
1/10
0 PC
A s
urfa
ce l
t pin
k gl
ossy
smal
l con
vex
~tra
ns li
ghte
r aro
und
edge
88
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
40-2
2 -1
1/
100
PCA
sur
face
tra
nslu
cent
~fla
t glo
ssy
rais
ed sp
ot in
cen
ter
822
Orn
ithin
icoc
cus h
orte
nsis
0.
94
Y17
869
AT0
3-40
-23
-1
1/10
0 PC
A s
urfa
ce v
ery
lt pi
nk (a
lmos
t tra
ns) c
onve
x gl
ossy
74
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-2
4 -1
1/
100
PCA
sur
face
lt p
ink
~tra
ns g
loss
y co
nvex
ligh
ter a
roun
d ed
ge
825
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-40
-25
-1
1/10
0 PC
A s
urfa
ce t
rans
luce
nt ~
flat g
loss
y ra
ised
spot
in c
ente
r 94
0 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1
129
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
C
olon
y M
orph
olog
y
Se
quen
ce L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T03-
40-2
6 -1
1/
100
PCA
sur
face
tin
y lt
pk g
loss
y co
nvex
~op
aque
81
2 Bl
asto
cocc
us sp
. BC
448
0.
96
AJ3
1657
1 A
T03-
40-2
8 -1
1/
100
PCA
sur
face
tra
nslu
cent
ver
y lt
pink
con
vex
glos
sy
851
Orn
ithin
icoc
cus h
orte
nsis
0.
94
Y17
869
AT0
3-40
-29
-1
1/10
PC
A
surf
ace
crea
my
lt pi
nk ~
trans
con
vex
glos
sy
811
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-30
-1
1/10
PC
A
surf
ace
lt pi
nk/lt
pea
ch ir
regu
lar r
ough
~tra
ns n
on-g
loss
y 83
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-3
1 -1
1/
10 P
CA
su
rfac
e cr
eam
y lt
pink
~op
aque
con
vex
glos
sy
780
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-32
-1
1/10
PC
A
surf
ace
dark
pin
k gl
ossy
~op
aque
con
vex
777
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-33
-1
1/10
PC
A
surf
ace
dark
pin
k gl
ossy
~op
aque
con
vex
833
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-34
-1
1/10
PC
A
surf
ace
crea
my
lt ta
n/lt
peac
h co
nvex
glo
ssy
~tra
ns li
ghte
r aro
und
edge
83
9 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
40-3
5 -1
1/
10 P
CA
su
rfac
e da
rk p
ink
glos
sy ~
opaq
ue c
onve
x 77
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-3
6 -1
1/
10 P
CA
su
rfac
e lt
peac
h gl
ossy
con
vex
~tra
ns
851
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-39
-2
1/10
0 PC
A s
urfa
ce p
inki
sh ta
n ~t
rans
~fla
t irre
gula
r glo
ssy
rais
ed sp
ot in
cen
ter
842
Orn
ithin
icoc
cus h
orte
nsis
0.
94
Y17
869
AT0
3-40
-40
-2
1/10
0 PC
A s
urfa
ce l
t pin
k tra
ns g
loss
y co
nvex
83
2 Bl
asto
cocc
us sp
. BC
521
0.
97
AJ3
1657
3 A
T03-
40-4
2 -2
1/
100
PCA
sur
face
cre
amy
very
lt p
ink
(alm
ost w
hite
) ~op
aque
glo
ssy
conv
ex
845
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-43
-2
1/10
PC
A
surf
ace
lt pe
ach
trans
glo
ssy
conv
ex
817
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-44
-2
1/10
PC
A
surf
ace
lt pe
ach
trans
glo
ssy
conv
ex
464
Blas
toco
ccus
sp. B
C52
1
0.97
A
J316
573
AT0
3-40
-45
-3
1/10
0 PC
A s
urfa
ce l
t tan
/lt p
ink
glos
sy ~
conv
ex ~
trans
84
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-4
6 -3
1/
10 P
CA
su
rfac
e ta
n lt
pink
~co
nvex
glo
ssy
~tra
ns
854
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-47
-3
1/10
PC
A
surf
ace
tan
lt pi
nk ~
conv
ex g
loss
y ~t
rans
irre
gula
r clu
ster
ed
790
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-48
-2
1/10
PC
A
surf
ace
lt ta
n da
rker
in c
ente
r lig
ht e
dges
~gl
ossy
~tra
ns c
onve
x 81
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-4
9 -2
1/
10 P
CA
su
rfac
e lt
tan
dark
er c
ente
r mor
e tra
ns e
dge
very
glo
ssy
~tra
ns c
onve
x ce
nter
82
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-5
0 -3
1/
10 P
CA
su
rfac
e ta
n/or
ange
dar
ker i
n ce
nter
ver
y lig
ht a
roun
d ed
ge v
ery
glos
sy ~
conv
ex
818
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-51
-2
1/10
PC
A
surf
ace
tan
lt pi
nk ~
conv
ex ~
glos
sy ~
trans
79
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-5
2 -1
PC
A
surf
ace
dark
er o
rang
e ve
ry c
onve
x irr
egul
ar o
paqu
e du
ll no
n-gl
ossy
75
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-5
3 -1
PC
A
surf
ace
lt or
ange
ver
y co
nvex
~tra
ns g
loss
y 82
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
40-5
4 -1
PC
A
surf
ace
oran
ge ~
glos
sy c
onve
x m
ore
conv
ex d
ome
in c
ente
r opa
que
711
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-55
-1
PCA
su
rfac
e or
ange
~gl
ossy
!con
vex
mor
e co
nvex
smal
l dom
e of
fsid
e of
cen
ter ~
trans
76
7 St
aphy
loco
ccus
sp. A
I-22
0.
98
AY
4376
31
AT0
3-40
-56
-1
PCA
su
rfac
e da
rker
ora
nge
conv
ex w
ith h
ole
in c
ente
r opa
que
dull
non-
glos
sy
819
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-57
-1
PCA
su
rfac
e lt
oran
ge c
onve
x gl
ossy
~op
aque
97
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-5
8 -1
N
A
surf
ace
lt pk
con
cave
cen
ter ~
dull
~opa
que
roun
d ra
ised
edg
es
729
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-59
-1
NA
su
rfac
e ta
n tra
ns g
loss
y co
nvex
79
7 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
40-6
0 -1
N
A
surf
ace
tan
trans
glo
ssy
conv
ex
927
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-40
-61
-1
MA
su
rfac
e lt
pk o
paqu
e gl
ossy
con
vex
768
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-62
-1
MA
su
rfac
e lt
pk o
paqu
e gl
ossy
con
vex
705
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-63
-2
MA
su
rfac
e lt
pk c
onve
x op
aque
~gl
ossy
87
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-6
4 -2
M
A
surf
ace
!lt p
k (a
lmos
t whi
te) i
rreg
ular
roug
h rig
id d
ull r
aise
d op
aque
81
2 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
40-6
6 -2
N
A
surf
ace
lt pk
tran
s glo
ssy
conv
ex
902
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-40
-67
-1
NA
su
rfac
e ta
n tra
ns g
loss
y co
nvex
67
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
40-6
8 -1
N
A
surf
ace
lt pk
irre
gula
r con
cave
cen
ter ~
glos
sy ra
ised
bum
py
902
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-40
-69
-1
NA
su
rfac
e lt
pk g
loss
y ra
ised
with
~co
ncav
e ce
nter
opa
que
622
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-1
-2
PCA
su
rfac
e or
ange
ver
y co
nvex
glo
ssy
spec
s in
colo
ny ~
trans
74
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-2
-2
PC
A
surf
ace
dark
ora
nge/
pink
irre
gula
r rou
gh ra
ised
non
-glo
ssy
614
Fran
kia
sp. (
stra
in A
VN
17s)
0.
93
L406
13
AT0
3-41
-3
-2
1/10
PC
A
surf
ace
pink
/ora
nge
big
~glo
ssy
opaq
ue ~
flat
781
Blas
toco
ccus
sp. B
C41
2
0.97
A
J316
574
AT0
3-41
-4
-3
1/10
0 PC
A s
urfa
ce l
t pin
k gl
ossy
flat
irre
gula
r dar
ker i
n ce
nter
tran
s aro
und
edge
s 85
0 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T03-
41-5
-1
PC
A
surf
ace
brow
n no
n-gl
ossy
irre
gula
r bum
py o
paqu
e 81
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-6
-1
PC
A
surf
ace
whi
te n
on-g
loss
y op
aque
con
vex
with
who
le in
cen
ter p
aste
y 82
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-7
-1
PC
A
surf
ace
big
purp
le/p
ink
conv
ex n
on-g
loss
y op
aque
ring
s in
colo
ny
841
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-8
-1
PCA
su
rfac
e irr
egul
ar b
umpy
ora
nge
~tra
ns g
loss
y ed
ges &
dar
ker ~
glos
sy o
paqu
e ce
nter
76
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1
130
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
C
olon
y M
orph
olog
y
S
eque
nce
Len
gth
B
LA
ST R
esul
t
Si
mila
rity
Acc
essi
on n
o.
AT0
3-41
-9
-1
PCA
su
rfac
e sm
all p
ink/
oran
ge g
loss
y tra
ns c
onve
x 86
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-1
0 -1
PC
A
surf
ace
dark
er p
ink
glos
sy o
paqu
e co
nvex
but
con
cave
in c
ente
r 82
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-1
1 -1
PC
A
surf
ace
smal
l lt p
ink/
oran
ge ~
trans
con
vex
~glo
ssy
roug
h 81
6 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
41-1
2 -1
PC
A
surf
ace
smal
l bro
wn
conv
ex n
on-g
loss
y ha
rd d
arke
r in
cent
er o
paqu
e 70
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-1
3 -1
PC
A
surf
ace
dark
er. p
ink
bum
py ro
ugh
opaq
ue c
onve
x no
n-gl
ossy
84
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-1
5 -2
1/
10 P
CA
su
rfac
e bi
g lt
pink
flat
non
-glo
ssy
opaq
ue c
onve
x do
t in
cent
er
827
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-41
-17
-1
1/10
PC
A
surf
ace
opaq
ue lt
pur
ple/
pink
~co
nvex
glo
ssy
arou
nd e
dge
dull
in c
ente
r 85
8 B
acte
rium
Elli
n500
4
0.98
A
Y23
4421
A
T03-
41-1
8 -1
1/
10 P
CA
su
rfac
e lt
tan
glos
sy ~
trans
edg
es &
dar
ker o
rang
e ~c
onve
x gl
ossy
opa
que
cent
er
810
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-41
-20
-1
1/10
PC
A
surf
ace
whi
te/ta
n ~t
rans
glo
ssy
arou
nd e
dges
& ta
n gl
ossy
~tra
ns c
onve
x ce
nter
71
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-2
1 -1
1/
10 P
CA
su
rfac
e tra
nslu
cent
flat
non
-glo
ssy
850
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-22
-1
1/10
PC
A
surf
ace
trans
~co
nvex
glo
ssy
ring
in c
ente
r 83
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-2
3 -1
1/
10 P
CA
su
rfac
e lt
pink
irre
gula
r con
vex
dull
non-
glos
sy ro
ugh
~tra
ns
800
Geo
rgen
ia sp
. 221
6.35
.28
0.
93
AB
0944
66
AT0
3-41
-24
-2
1/10
0 PC
A s
urfa
ce l
t tan
glo
ssy
~con
vex
~tra
ns
830
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-41
-26
-1
1/10
0 PC
A s
urfa
ce w
hite
tran
s glo
ssy
flat w
ith ra
ised
cen
ter s
mal
l 80
6 Bl
asto
cocc
us sp
. BC
521
0.
98
AJ3
1657
3 A
T03-
41-2
7 -1
1/
100
PCA
sur
face
lt p
ink
irreg
ular
glo
ssy
~con
vex
~tra
ns
837
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-28
-1
1/10
0 PC
A s
urfa
ce l
t pin
k irr
egul
ar n
on-g
loss
y ~c
onve
x ~t
rans
81
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-2
9 -1
1/
100
PCA
sur
face
lt p
k tra
ns e
dge
glos
sy ra
ised
cen
ter d
arke
r pin
k &
mor
e op
aque
cen
ter
622
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-30
-1
PCA
su
rfac
e lt
pink
/lt b
row
n gl
ossy
smal
l con
vex
~tra
ns
894
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-31
-2
1/10
PC
A
surf
ace
~lt p
ink
~fla
t dul
l non
-glo
ssy
big
past
ey o
paqu
e 84
2 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
41-3
2 -2
1/
10 P
CA
su
rfac
e da
rker
pin
k ~f
lat g
loss
y ~d
ull b
ig p
aste
y op
aque
82
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-3
3 -2
1/
100
PCA
sur
face
whi
te ~
opaq
ue g
loss
y co
nvex
70
1 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T03-
41-3
5 -2
PC
A
surf
ace
crea
my
brig
ht p
k op
aque
glo
ssy
conv
ex
827
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-36
-1
1/10
PC
A
surf
ace
dark
er o
rang
e gl
ossy
~tra
ns c
onve
x 60
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-3
7 -1
1/
10 P
CA
su
rfac
e cr
eam
y lt
pk/ta
n ~o
paqu
e gl
ossy
con
vex
825
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-38
-1
1/10
PC
A
surf
ace
purp
le ~
trans
glo
ssy
conv
ex
825
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-41
-39
-1
1/10
PC
A
surf
ace
crea
my
dark
er p
k/or
ange
~op
aque
con
vex
glos
sy
834
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-40
-1
1/10
PC
A
surf
ace
crea
my
dark
er o
rang
e/da
rker
pk
opaq
ue c
onve
x gl
ossy
77
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-4
1 -1
1/
10 P
CA
su
rfac
e cr
eam
y ta
n ~o
paqu
e co
nvex
glo
ssy
729
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-41
-42
-1
1/10
PC
A
surf
ace
lt pk
non
-glo
ssy
dull
conv
ex o
paqu
e te
xtur
ed
847
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-43
-1
1/10
PC
A
surf
ace
brig
ht p
ink
tiny
opaq
ue c
onve
x ~g
loss
y 86
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-4
4 -1
1/
10 P
CA
su
rfac
e da
rker
red
tiny
trans
glo
ssy
conv
ex
783
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-45
-1
1/10
PC
A
surf
ace
yello
w c
ream
tran
s glo
ssy
~con
vex
788
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-41
-46
-1
1/10
PC
A
surf
ace
dark
er tr
ans g
loss
y co
nvex
77
7 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T03-
41-4
7 -1
1/
10 P
CA
su
rfac
e br
ight
pin
k op
aque
glo
ssy
conv
ex
840
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-48
-1
1/10
PC
A
surf
ace
crea
my
tan
~opa
que
conv
ex g
loss
y 81
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-4
9 -1
1/
10 P
CA
su
rfac
e lt
pk n
on-g
loss
y du
ll co
nvex
opa
que
text
ured
72
2 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T03-
41-5
0 -1
1/
10 P
CA
su
rfac
e lt
pk/ta
n gl
ossy
con
vex
~tra
ns
759
Blas
toco
ccus
sp. B
C44
8
0.96
A
J316
571
AT0
3-41
-51
-1
1/10
PC
A
surf
ace
dark
er p
k/lt
oran
ge g
loss
y co
nvex
~op
aque
80
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-5
2 -1
1/
10 P
CA
su
rfac
e ye
llow
cre
am tr
ans g
loss
y ~c
onve
x 73
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-5
3 -2
1/
10 P
CA
su
rfac
e w
hite
tran
s glo
ssy
conv
ex d
ark
spot
in c
ente
r 79
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-5
4 -2
1/
10 P
CA
su
rfac
e lt
pk n
on-g
loss
y du
ll co
nvex
opa
que
text
ured
77
7 Bl
asto
cocc
us sp
. BC
448
0.
95
AJ3
1657
1 A
T03-
41-5
5 -2
1/
10 P
CA
su
rfac
e cr
eam
y lt
pk ~
trans
con
vex
glos
sy
805
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-41
-56
-1
PCA
su
rfac
e br
ight
pin
k op
aque
irre
gula
r rai
sed
smoo
th (l
ooks
like
a n
erd)
81
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-5
7 -1
PC
A
surf
ace
lt pk
/tan
~tra
ns c
onve
x gl
ossy
83
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-5
8 -1
PC
A
surf
ace
brig
ht p
k op
aque
smal
l con
vex
~glo
ssy
767
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-41
-59
-1
1/10
0 PC
A s
urfa
ce w
hite
tran
s glo
ssy
conv
ex
816
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-60
-1
1/10
0 PC
A s
urfa
ce t
rans
glo
ssy
~con
vex
746
Blas
toco
ccus
sp. B
C52
1
0.97
A
J316
573
131
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
C
olon
y M
orph
olog
y
Se
quen
ce L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T03-
41-6
1 -1
1/
100
PCA
sur
face
cre
amy
lt pk
glo
ssy
conv
ex ~
trans
73
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-6
2 -1
1/
100
PCA
sur
face
tra
ns g
loss
y ~c
onve
x 81
0 Pr
omic
rom
onos
pora
ent
erop
hila
0.
95
X83
807
AT0
3-41
-63
-1
1/10
0 PC
A s
urfa
ce y
ello
w fl
at g
loss
y tra
ns ir
regu
lar
731
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-64
-1
1/10
0 PC
A s
urfa
ce l
t pk
glos
sy c
onve
x ~t
rans
79
1 C
ellu
lom
onas
car
tae
MSD
201
06
0.95
X
7945
6 A
T03-
41-6
5 -1
1/
100
PCA
sur
face
cre
amy
lt pk
glo
ssy
conv
ex ~
trans
82
6 O
xalo
bact
er sp
. Es2
-1
0.98
A
Y36
7029
A
T03-
41-6
7 -2
1/
100
PCA
sur
face
cre
amy
very
lt p
k gl
ossy
con
vex
~opa
que
810
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-68
-1
1/10
0 PC
A s
urfa
ce c
ream
y ve
ry lt
pk
glos
sy c
onve
x ~o
paqu
e 61
2 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
41-6
9 -1
1/
100
PCA
sur
face
tra
ns g
loss
y co
nvex
tiny
46
3 Bl
asto
cocc
us sp
. BC
521
0.
98
AJ3
1657
3 A
T03-
41-7
0 -1
1/
100
PCA
sur
face
tra
ns lt
pk
glos
sy c
onve
x 60
1 Bl
asto
cocc
us sp
. BC
521
0.
98
AJ3
1657
3 A
T03-
41-7
1 -1
1/
100
PCA
sur
face
lt p
k gl
ossy
con
vex
~tra
ns
807
Cel
lulo
mon
as c
arta
e M
SD 2
0106
0.
95
X79
456
AT0
3-41
-73
-1
1/10
0 PC
A s
urfa
ce c
ream
y tra
ns g
loss
y co
nvex
82
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-7
4 -2
1/
100
PCA
sur
face
lt p
k gl
ossy
con
vex
~opa
que
659
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-41
-76
-1
MA
su
rfac
e lt
pk g
loss
y co
nvex
opa
que
826
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-41
-77
-1
MA
su
rfac
e of
fwhi
te g
loss
y co
nvex
opa
que
762
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-78
-1
MA
su
rfac
e of
fwhi
te ~
trans
glo
ssy
conv
ex
765
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-79
-1
MA
su
rfac
e da
rker
pea
ch/o
rang
e gl
ossy
rais
ed o
paqu
e bu
mpy
surf
ace
660
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-41
-80
-1
MA
su
rfac
e of
fwhi
te ir
regu
lar r
igid
roug
h su
rfac
e du
ll ra
ised
opa
que
481
Cel
lulo
mon
as c
arta
e M
SD 2
0106
0.
94
X79
456
AT0
3-41
-81
-1
MA
su
rfac
e da
rker
pea
ch/o
rang
e irr
egul
ar ri
gid
roug
h su
rfac
e du
ll ra
ised
opa
que
705
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-82
-1
MA
su
rfac
e lt
pk ~
conv
ex g
loss
y ~t
rans
75
0 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
41-8
3 -1
M
A
surf
ace
trans
~of
fwhi
te g
loss
y co
nvex
75
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-8
4 -2
M
A
surf
ace
~yel
low
tran
s glo
ssy
conv
ex
706
Mod
esto
bact
er m
ultis
epta
tus
0.98
A
J871
304
AT0
3-41
-85
-2
MA
su
rfac
e pk
~irr
egul
ar ~
dull
rais
ed b
umpy
rigi
d su
rfac
e 75
1 Pr
omic
rom
onos
pora
ent
erop
hila
0.
95
X83
807
AT0
3-41
-86
-2
MA
su
rfac
e ye
llow
~tra
ns g
loss
y co
nvex
74
2 Pr
omic
rom
onos
pora
ent
erop
hila
0.
95
X83
807
AT0
3-41
-87
-2
MA
su
rfac
e m
ed lt
pk
with
dar
ker c
ente
r con
vex
(con
vex
bum
p in
cen
ter)
glo
ssy
opaq
ue
779
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-88
-2
NA
su
rfac
e pe
ach/
pk c
onve
x op
aque
glo
ssy
764
Cel
lulo
mon
as c
ella
sea
0.
98
X83
804
AT0
3-41
-89
-1
NA
su
rfac
e or
ange
~op
aque
con
vex
glos
sy
606
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-90
-1
NA
su
rfac
e of
fwhi
te o
paqu
e ~f
lat g
loss
y 80
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-9
1 -1
N
A
surf
ace
trans
~pu
rple
/pk
glos
sy c
onve
x 79
1 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T03-
41-9
2 -1
N
A
surf
ace
offw
hite
/tan
dark
er c
ente
r glo
ssy
conv
ex ~
trans
77
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-9
3 -1
N
A
surf
ace
brig
ht o
rang
e irr
egul
ar ra
ised
opa
que
dull
rigid
roug
h 77
0 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T03-
41-9
4 -1
N
A
surf
ace
lt pk
opa
que
~dul
l ~irr
egul
ar ra
ised
78
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-9
5 -1
N
A
surf
ace
lt pk
opa
que
~glo
ssy
conv
ex
775
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
3-41
-96
-1
NA
su
rfac
e pe
ach
crea
msa
ver g
loss
y ra
ised
~op
aque
77
6 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
41-9
7 -1
N
A
surf
ace
pk g
loss
y co
nvex
opa
que
702
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
3-41
-98
-2
NA
su
rfac
e bi
g/m
ed lt
pk/
peac
h ~c
onve
x gl
ossy
opa
que
781
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-99
-1
NA
su
rfac
e tra
ns g
loss
y co
nvex
~ta
n/pu
rple
78
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-1
00
-1
NA
su
rfac
e ra
ised
ora
nge/
pk ir
regu
lar o
paqu
e ~g
loss
y 76
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-1
01
-1
NA
su
rfac
e pe
ach/
pk g
loss
y ~o
paqu
e co
nvex
80
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
41-1
03
-2
NA
su
rfac
e ho
t pk
conv
ex o
paqu
e ~d
ull
630
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-41
-104
-2
N
A
surf
ace
trans
tan
glos
sy c
onve
x 96
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-1
05
-2
NA
su
rfac
e ho
t pk
conv
ex c
ente
r opa
que
~dul
l rai
sed
edge
s 10
27
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
3-41
-106
-2
N
A
surf
ace
trans
tan
glos
sy c
onve
x 92
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
41-1
07
-2
NA
su
rfac
e ho
t pk
conc
ave
cent
er o
paqu
e ~d
ull r
aise
d ed
ges
821
Blas
toco
ccus
sp. B
C41
2
0.98
A
J316
574
AT0
3-41
-108
-2
N
A
surf
ace
~lt p
k gl
ossy
con
vex
~opa
que
854
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-42
-1
-2
PCA
su
rfac
e bi
g w
hite
irre
gula
r ~co
nvex
opa
que
~glo
ssy
849
Baci
llus c
ereu
s 0.
99
AJ5
7728
1 A
T03-
42-2
-2
M
A
surf
ace
roun
d ye
llow
glo
ssy
flat o
paqu
e 80
4 N
ocar
dioi
des s
p. M
WH
-CaK
6
0.99
A
J565
419
AT0
3-43
-1
-1
1/10
0 PC
A s
urfa
ce p
each
/pin
k irr
egul
ar n
on-g
loss
y ~c
onve
x ro
ugh
827
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
132
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
C
olon
y M
orph
olog
y
S
eque
nce
Len
gth
B
LA
ST R
esul
t
Si
mila
rity
Acc
essi
on n
o.
AT0
3-43
-3
-1
1/10
PC
A
surf
ace
oran
ge/p
ink
conv
ex g
loss
y op
aque
75
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
43-4
-2
PC
A
surf
ace
dark
er o
rang
e ve
ry c
onve
x ~d
ull ~
glos
sy o
paqu
e 82
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
43-5
-2
1/
10 P
CA
su
rfac
e lt
pink
~fla
t ~tra
ns ~
glos
sy
783
Blas
toco
ccus
sp. B
C52
1
0.97
A
J316
573
AT0
3-43
-6
-1
PCA
su
rfac
e da
rker
ora
nge
very
con
vex
very
glo
ssy
opaq
ue
543
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-43
-7
-1
MA
su
rfac
e lt
oran
ge ~
irreg
ular
~fla
t opa
que
glos
sy ~
rigid
/roug
h 10
36
Kin
eoco
ccus
-like
bac
teriu
m A
S313
8
0.99
A
F060
686
AT0
3-43
-8
-1
MA
su
rfac
e ho
t pk/
oran
ge o
paqu
e ra
ised
roug
h/rig
id b
umpy
irre
gula
r 10
52
Cel
lulo
mon
as c
ella
sea
0.
98
X83
804
AT0
3-43
-9
-1
MA
su
rfac
e lt
oran
ge ~
irreg
ular
rais
ed g
loss
y ~u
neve
n su
rface
~op
aque
10
03
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-43
-10
-1
MA
su
rfac
e ho
t ora
nge
big
opaq
ue g
loss
y co
ncav
e irr
egul
ar u
neve
n ce
nter
edg
es ra
ised
10
17
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-43
-11
-1
MA
su
rfac
e irr
egul
ar m
ed lt
ora
nge/
hot y
ello
w ra
ised
bum
py ri
gid
roug
h ~t
rans
10
42
Kin
eoco
ccus
-like
bac
teriu
m A
S313
8
0.98
A
F060
686
AT0
3-43
-12
-1
MA
su
rfac
e pk
irre
gula
r rai
sed
opaq
ue b
umpy
rigi
d ro
ugh
1043
C
ellu
lom
onas
cel
lase
a
0.98
X
8380
4 A
T03-
43-1
3 -2
N
A
surf
ace
med
ora
nge
opaq
ue c
ente
r glo
ssy
conv
ex ~
trans
off
whi
te e
dges
10
44
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-43
-14
-1
NA
su
rfac
e pk
/ora
nge
opaq
ue ~
umbo
nate
glo
ssy
1034
Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
43-1
5 -1
N
A
surf
ace
pk/o
rang
e gl
ossy
~op
aque
con
vex
1044
Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
43-1
6 -1
N
A
surf
ace
pk/o
rang
e gl
ossy
um
bona
te o
paqu
e 90
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
43-1
7 -2
1/
100
PCA
sur
face
pk
glos
sy ~
conv
ex ~
opaq
ue
1000
K
ineo
cocc
us-li
ke b
acte
rium
AS3
138
0.
98
AF0
6068
6 A
T03-
43-1
8 -1
1/
100
PCA
sur
face
pk/
purp
le g
loss
y ~o
paqu
e ~c
onve
x ~i
rreg
ular
10
45
Blas
toco
ccus
sp. B
C41
2
0.98
A
J316
574
AT0
3-43
-19
-1
1/10
0 PC
A s
urfa
ce l
t pk
~tra
ns g
loss
y ~c
onve
x ~d
arke
r in
cent
er
966
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-43
-22
-1
1/10
0 PC
A s
urfa
ce h
ot o
rang
e/ho
t pin
k ~i
rreg
ular
glo
ssy
~tra
ns ~
conv
ex
1023
Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
43-2
3 -1
1/
100
PCA
sur
face
pk
trans
glo
ssy
irreg
ular
~ra
ised
85
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
43-2
5 -1
1/
100
PCA
sur
face
lt p
k ~i
rreg
ular
~ra
ised
glo
ssy
~tra
ns
932
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-43
-26
-1
1/10
PC
A
surf
ace
pk/o
rang
e ~c
onve
x op
aque
glo
ssy
roun
d 92
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
43-2
7 -1
1/
10 P
CA
su
rfac
e pk
/ora
nge
conv
ex g
loss
y ro
und
~opa
que
980
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-43
-28
-1
1/10
PC
A
surf
ace
brow
nish
ora
nge
~tra
ns g
loss
y co
nvex
84
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
43-2
9 -2
M
A
surf
ace
dark
er b
right
ora
nge
roug
h rig
id b
umpy
irre
gula
r opa
que
glos
sy
1071
K
ineo
cocc
us-li
ke b
acte
rium
AS3
138
0.
98
AF0
6068
6 A
T03-
43-3
0 -2
M
A
surf
ace
gold
en y
ello
w) ~
irreg
ular
rais
ed g
loss
y ~u
neve
n su
rfac
e ~t
rans
10
03
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-43
-31
-2
MA
su
rfac
e bi
g or
ange
/pk
opaq
ue ~
glos
sy c
onca
ve u
neve
n ce
nter
edg
es ra
ised
roun
d 95
9 K
ocur
ia e
ryth
rom
yxa
0.
99
Y11
330
AT0
3-43
-32
-1
1/10
PC
A
surf
ace
med
pk/
oran
ge ro
und
~con
vex
glos
sy ~
trans
94
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
43-3
3 -1
1/
10 P
CA
su
rfac
e sm
all l
t tan
/lt p
k gl
ossy
con
vex
~tra
ns
961
Koc
uria
ery
thro
myx
a
0.99
Y
1133
0 A
T03-
43-3
4 -1
1/
10 P
CA
su
rfac
e da
rker
brig
ht o
rang
e ro
und
conv
ex g
loss
y op
aque
96
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
44-1
-2
1/
10 P
CA
su
rfac
e lt
peac
h co
nvex
ver
y gl
ossy
~op
aque
in c
ente
r tra
ns e
dges
84
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
45-1
-1
PC
A
surf
ace
lt pi
nk ir
regu
lar ~
glos
sy ra
ised
~op
aque
85
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
45-2
-1
1/
100
PCA
sur
face
lt p
ink
glos
sy ~
conv
ex ~
irreg
ular
~tra
ns
851
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-45
-3
-1
1/10
0 PC
A s
urfa
ce l
t pin
k gl
ossy
~co
nvex
~irr
egul
ar ~
trans
76
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
45-4
-1
1/
100
PCA
sur
face
dar
ker p
k gl
ossy
con
vex
~tra
ns
809
Unc
ult a
ctin
obac
teriu
m c
lone
AC
TIN
O2B
0.
96
AY
4946
41
AT0
3-45
-5
-1
1/10
0 PC
A s
urfa
ce d
arke
r pk
glos
sy c
onve
x ~t
rans
84
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
45-7
-1
1/
100
PCA
sur
face
lt p
ink
glos
sy c
onve
x ~t
rans
72
0 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
45-9
-1
1/
10 P
CA
su
rfac
e cr
eam
y lt
pk g
loss
y co
nvex
~tra
ns
812
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-45
-10
-1
1/10
PC
A
surf
ace
dark
er p
k gl
ossy
con
vex
~tra
ns
839
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-45
-11
-1
1/10
PC
A
surf
ace
tan/
lt pk
glo
ssy
conv
ex ~
trans
77
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
45-1
2 -1
1/
10 P
CA
su
rfac
e cr
eam
y lt
pk g
loss
y co
nvex
~tra
ns
858
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-45
-13
-1
1/10
PC
A
surf
ace
trans
~co
nvex
glo
ssy
945
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-45
-14
-1
1/10
PC
A
surf
ace
crea
my
lt pe
ach/
pk g
loss
y co
nvex
~tra
ns
852
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-45
-15
-2
1/10
PC
A
surf
ace
trans
ver
y lt
pk g
loss
y co
nvex
84
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
45-1
6 -1
N
A
surf
ace
roun
d pi
nk o
paqu
e gl
ossy
con
vex
837
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-45
-17
-2
MA
su
rfac
e sm
all r
ound
yel
low
opa
que
glos
sy c
onve
x 84
3 Pr
omic
rom
onos
pora
ent
erop
hila
0.
95
X83
807
AT0
3-45
-18
-2
MA
su
rfac
e sm
all p
ink
irreg
ular
gro
wth
con
vex
~tra
ns
834
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-45
-19
-1
MA
su
rfac
e w
hite
glo
ssy
opaq
ue c
onve
x 93
3 Pr
omic
rom
onos
pora
ent
erop
hila
0.
96
X83
807
133
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
C
olon
y M
orph
olog
y
S
eque
nce
Len
gth
B
LA
ST R
esul
t
Sim
ilari
ty A
cces
sion
no.
A
T03-
45-2
0 -1
M
A
surf
ace
lt pk
glo
ssy
conv
ex o
paqu
e 93
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
45-2
2 -1
M
A
surf
ace
lt pk
smoo
th ~
irreg
ular
~gl
ossy
rais
ed o
paqu
e 89
7 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
45-2
3 -1
M
A
surf
ace
hot o
rang
e ~c
onve
x op
aque
~gl
ossy
65
6 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
45-2
4 -1
M
A
surf
ace
brig
ht y
ello
w ~
flat g
loss
y op
aque
~ro
ugh
962
Act
inom
ycet
acea
e
0.99
X
8731
0 A
T03-
45-2
5 -1
M
A
surf
ace
yello
w c
onve
x ro
und
opaq
ue ~
glos
sy ri
ngs
872
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-45
-26
-1
MA
su
rfac
e ho
t ora
nge/
pk g
loss
y co
nvex
opa
que
924
Mod
esto
bact
er m
ultis
epta
tus
0.99
A
J871
304
AT0
3-45
-27
-1
MA
su
rfac
e of
fwhi
te g
loss
y ~c
onve
x op
aque
89
5 Pr
omic
rom
onos
pora
ent
erop
hila
0.
95
X83
807
AT0
3-45
-28
-1
NA
su
rfac
e ho
t pea
ch g
loss
y ~c
onve
x op
aque
94
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
45-2
9 -1
N
A
surf
ace
trans
tan
glos
sy c
onve
x 80
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-2
-1
1/
100
PCA
sur
face
cre
amy
lt pk
~tra
ns g
loss
y co
nvex
smal
l 97
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-3
-1
1/
100
PCA
sur
face
dar
ker p
k tra
ns g
loss
y co
nvex
smal
l 85
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-4
-1
1/
100
PCA
sur
face
tra
ns g
loss
y ~c
onve
x sm
all
792
Cel
lulo
mon
as c
ella
sea
0.
96
X83
804
AT0
3-46
-7
-1
1/10
0 PC
A s
urfa
ce w
hite
tran
s glo
ssy
conv
ex sm
all
813
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-8
-1
1/10
0 PC
A s
urfa
ce c
ream
y lt
pk ~
trans
glo
ssy
conv
ex sm
all
852
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-9
-1
1/10
0 PC
A s
urfa
ce c
ream
y lt
pk ~
trans
glo
ssy
conv
ex sm
all
800
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-10
-1
1/10
0 PC
A s
urfa
ce c
ream
y lt
pk ~
trans
glo
ssy
conv
ex sm
all
860
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-11
-1
1/10
0 PC
A s
urfa
ce w
hite
tran
s glo
ssy
conv
ex sm
all
770
Prom
icro
mon
ospo
ra e
nter
ophi
la
0.93
X
8380
7 A
T03-
46-1
2 -1
1/
100
PCA
sur
face
dar
ker p
k tra
ns g
loss
y co
nvex
smal
l 82
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-1
4 -2
1/
100
PCA
sur
face
dar
ker p
k/pe
ach
glos
sy c
onve
x ~t
rans
84
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-1
5 -2
1/
100
PCA
sur
face
dar
ker r
ed/o
rang
e gl
ossy
con
vex
trans
59
0 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
46-1
6 -2
1/
100
PCA
sur
face
cre
amy
lt pk
~tra
ns g
loss
y co
nvex
78
9 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
46-1
8 -2
1/
100
PCA
sur
face
whi
te tr
ans g
loss
y co
nvex
smal
l 70
9 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
46-1
9 -2
1/
100
PCA
sur
face
dar
ker p
k/pu
rple
tran
s glo
ssy
conv
ex sm
all
859
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-20
-2
1/10
0 PC
A s
urfa
ce w
hite
/lt p
k gl
ossy
con
vex
~opa
que
846
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-46
-21
-2
1/10
0 PC
A s
urfa
ce c
ream
y lt
pk g
loss
y co
nvex
~tra
ns
856
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-22
-2
1/10
0 PC
A s
urfa
ce d
arke
r pk/
oran
ge g
loss
y co
nvex
tran
s 73
2 Bl
asto
cocc
us sp
. BC
448
0.
96
AJ3
1657
1 A
T03-
46-2
3 -2
1/
100
PCA
sur
face
pk/
peac
h tra
ns g
loss
y co
nvex
85
4 C
ellu
lom
onas
car
tae
MSD
201
06
0.94
X
7945
6 A
T03-
46-2
4 -2
1/
100
PCA
sur
face
pk/
peac
h tra
ns g
loss
y co
nvex
mor
e co
nvex
poi
nt in
cen
ter
805
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-25
-2
1/10
0 PC
A s
urfa
ce w
hite
glo
ssy
conv
ex ~
opaq
ue
828
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-26
-2
1/10
0 PC
A s
urfa
ce p
urpl
e tra
ns g
loss
y co
nvex
84
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-2
7 -1
PC
A
surf
ace
crea
m n
on-g
loss
y co
nvex
pas
tey
~opa
que
852
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-28
-1
PCA
su
rfac
e lt
pk/c
ream
glo
ssy
conv
ex ~
opaq
ue
769
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-29
-1
PCA
su
rfac
e br
ight
pk
rais
ed ir
regu
lar s
moo
th o
paqu
e (lo
oks l
ike
a ne
rd)
792
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-30
-1
PCA
su
rfac
e lt
pk/ta
n ~g
loss
y co
nvex
~op
aque
83
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-3
1 -1
PC
A
surf
ace
oran
ge/ta
n du
ll no
n-gl
ossy
smoo
th c
onve
x ~o
paqu
e 64
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-3
2 -1
PC
A
surf
ace
oran
ge/p
k ~g
loss
y co
nvex
opa
que
727
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-33
-1
PCA
su
rfac
e tra
ns ta
n co
nvex
glo
ssy
844
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-34
-1
PCA
su
rfac
e tra
ns c
ream
con
vex
glos
sy
810
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-36
-1
PCA
su
rfac
e cr
eam
non
-glo
ssy
conv
ex p
aste
y ~o
paqu
e 86
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-3
7 -1
PC
A
surf
ace
lt pk
/tan
~glo
ssy
conv
ex ~
opaq
ue
748
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-38
-1
PCA
su
rfac
e or
ange
/dar
ker r
ed g
loss
y ~t
rans
con
vex
811
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-39
-1
PCA
su
rfac
e cr
eam
~op
aque
non
-glo
ssy
conv
ex p
aste
y 82
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-4
0 -1
PC
A
surf
ace
tan
~glo
ssy
conv
ex ~
opaq
ue so
ft 81
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-4
1 -1
PC
A
surf
ace
crea
my
brig
ht p
k/or
ange
opa
que
glos
sy c
onve
x 77
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-4
3 -1
PC
A
surf
ace
dark
er o
rang
e gl
ossy
~tra
ns c
onve
x 71
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-4
4 -1
PC
A
surf
ace
tan
~glo
ssy
conv
ex ~
opaq
ue so
ft 80
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-4
5 -1
PC
A
surf
ace
dark
er o
rang
e ~g
loss
y co
nvex
~op
aque
95
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1
134
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
C
olon
y M
orph
olog
y
S
eque
nce
Len
gth
B
LA
ST R
esul
t
Si
mila
rity
Acc
essi
on n
o.
AT0
3-46
-46
-1
PCA
su
rfac
e br
ight
dar
ker p
k ve
ry g
loss
y co
nvex
opa
que
606
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-47
-1
1/10
PC
A
surf
ace
dark
er p
k/pu
rple
big
tran
s edg
es c
olor
cen
ter ~
trans
cen
ter g
loss
y co
nvex
83
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-4
8 -1
1/
10 P
CA
su
rfac
e da
rker
pk/
purp
le ir
regu
lar t
rans
glo
ssy
flat
775
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-49
-1
1/10
PC
A
surf
ace
lt pu
rple
tran
s glo
ssy
conv
ex
862
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-50
-1
1/10
PC
A
surf
ace
trans
tan
glos
sy c
onve
x 78
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-5
1 -1
1/
10 P
CA
su
rfac
e tra
ns lt
pk
glos
sy ~
conv
ex
818
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-52
-1
1/10
PC
A
surf
ace
big
lt pk
/ora
nge
smal
l con
vex
~opa
que
~glo
ssy
841
Blas
toco
ccus
sp. B
C41
2
0.98
A
J316
574
AT0
3-46
-53
-1
1/10
PC
A
surf
ace
pink
irre
gula
r ~co
nvex
~op
aque
glo
ssy
772
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-54
-1
1/10
PC
A
surf
ace
purp
le/ta
n tra
ns ~
flat g
loss
y 82
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-5
5 -1
1/
10 P
CA
su
rfac
e cr
eam
y lt
pk/p
each
glo
ssy
conv
ex ~
opaq
ue
560
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-56
-1
1/10
PC
A
surf
ace
tang
erin
e ~o
paqu
e gl
ossy
con
vex
815
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-57
-1
1/10
PC
A
surf
ace
crea
my
trans
glo
ssy
conv
ex
862
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-58
-1
1/10
PC
A
surf
ace
brig
ht p
k/pe
ach
trans
edg
e m
ore
colo
r cen
ter ~
tarn
s cen
ter g
loss
y co
nvex
83
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-5
9 -1
1/
10 P
CA
su
rfac
e tra
ns g
loss
y ~c
onve
x 78
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-6
2 -1
1/
10 P
CA
su
rfac
e
839
Cel
lulo
mon
as c
arta
e M
SD 2
0106
0.
94
X79
456
AT0
3-46
-63
-1
1/10
PC
A
surf
ace
crea
my
lt pe
ach/
lt pk
glo
ssy
conv
ex ~
opaq
ue
830
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-64
-1
1/10
PC
A
surf
ace
dark
er o
rang
e/da
rker
red
trans
glo
ssy
conv
ex
766
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-65
-1
1/10
PC
A
surf
ace
brig
ht p
k/pe
ach
trans
edg
e m
ore
colo
r cen
ter ~
trans
cen
ter g
loss
y co
nvex
85
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-6
6 -1
1/
10 P
CA
su
rfac
e br
ight
ora
nge
irreg
ular
text
ured
~op
aque
flat
aro
und
edge
~co
nvex
cen
ter
800
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-67
-1
1/10
PC
A
surf
ace
crea
my
lt pe
ach/
lt pk
glo
ssy
conv
ex ~
opaq
ue
837
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-68
-1
1/10
PC
A
surf
ace
trans
dar
ker p
urpl
e/da
rker
pk
glos
sy c
onve
x 65
5 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
46-6
9 -1
1/
10 P
CA
su
rfac
e da
rker
ora
nge/
dark
er re
d tra
ns g
loss
y co
nvex
85
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
46-7
1 -1
1/
10 P
CA
su
rfac
e tra
ns lt
pk
glos
sy c
onve
x 70
9 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
46-7
2 -1
1/
10 P
CA
su
rfac
e
844
Cel
lulo
sim
icro
bium
cel
lula
ns
0.95
A
B16
6888
A
T03-
46-7
3 -1
1/
10 P
CA
su
rfac
e lt
pk ~
glos
sy c
onve
x ~o
paqu
e 80
4 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T03-
46-7
4 -1
1/
10 P
CA
su
rfac
e cr
eam
y lt
pk g
loss
y co
nvex
~tra
ns
858
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-75
-1
NA
su
rfac
e cr
eam
y/w
hite
~tra
ns g
loss
y co
nvex
80
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-7
6 -1
N
A
surf
ace
lt pk
~op
aque
glo
ssy
conv
ex
545
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
3-46
-79
-1
NA
su
rfac
e pk
opa
que
conv
ex ~
glos
sy fr
ayed
edg
es
897
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-80
-1
NA
su
rfac
e pk
~op
aque
con
cave
irre
gula
r ~du
ll 80
7 Bl
asto
cocc
us sp
. BC
412
0.
98
AJ3
1657
4 A
T03-
46-8
1 -1
N
A
surf
ace
crea
m e
dges
con
vex
glos
sy ~
trans
dar
ker (
brow
n/ta
n) c
ente
r 95
7 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-8
2 -1
N
A
surf
ace
very
lt ta
n/pk
glo
ssy
conv
ex ~
trans
89
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-8
3 -1
N
A
surf
ace
irreg
ular
tran
s cle
ar g
loss
y ed
ges &
ora
nge
cent
er ~
conv
ex
848
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-84
-1
NA
su
rfac
e tra
ns g
loss
y co
nvex
10
30
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-85
-1
NA
su
rfac
e tra
ns g
loss
y co
nvex
fray
ed e
dges
ora
nge
trans
cen
ter
701
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-86
-1
NA
su
rfac
e pe
ach/
oran
ge ~
opaq
ue c
onve
x gl
ossy
96
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-8
9 -1
M
A
surf
ace
brig
ht o
rang
e/ho
t pk
irreg
ular
bum
py !r
aise
d op
aque
~gl
ossy
86
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-9
0 -1
M
A
surf
ace
!lt p
k gl
ossy
con
vex
opaq
ue
982
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-91
-1
MA
su
rfac
e tra
ns fl
at g
loss
y irr
egul
ar
866
Prom
icro
mon
ospo
ra e
nter
ophi
la
0.96
X
8380
7 A
T03-
46-9
2 -1
M
A
surf
ace
lt pk
dul
l irr
egul
ar ro
ugh
rigid
bum
py ra
ised
opa
que
978
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-93
-1
MA
su
rfac
e pk
~op
aque
irre
gula
r glo
ssy
fray
ed e
dges
rais
ed
948
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-94
-1
MA
su
rfac
e pk
con
vex
glos
sy ~
opaq
ue sm
all b
umps
on
surf
ace
in c
ente
r 94
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-9
5 -2
M
A
surf
ace
lt ye
llow
opa
que
glos
sy c
onve
x 85
5 Pr
omic
rom
onos
pora
ent
erop
hila
0.
96
X83
807
AT0
3-46
-96
-2
MA
su
rfac
e lt
pk o
paqu
e ~g
loss
y co
nvex
94
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-9
7 -2
M
A
surf
ace
hot p
k irr
egul
ar ra
ised
bum
py sm
ooth
opa
que
~dul
l 94
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-9
8 -2
M
A
surf
ace
hot p
k irr
egul
ar d
ull r
aise
d ro
ugh
rigid
opa
que
961
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-46
-100
-2
N
A
surf
ace
trans
ora
nge
glos
sy c
onve
x 97
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1
135
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T03-
46-1
01
-2
NA
su
rfac
e tra
ns !~
oran
ge g
loss
y co
nvex
80
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-1
02
-2
NA
su
rfac
e pk
irre
gula
r hol
e in
cen
ter o
paqu
e ra
ised
smoo
th ~
glos
sy
953
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-46
-103
-2
N
A
surf
ace
flat o
ffw
hite
opa
que
oran
ge c
ente
r ~du
ll cu
rled
up e
dges
95
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
46-1
04
-2
NA
su
rfac
e tra
ns ~
pk/p
each
glo
ssy
conv
ex
911
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-48
-1
-1
PCA
su
rfac
e pe
ach
non-
glos
sy o
paqu
e ve
ry c
onve
x 74
4 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T03-
48-2
-2
1/
10 P
CA
su
rfac
e fla
t pur
ple/
pink
glo
ssy
~tra
ns
848
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-48
-4
-1
1/10
PC
A
surf
ace
tan
glos
sy c
onve
x ~t
rans
dar
ker s
pot i
n ce
nter
74
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
48-5
-1
1/
10 P
CA
su
rfac
e pi
nk n
on-g
loss
y du
ll irr
egul
ar c
onve
x ~o
paqu
e 86
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
48-6
-1
1/
10 P
CA
su
rfac
e pi
nk n
on-g
loss
y du
ll co
nvex
~op
aque
97
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
48-7
-2
N
A
surf
ace
smal
l rou
nd p
each
smoo
th c
onve
x op
aque
67
0 Bl
asto
cocc
us sp
. BC
448
0.
95
AJ3
1657
1 A
T03-
48-8
-1
N
A
surf
ace
larg
e ro
und
crea
m sm
ooth
flat
~tra
ns
795
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-48
-9
-1
NA
su
rfac
e ro
und
crea
m sm
ooth
con
vex
~tra
ns
759
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-48
-10
-1
NA
su
rfac
e ro
und
pink
tran
s con
vex
smoo
th
701
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
3-48
-11
-2
1/10
PC
A
surf
ace
roun
d ye
llow
flat
~tra
ns sm
ooth
76
7 St
aphy
loco
ccus
sp. 1
6b-5
A
0.99
A
Y56
1556
A
T03-
48-1
2 -2
M
A
surf
ace
roun
d pi
nk ~
trans
kno
blik
e pr
otub
eran
ce
698
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-48
-13
-1
MA
su
rfac
e lt
pk w
ith b
lack
spec
kles
on
surf
ace
opaq
ue ~
glos
sy c
onve
x 95
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
48-1
4 -1
M
A
surf
ace
lt pk
opa
que
umbo
nate
~gl
ossy
91
0 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
48-1
5 -1
M
A
surf
ace
lt pk
irre
gula
r rig
id ro
ugh
opaq
ue c
onca
ve c
ente
r dul
l 91
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
48-1
8 -1
1/
10 P
CA
su
rfac
e br
owni
sh o
rang
e ~t
rans
glo
ssy
conv
ex
957
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-48
-19
-1
1/10
PC
A
surf
ace
brow
nish
ora
nge
opaq
ue ~
dull
conv
ex
858
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-48
-20
-1
1/10
PC
A
surf
ace
brow
nish
ora
nge
~irr
egul
ar g
loss
y fr
ayed
edg
es ~
trans
rais
ed
954
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-48
-21
-1
1/10
PC
A
surf
ace
dark
er o
rang
e ~o
paqu
e gl
ossy
con
vex
951
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-48
-22
-1
1/10
PC
A
surf
ace
dark
er o
rang
e ~t
rans
glo
ssy
conv
ex (s
ome
are
umbo
nate
) 95
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
48-2
4 -1
1/
10 P
CA
su
rfac
e !lt
pk
~con
vex
opaq
ue g
loss
y 97
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
48-2
7 -2
1/
10 P
CA
su
rfac
e tra
ns !l
t pk
glos
sy c
onve
x 92
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T03-
48-2
8 -2
1/
10 P
CA
su
rfac
e of
fwhi
te/!l
t pk
~tra
ns g
loss
y co
nvex
98
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
49-2
-2
1/
100
PCA
sur
face
lt y
ello
w ~
flat g
loss
y irr
egul
ar ~
trans
82
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
49-3
-1
PC
A
surf
ace
big
yello
w c
onve
x no
n-gl
ossy
text
ured
(bum
py) o
paqu
e 80
0 M
icro
cocc
us lu
teus
0.
99
AJ4
0909
6 A
T03-
50-1
-1
PC
A
surf
ace
dark
er o
rang
e ve
ry c
onve
x gl
ossy
big
opa
que
797
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-50
-2
-2
1/10
PC
A
surf
ace
peac
h gl
ossy
tran
s rai
sed
in c
ente
r mor
e op
aque
in c
ente
r 84
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T03-
50-3
-2
1/
100
PCA
sur
face
lt p
k ~
conv
ex n
on-g
loss
y ro
ugh
838
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-50
-4
-1
1/10
PC
A
surf
ace
lt pk
flat
glo
ssy
~opa
que
837
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-50
-5
-1
PCA
su
rfac
e da
rk o
rang
e co
nvex
glo
ssy
~opa
que
871
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-50
-6
-1
PCA
su
rfac
e w
hite
opa
que
over
grow
th o
ver 1
/2 p
late
irre
gula
r ~fla
t 78
3 Pa
ntoe
a an
anat
is st
rain
BD
390
0.
96
AY
5307
95
AT0
3-50
-8
-1
NA
su
rfac
e sm
all r
ound
pin
k co
nvex
~tra
ns
793
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
3-50
-9
-2
MA
su
rfac
e sm
all r
ound
bro
wn
opaq
ue c
onve
x 57
0 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.95
L4
0620
A
T03-
50-1
0 -2
M
A
surf
ace
smal
l rou
nd b
row
n op
aque
con
vex
768
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
98
L406
20
AT0
3-50
-11
-1
MA
su
rfac
e sm
all r
ound
pin
k co
nvex
~tra
ns
928
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-50
-12
-1
MA
su
rfac
e sm
all r
ound
bro
wn
opaq
ue c
onve
x 52
4 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.96
L4
0620
A
T03-
50-1
3 -1
M
A
surf
ace
lt pk
with
bla
ck sp
eckl
ed su
rfac
e ~o
paqu
e ~d
ull c
onve
x 86
9 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.98
L4
0620
A
T03-
50-1
4 -1
M
A
surf
ace
hot o
rang
e op
aque
~gl
ossy
con
vex
867
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
3-50
-15
-1
MA
su
rfac
e ho
t ora
nge
opaq
ue ~
glos
sy c
onve
x 76
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
151-
1 -1
M
A
surf
ace
smal
l rou
nd p
ink
smoo
th c
onca
ve ~
trans
79
1 Bl
asto
cocc
us sp
. BC
521
0.
98
AJ3
1657
3 A
T04-
151-
2 -1
M
A
surf
ace
smal
l rou
nd p
ink
conv
ex o
paqu
e 83
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
151-
3 -2
1/
10 P
CA
su
rfac
e sm
all r
ound
pin
k co
nvex
~tra
ns
802
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
1-4
-1
1/10
PC
A
surf
ace
smal
l rou
nd p
ink
conv
ex ~
trans
45
6 Bl
asto
cocc
us sp
. BC
521
0.
98
AJ3
1657
3 A
T04-
151-
5 -1
1/
100
PCA
sur
face
off
whi
te ~
opaq
ue c
onve
x fr
ayed
edg
es
889
Blas
toco
ccus
sp. B
C41
2
0.98
A
J316
574
136
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
154-
1 -1
M
A
surf
ace
smal
l rou
nd g
rey
trans
con
vex
862
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
97
L406
20
AT0
4-15
4-4
-1
1/10
0 PC
A s
urfa
ce l
t pk
tiny
trans
glo
ssy
conv
ex
780
Blas
toco
ccus
sp. B
C42
1
0.96
A
J316
574
AT0
4-15
6-2
-1
1/10
0 PC
A s
urfa
ce p
k ~i
rreg
ular
~op
aque
glo
ssy
rais
ed
852
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
7-3
-2
1/10
PC
A
surf
ace
dark
er b
row
n du
ll op
aque
in a
gar m
aybe
fung
us
959
Stre
ptom
yces
sp. X
13
0.99
A
F060
793
AT0
4-15
8-1
-1
NA
su
rfac
e pe
ach
roun
d ~t
rans
con
vex
unev
en e
dges
83
3 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
158-
2 -1
N
A
surf
ace
smal
l pea
ch ro
und
~tra
ns
735
Kin
eoco
ccus
-like
bac
teriu
m A
S297
8
0.98
A
F060
676
AT0
4-15
8-3
-1
1/10
PC
A
surf
ace
smal
l pin
k ro
und
conv
ex o
paqu
e 97
7 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T04-
158-
4 -1
M
A
surf
ace
smal
l pin
k ro
und
rais
ed ~
trans
con
vex
797
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
4-15
8-6
-1
MA
su
rfac
e ro
und
pink
con
vex
bum
py
786
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
8-7
-1
MA
su
rfac
e ro
und
yello
w fl
at o
paqu
e 75
9 C
ellu
lom
onas
cel
lase
a
0.98
X
8380
4 A
T04-
158-
8 -1
1/
100
PCA
sur
face
hot
pk
glos
sy c
onve
x ~o
paqu
e 82
9 Bl
asto
cocc
us sp
. BC
412
0.
97
AJ3
1657
4 A
T04-
158-
9 -1
1/
100
PCA
sur
face
pea
ch ~
trans
glo
ssy
conv
ex (~
dark
er in
cen
ter)
67
3 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
158-
10
-2
1/10
0 PC
A s
urfa
ce t
ange
rine
glos
sy c
onve
x ~t
rans
66
3 B
acte
rium
Elli
n602
3
0.99
A
Y23
4675
A
T04-
161-
3 -1
M
A
surf
ace
whi
te ~
opaq
ue ~
irreg
ular
bum
py ri
gid
roug
h ra
ised
84
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
4 -1
M
A
surf
ace
brow
n/ta
n gl
ossy
~tra
ns c
onve
x 75
4 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
161-
5 -1
M
A
surf
ace
lt pk
/pea
ch ~
opaq
ue g
loss
y co
nvex
95
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
6 -1
M
A
surf
ace
whi
te o
paqu
e gl
ossy
con
vex
860
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-7
-1
MA
su
rfac
e lt
pk/p
each
~op
aque
~irr
egul
ar b
umpy
rigi
d ro
ugh
rais
ed
965
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-8
-1
MA
su
rfac
e lt
pk ~
opaq
ue c
onve
x gl
ossy
87
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
9 -1
M
A
surf
ace
lt ye
llow
~tra
ns g
loss
y co
nvex
87
6 Pr
omic
rom
onos
pora
ent
erop
hila
0.
95
X83
807
AT0
4-16
1-10
-1
M
A
surf
ace
pk/o
rang
e gl
ossy
con
vex
~opa
que
463
Bac
teriu
m E
llin6
023
0.
98
AY
2346
75
AT0
4-16
1-11
-1
M
A
surf
ace
trans
tan
glos
sy c
onve
x 75
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
12
-1
MA
su
rfac
e of
fwhi
te/lt
pk
glos
sy ~
opaq
ue c
onve
x (m
ore
conv
ex c
ente
r)
1003
Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
161-
13
-1
MA
su
rfac
e w
hite
glo
ssy
~opa
que
conv
ex
1003
Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
14
-1
MA
su
rfac
e pk
/ora
nge
~tra
ns g
loss
y co
nvex
83
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
15
-1
MA
su
rfac
e of
fwhi
te ~
trans
~irr
egul
ar b
umpy
rigi
d ro
ugh
rais
ed
850
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-16
-1
M
A
surf
ace
oran
ge g
loss
y ~o
paqu
e co
nvex
88
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
17
-1
MA
su
rfac
e or
ange
~tra
ns g
loss
y co
nvex
(mor
e co
nvex
in c
ente
r)
774
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-18
-1
M
A
surf
ace
yello
w/ta
n tra
ns g
loss
y co
nvex
70
4 C
ellu
lom
onas
car
tae
MSD
201
06
0.95
X
7945
6 A
T04-
161-
19
-1
MA
su
rfac
e of
fwhi
te g
loss
y co
nvex
~op
aque
75
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
20
-1
MA
su
rfac
e lt
pk ~
opaq
ue c
onve
x gl
ossy
85
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
21
-1
MA
su
rfac
e lt
pk ~
opaq
ue c
onve
x gl
ossy
83
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
22
-1
MA
su
rfac
e ve
ry lt
pk
~tra
ns ~
irreg
ular
bum
py ri
gid
roug
h ra
ised
98
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
23
-1
MA
su
rfac
e w
hite
~op
aque
glo
ssy
conv
ex
751
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-25
-1
M
A
surf
ace
tan/
brow
n ~o
paqu
e gl
ossy
con
vex
774
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-26
-3
M
A
surf
ace
crea
m c
onve
x op
aque
~gl
ossy
72
1 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
161-
27
-3
MA
su
rfac
e cr
eam
con
vex
wrin
kly
clum
py ~
trans
dul
l 77
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
28
-3
MA
su
rfac
e lt
peac
h co
nvex
clu
mpy
~tra
ns d
ull
694
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
4-16
1-29
-3
M
A
surf
ace
peac
h/lt
pk c
onve
x du
ll op
aque
clu
mpy
76
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
30
-1
MA
su
rfac
e or
ange
con
vex
opaq
ue g
loss
y sm
all d
ip in
cen
ter
736
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-31
-1
M
A
surf
ace
lt or
ange
/pea
ch c
onve
x gl
ossy
opa
que
945
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-32
-1
M
A
surf
ace
offw
hite
glo
ssy
~con
vex
~opa
que
635
Cel
lulo
sim
icro
bium
funk
ei st
rain
CD
C#J
B40
83ot
0.
92
AY
5237
89
AT0
4-16
1-33
-1
M
A
surf
ace
crea
m o
paqu
e co
nvex
glo
ssy
691
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-34
-1
M
A
surf
ace
crea
m c
onve
x op
aque
~du
ll ~c
lum
py
854
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-35
-1
M
A
surf
ace
lt pk
/pea
ch o
paqu
e co
nvex
~gl
ossy
84
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
36
-1
MA
su
rfac
e lt
yello
w g
loss
y ~c
onve
x ~t
rans
76
9 Pr
omic
rom
onos
pora
ent
erop
hila
0.
95
X83
807
AT0
4-16
1-37
-1
M
A
surf
ace
peac
h gl
ossy
opa
que
~fla
t 77
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1
137
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
161-
38
-1
MA
su
rfac
e lt
pk ~
dull
conv
ex ~
trans
~cl
umpy
10
39
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-39
-3
M
A
surf
ace
flat y
ello
w g
loss
y ~o
paqu
e 80
3 Pr
omic
rom
onos
pora
ent
erop
hila
0.
94
X83
807
AT0
4-16
1-40
-3
M
A
surf
ace
grey
con
vex
opaq
ue ~
glos
sy m
ore
conv
ex in
cen
ter t
op (l
ike
crea
msa
ver)
75
8 Bl
asto
cocc
us sp
. BC
412
0.
97
AJ3
1657
4 A
T04-
161-
41
-3
MA
su
rfac
e of
fwhi
te c
onve
x cl
umpy
~du
ll op
aque
94
3 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T04-
161-
42
-3
MA
su
rfac
e pe
ach
wrin
kly
conv
ex o
paqu
e ~g
loss
y 64
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
43
-2
MA
su
rfac
e pk
/ora
nge
clum
py c
onve
x ~d
ull ~
opaq
ue
520
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-44
-2
M
A
surf
ace
crea
m/y
ello
w ~
conv
ex ~
glos
sy o
paqu
e 64
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
45
-2
MA
su
rfac
e of
fwhi
te c
lum
py c
onve
x du
ll ~o
paqu
e 76
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
46
-2
MA
su
rfac
e lt
pk ~
conv
ex o
paqu
e ~g
loss
y 78
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
47
-2
MA
su
rfac
e or
ange
flat
with
rais
ed ri
ng in
cen
ter o
paqu
e ~g
loss
y 93
2 Bl
asto
cocc
us a
ggre
gatu
s 0.
99
AJ4
3019
3 A
T04-
161-
48
-2
MA
su
rfac
e lt
pk c
lum
py o
paqu
e du
ll 10
20
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-49
-2
M
A
surf
ace
lt or
ange
flat
opa
que
~dul
l 61
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
50
-2
MA
su
rfac
e or
ange
con
vex
opaq
ue g
loss
y
614
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-51
-2
M
A
surf
ace
offw
hite
glo
ssy
opaq
ue c
onve
x (m
ore
conv
ex in
cen
ter l
ike
crea
msa
ver)
63
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
52
-2
MA
su
rfac
e ye
llow
glo
ssy
~con
vex
trans
84
1 Pr
omic
rom
onos
pora
ent
erop
hila
0.
94
X83
807
AT0
4-16
1-53
-2
M
A
surf
ace
pk ~
opaq
ue ~
irreg
ular
bum
py ri
gid
roug
h ra
ised
86
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
54
-2
MA
su
rfac
e of
fwhi
te g
loss
y op
aque
con
vex
(mor
e co
nvex
in c
ente
r lik
e cr
eam
save
r)
836
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-55
-2
M
A
surf
ace
pk/p
each
con
vex
glos
sy o
paqu
e 98
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
56
-2
MA
su
rfac
e lt
pk ~
trans
~co
nvex
glo
ssy
unsm
ooth
edg
es
631
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-57
-2
M
A
surf
ace
lt pk
glo
ssy
conv
ex o
paqu
e (li
ke c
ream
life
save
r)
695
Blas
toco
ccus
sp. B
C41
2
0.98
A
J316
574
AT0
4-16
1-58
-2
M
A
surf
ace
pk w
ith d
arke
r spe
cs g
loss
y co
nvex
~op
aque
75
2 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
161-
59
-2
MA
su
rfac
e w
hite
opa
que
glos
sy c
onve
x 77
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
60
-2
MA
su
rfac
e lt
pk tr
ans g
loss
y co
nvex
64
1 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
161-
61
-2
MA
su
rfac
e lt
pk g
loss
y op
aque
con
vex
(mor
e co
nvex
in c
ente
r lik
e cr
eam
life
save
r)
739
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-62
-2
M
A
surf
ace
lt pk
clu
mpy
~op
aque
irre
gula
r bum
py ri
gid
roug
h ra
ised
55
1 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
161-
63
-2
MA
su
rfac
e da
rker
ora
nge
(~re
d) g
loss
y co
nvex
~op
aque
73
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
64
-2
MA
su
rfac
e lt
pk ~
glos
sy c
onve
x ~o
paqu
e 69
9 Bl
asto
cocc
us sp
. BC
412
0.
97
AJ3
1657
4 A
T04-
161-
65
-2
MA
su
rfac
e bu
bble
gum
pk
opaq
ue g
loss
y co
nvex
75
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
67
-2
MA
su
rfac
e of
fwhi
te g
loss
y op
aque
con
vex
(mor
e co
nvex
in c
ente
r lik
e cr
eam
save
r)
677
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-68
-2
M
A
surf
ace
lt pk
/off
whi
te w
ith d
arke
r spe
cs o
n su
rfac
e co
nvex
glo
ssy
~opa
que
731
Blas
toco
ccus
sp. B
C41
2
0.96
A
J316
574
AT0
4-16
1-69
-2
M
A
surf
ace
lt pk
with
dar
k (r
ust)
cent
er g
loss
y co
nvex
~op
aque
78
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
70
-2
MA
su
rfac
e lt
pk g
loss
y co
nvex
~op
aque
bum
py su
rfac
e 78
3 Bl
asto
cocc
us sp
. BC
448
0.
96
AJ3
1657
1 A
T04-
161-
71
-2
MA
su
rfac
e pk
/pea
ch ir
regu
lar ~
opaq
ue ri
gid
roug
h ra
ised
~du
ll 75
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
72
-2
MA
su
rfac
e of
fwhi
te o
paqu
e gl
ossy
con
vex
(dip
in c
ente
r)
783
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-73
-1
1/
10 P
CA
su
rfac
e pk
/pur
ple
glos
sy c
onve
x tra
ns
794
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-74
-1
1/
10 P
CA
su
rfac
e lt
pk g
loss
y co
nvex
~tra
ns
715
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-75
-1
1/
10 P
CA
su
rfac
e da
rker
ora
nge
~irr
egul
ar in
aga
r opa
que
~glo
ssy
672
Blas
toco
ccus
sp. B
C44
8
0.96
A
J316
571
AT0
4-16
1-76
-1
1/
10 P
CA
su
rfac
e or
ange
/pk
glos
sy c
onve
x ~t
rans
55
0 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
78
-1
1/10
PC
A
surf
ace
lt pk
~tra
ns in
aga
r irr
egul
ar b
umpy
rigi
d ro
ugh
rais
ed
760
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-79
-1
1/
10 P
CA
su
rfac
e ve
ry lt
pk
trans
glo
ssy
conv
ex
761
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-81
-1
1/
10 P
CA
su
rfac
e of
fwhi
te/ta
n gl
ossy
con
vex
~tra
ns
692
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-82
-1
1/
10 P
CA
su
rfac
e pe
ach/
pk ~
trans
glo
ssy
conv
ex
799
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-83
-1
1/
10 P
CA
su
rfac
e ho
t pk/
oran
ge ~
opaq
ue g
loss
y co
nvex
73
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
84
-1
1/10
PC
A
surf
ace
lt pk
/pea
ch g
loss
y co
nvex
~tra
ns
951
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-85
-1
1/
10 P
CA
su
rfac
e tra
ns ~
whi
te g
loss
y ~c
onve
x (c
onve
x ce
nter
/edg
es ~
flat)
754
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-86
-1
1/
10 P
CA
su
rfac
e pe
ach/
tan
glos
sy c
onve
x ~t
rans
74
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
87
-1
1/10
PC
A
surf
ace
hot p
k/or
ange
~op
aque
glo
ssy
conv
ex
614
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
138
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
161-
88
-1
1/10
PC
A
surf
ace
very
lt p
k ~t
rans
~irr
egul
ar g
loss
y ra
ised
bum
py su
rfac
e 73
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
89
-1
1/10
PC
A
surf
ace
pk/p
each
~du
ll co
nvex
~op
aque
uns
moo
th e
dges
73
7 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
161-
90
-1
1/10
PC
A
surf
ace
pk/p
each
tran
s glo
ssy
conv
ex
517
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-91
-1
1/
10 P
CA
su
rfac
e ru
st g
loss
y co
nvex
~tra
ns
740
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-92
-1
1/
10 P
CA
su
rfac
e lt
pk g
loss
y co
nvex
~tra
ns
670
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-93
-1
1/
10 P
CA
su
rfac
e ve
ry lt
pk/
offw
hite
glo
ssy
conv
ex ~
trans
79
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
94
-1
1/10
PC
A
surf
ace
lt pk
glo
ssy
conv
ex tr
ans
680
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-95
-1
1/
10 P
CA
su
rfac
e br
own/
purp
le tr
ans g
loss
y co
nvex
40
9 B
acte
rium
Elli
n602
3
0.98
A
Y23
4675
A
T04-
161-
96
-1
1/10
PC
A
surf
ace
purp
le/ta
n tra
ns g
loss
y co
nvex
80
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
97
-1
1/10
PC
A
surf
ace
lt pk
~op
aque
con
vex
glos
sy
653
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-98
-1
1/
10 P
CA
su
rfac
e w
hite
~op
aque
glo
ssy
conv
ex
765
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-99
-1
1/
10 P
CA
su
rfac
e pk
glo
ssy
conv
ex ~
opaq
ue (u
nsm
ooth
edg
es)
689
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-10
0 -1
1/
10 P
CA
su
rfac
e lt
pk/p
each
~du
ll co
nvex
~op
aque
98
4 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T04-
161-
101
-1
1/10
PC
A
surf
ace
lt pk
~tra
ns g
loss
y co
nvex
61
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
103
-1
1/10
PC
A
surf
ace
very
lt p
k/of
fwhi
te ~
dull
~fla
t ~op
aque
in a
gar
793
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-10
4 -1
1/
10 P
CA
su
rfac
e lt
pk/ta
n gl
ossy
con
vex
~tra
ns
701
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-10
5 -1
1/
10 P
CA
su
rfac
e or
ange
~tra
ns ~
irreg
ular
~du
ll ra
ised
rigi
d bu
mpy
79
7 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
161-
106
-1
1/10
PC
A
surf
ace
whi
te tr
ans ~
conv
ex g
loss
y (c
onve
x ce
nter
/edg
es ~
flat)
731
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-10
7 -1
1/
10 P
CA
su
rfac
e pu
rple
/bro
wn
trans
glo
ssy
conv
ex
681
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-10
8 -1
1/
10 P
CA
su
rfac
e or
ange
glo
ssy
conv
ex ~
opaq
ue
649
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-10
9 -1
1/
10 P
CA
su
rfac
e pk
glo
ssy
conv
ex o
paqu
e 61
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
110
-1
1/10
PC
A
surf
ace
lt pk
/pea
ch g
loss
y co
nvex
~op
aque
65
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
111
-1
1/10
PC
A
surf
ace
lt pk
tran
s glo
ssy
conv
ex
682
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-11
2 -1
1/
10 P
CA
su
rfac
e lt
oran
ge/ta
n tra
ns g
loss
y co
nvex
65
5 Bl
asto
cocc
us sp
. BC
448
0.
96
AJ3
1657
1 A
T04-
161-
113
-1
1/10
PC
A
surf
ace
lt pk
~du
ll ~o
paqu
e co
nvex
57
1 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
161-
114
-1
1/10
PC
A
surf
ace
offw
hite
/lt p
k w
ith d
ark
spot
in c
ente
r con
vex
glos
sy ~
opaq
ue
785
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-11
5 -3
1/
10 P
CA
su
rfac
e or
ange
glo
ssy
conv
ex ~
opaq
ue
630
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-11
6 -3
1/
10 P
CA
su
rfac
e ta
n ~t
rans
glo
ssy
conv
ex
748
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-11
7 -3
1/
10 P
CA
su
rfac
e lt
oran
ge ~
trans
glo
ssy
conv
ex
802
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-11
8 -3
1/
10 P
CA
su
rfac
e lt
pk ~
trans
glo
ssy
conv
ex
770
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-11
9 -3
1/
10 P
CA
su
rfac
e or
ange
/hot
pk
~opa
que
glos
sy c
onve
x 75
3 G
eode
rmat
ophi
lus s
p. B
C51
8
0.99
A
J296
064
AT0
4-16
1-12
0 -3
1/
10 P
CA
su
rfac
e pk
/pea
ch ~
opaq
ue g
loss
y co
nvex
73
5 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
161-
121
-3
1/10
PC
A
surf
ace
tan
/ora
nge
~tra
ns g
loss
y co
nvex
80
4 G
eode
rmat
ophi
lus s
p. B
C51
8
0.98
A
J296
064
AT0
4-16
1-12
2 -3
1/
10 P
CA
su
rfac
e lt
yello
w/ta
n ~i
rreg
ular
big
glo
ssy
rippl
ed b
umpy
opa
que
905
Mic
roco
ccus
sp. R
G-6
4
0.99
A
Y56
1623
A
T04-
161-
123
-3
1/10
PC
A
surf
ace
lt or
ange
/pk
~tra
ns g
loss
y co
nvex
63
7 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
161-
124
-3
1/10
PC
A
surf
ace
tan
with
dar
ker t
an in
cen
ter g
loss
y ~t
rans
con
vex
821
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-12
5 -2
1/
10 P
CA
su
rfac
e or
ange
/tan
glos
sy ~
trans
con
vex
838
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-12
6 -2
1/
10 P
CA
su
rfac
e or
ange
/pk
opaq
ue g
loss
y co
nvex
74
6 Bl
asto
cocc
us sp
. BC
412
0.
97
AJ3
1657
4 A
T04-
161-
127
-2
1/10
PC
A
surf
ace
pk/p
each
~tra
ns g
loss
y co
nvex
76
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
129
-2
1/10
PC
A
surf
ace
pk/p
each
glo
ssy
opaq
ue c
onve
x ce
nter
uns
moo
th fl
at e
dges
76
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
130
-2
1/10
PC
A
surf
ace
oran
ge/p
k ~t
rans
glo
ssy
~con
vex
!~irr
egul
ar
751
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-13
1 -2
1/
10 P
CA
su
rfac
e sm
all l
t pk
opaq
ue ~
glos
sy c
onve
x 79
5 Bl
asto
cocc
us sp
. BC
412
0.
98
AJ3
1657
4 A
T04-
161-
133
-2
1/10
PC
A
surf
ace
peac
h/pk
~tra
ns g
loss
y co
nvex
68
3 G
eorg
enia
sp. T
04-0
4
0.95
A
Y88
0044
A
T04-
161-
134
-2
1/10
PC
A
surf
ace
trans
off
whi
te/ta
n gl
ossy
con
vex
633
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-13
5 -2
1/
10 P
CA
su
rfac
e da
rker
ora
nge
glos
sy m
ore
opaq
ue a
nd c
onve
x ce
nter
(~tra
ns fl
at e
dges
) 74
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
137
-2
1/10
PC
A
surf
ace
trans
glo
ssy
conv
ex
700
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-13
8 -2
1/
10 P
CA
su
rfac
e pk
/pea
ch ~
trans
glo
ssy
conv
ex (m
ore
conv
ex in
cen
ter)
79
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1
139
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
161-
140
-2
1/10
PC
A
surf
ace
peac
h/pk
~tra
ns g
loss
y co
nvex
74
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
141
-2
1/10
PC
A
surf
ace
yello
w /o
rang
e ~t
rans
glo
ssy
conv
ex
794
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
1-14
2 -2
1/
10 P
CA
su
rfac
e or
ange
/pk
~fla
t glo
ssy
~tra
ns
772
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
1-14
3 -2
1/
10 P
CA
su
rfac
e lt
pk ~
trans
glo
ssy
conv
ex (h
ard)
60
0 Bl
asto
cocc
us sp
. BC
448
0.
96
AJ3
1657
1 A
T04-
161-
144
-2
1/10
PC
A
surf
ace
oran
ge/h
ot p
k op
aque
~gl
ossy
rais
ed ir
regu
lar r
igid
bum
py ro
ugh
738
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
4-16
1-14
5 -2
1/
10 P
CA
su
rfac
e bu
rnt o
rang
e ~c
onve
x gl
ossy
~op
aque
83
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
146
-2
1/10
PC
A
surf
ace
pk o
paqu
e co
nvex
glo
ssy
979
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
1-14
7 -2
1/
10 P
CA
su
rfac
e of
fwhi
te fl
at b
ut ra
ised
mid
dle
opaq
ue g
loss
y 63
2 Bl
asto
cocc
us sp
. BC
448
0.
95
AJ3
1657
1 A
T04-
161-
148
-2
1/10
PC
A
surf
ace
lt or
ange
opa
que
conv
ex g
loss
y 73
0 Bl
asto
cocc
us sp
. BC
448
0.
96
AJ3
1657
1 A
T04-
161-
149
-2
1/10
PC
A
surf
ace
pink
opa
que
conv
ex g
loss
y 97
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
161-
150
-2
1/10
PC
A
surf
ace
peac
h ~c
onve
x gl
ossy
~op
aque
78
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
161-
151
-2
1/10
0 PC
A s
urfa
ce l
t pk
~tra
ns g
loss
y co
nvex
79
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
162-
1 -1
1/
10 P
CA
su
rfac
e lt
pk ~
trans
~irr
egul
ar b
umpy
rigi
d ro
ugh
rais
ed
769
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
97
L406
20
AT0
4-16
2-3
-1
MA
su
rfac
e bl
ack
~tra
ns ~
irreg
ular
bum
py ri
gid
roug
h ra
ised
66
0 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.93
L4
0620
A
T04-
162-
5 -1
M
A
surf
ace
blac
k ~t
rans
~irr
egul
ar b
umpy
rigi
d ro
ugh
rais
ed
569
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
96
L406
20
AT0
4-16
2-6
-1
MA
su
rfac
e bl
ack
~tra
ns ~
irreg
ular
bum
py ri
gid
roug
h ra
ised
77
6 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.97
L4
0620
A
T04-
162-
7 -2
M
A
surf
ace
blac
k ~t
rans
~irr
egul
ar b
umpy
rigi
d ro
ugh
rais
ed
750
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
97
L406
20
AT0
4-16
2-8
-2
MA
su
rfac
e bl
ack
~tra
ns ~
irreg
ular
bum
py ri
gid
roug
h ra
ised
78
2 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.97
L4
0620
A
T04-
162-
9 -1
M
A
surf
ace
blac
k ~t
rans
~irr
egul
ar b
umpy
rigi
d ro
ugh
rais
ed
744
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
96
L406
20
AT0
4-16
2-10
-2
M
A
surf
ace
blac
k ~t
rans
~irr
egul
ar b
umpy
rigi
d ro
ugh
rais
ed
776
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
97
L406
20
AT0
4-16
2-12
-1
PC
A
surf
ace
brig
ht y
ello
w ~
irreg
ular
opa
que
glos
sy b
ig c
onve
x 98
3 M
icro
cocc
us lu
teus
0.
99
AJ4
0909
6 A
T04-
162-
13
-2
PCA
su
rfac
e lt
yello
w ~
glos
sy ~
irreg
ular
opa
que
conv
ex b
ig
959
Mic
roco
ccus
lute
us
0.99
A
J409
096
AT0
4-16
4-1
-2
MA
su
rfac
e gl
ossy
flat
opa
que
crea
m y
ello
w
764
Hal
obac
illus
sp. M
O22
0.
96
AY
5531
13
AT0
4-16
4-2
-1
MA
su
rfac
e gl
ossy
con
vex
opaq
ue p
ink
749
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-3
-1
MA
su
rfac
e gl
ossy
con
vex
opaq
ue c
ream
78
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
5 -1
M
A
surf
ace
dull
clum
py o
ff-w
hite
con
vex
opaq
ue
720
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-6
-1
MA
su
rfac
e ~d
ull c
lum
py c
onve
x lt
pink
ish
crea
m
767
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
4-7
-1
MA
su
rfac
e gl
ossy
flat
tran
s cre
am c
olor
ed
749
Bac
teriu
m K
2-24
0.
96
AY
3454
29
AT0
4-16
4-8
-1
MA
su
rfac
e ~g
loss
y ~o
paqu
e ~c
onve
x lt
pk
762
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
4-9
-1
MA
su
rfac
e gl
ossy
offw
hite
flat
~op
aque
77
0 G
laci
al ic
e ba
cter
ium
G50
0K-1
9
0.98
A
F479
330
AT0
4-16
4-10
-1
M
A
surf
ace
smal
l ~co
nvex
lt p
ink
glos
sy o
paqu
e 72
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
11
-1
MA
su
rfac
e fla
t glo
ssy
lt ye
llow
ish
opaq
ue
570
Bac
teriu
m K
2-24
0.
96
AY
3454
29
AT0
4-16
4-12
-1
M
A
surf
ace
flat g
loss
y op
aque
off
whi
te p
inki
sh
735
Hal
obac
illus
sp. M
O22
0.
97
AY
5531
13
AT0
4-16
4-13
-2
1/
10 P
CA
su
rfac
e gl
ossy
lt p
ink
conv
ex ~
opaq
ue
640
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
4-14
-1
1/
10 P
CA
su
rfac
e gl
ossy
lt p
ink
conv
ex o
paqu
e 77
7 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
16
-1
1/10
PC
A
surf
ace
glos
sy ~
conv
ex o
rang
e/pi
nk o
paqu
e 75
8 M
odes
toba
cter
sp. E
llin1
65
0.98
A
F409
007
AT0
4-16
4-17
-1
1/
10 P
CA
su
rfac
e gl
ossy
flat
opa
que
whi
te
624
Gla
cial
ice
bact
eriu
m G
500K
-9
0.93
A
F479
338
AT0
4-16
4-18
-1
1/
10 P
CA
su
rfac
e gl
ossy
~co
nvex
lt p
ink
opaq
ue
747
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
4-19
-1
1/
10 P
CA
su
rfac
e du
ll ye
llow
flat
opa
que
grow
s in
agar
74
8 St
rept
omyc
es c
hung
whe
nsis
stra
in A
A-9
8
0.99
A
Y38
2292
A
T04-
164-
20
-1
1/10
PC
A
surf
ace
brow
nish
opa
que
flat g
row
ing
arou
nd b
ubbl
e in
aga
r 78
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
164-
21
-3
MA
su
rfac
e of
f whi
te fl
at o
paqu
e gl
ossy
76
6 St
aphy
loco
ccus
epi
derm
idis
0.
99
AY
0303
42
AT0
4-16
4-22
-3
M
A
surf
ace
peac
h gl
ossy
con
vex
~clu
mpy
opa
que
1005
Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
164-
23
-2
MA
su
rfac
e lig
ht o
rang
e/pi
nk c
onve
x op
aque
glo
ssy
768
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-24
-2
M
A
surf
ace
light
yel
low
ish
peac
h co
nvex
clu
mpy
~gl
ossy
opa
que
786
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-25
-1
M
A
surf
ace
~con
vex
with
dip
in th
e ce
nter
lt p
ink
opaq
ue g
loss
y 78
0 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
26
-1
MA
su
rfac
e fla
t opa
que
off w
hite
/yel
low
glo
ssy
964
Hal
obac
illus
sp. M
O22
0.
97
AY
5531
13
AT0
4-16
4-27
-1
M
A
surf
ace
light
pea
ch o
paqu
e cl
umpy
con
vex
760
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
140
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
164-
28
-1
MA
su
rfac
e lig
ht p
ink
opaq
ue c
lum
py c
onve
x 97
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
29
-1
MA
su
rfac
e of
f whi
te ~
dull
conv
ex g
reen
ish
cent
er o
paqu
e 77
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
30
-2
MA
su
rfac
e fla
t opa
que
crea
m g
loss
y 62
5 Ba
cillu
s lito
ralis
0.
97
AY
6086
05
AT0
4-16
4-31
-2
M
A
surf
ace
peac
h co
nvex
opa
que
clum
py
754
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-32
-2
M
A
surf
ace
light
pin
k co
nvex
glo
ssy
opaq
ue
986
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
4-34
-2
M
A
surf
ace
light
pin
k co
nvex
glo
ssy
opaq
ue
763
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-35
-2
M
A
surf
ace
flesh
col
ored
clu
mpy
~gl
ossy
opa
que
conv
ex
739
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-36
-1
M
A
surf
ace
peac
h co
nvex
clu
mpy
opa
que
~glo
ssy
771
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-37
-1
M
A
surf
ace
light
pin
k co
nvex
glo
ssy
opaq
ue
987
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
4-38
-1
M
A
surf
ace
peac
h cl
umpy
~du
ll co
nvex
opa
que
772
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
4-39
-1
M
A
surf
ace
flat g
loss
y ~t
rans
yel
low
77
1 B
acte
rium
K2-
24
0.97
A
Y34
5429
A
T04-
164-
40
-1
MA
su
rfac
e pi
nk c
onve
x op
aque
glo
ssy
774
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
4-41
-1
1/
10 P
CA
su
rfac
e pi
nk g
loss
y op
aque
con
vex
792
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-42
-1
1/
10 P
CA
su
rfac
e pi
nk g
loss
y op
aque
~co
nvex
79
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
164-
43
-1
1/10
PC
A
surf
ace
yello
w g
loss
y op
aque
~co
nvex
77
1 K
ocur
ia e
ryth
rom
yxa
0.
98
Y11
330
AT0
4-16
4-44
-3
1/
10 P
CA
su
rfac
e fla
t whi
te tr
ans g
loss
y 73
7 Sp
hing
omon
as sp
. SIA
181-
1A1
0.
97
AF3
9503
2 A
T04-
164-
45
-2
1/10
PC
A
surf
ace
oran
ge fl
at o
paqu
e gl
ossy
61
1 Sp
hing
omon
as sp
. SIA
181-
1A1
0.
97
AF3
9503
2 A
T04-
164-
46
-2
1/10
PC
A
surf
ace
light
pin
k gl
ossy
opa
que
~con
vex
843
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-47
-2
1/
10 P
CA
su
rfac
e lig
ht p
ink
glos
sy o
paqu
e ~c
onve
x 77
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
48
-4
1/10
PC
A
surf
ace
whi
te tr
ans f
lat
795
Stap
hylo
cocc
us e
pide
rmid
is
0.99
A
Y03
0342
A
T04-
164-
49
-4
1/10
PC
A
surf
ace
pink
glo
ssy
conv
ex o
paqu
e 80
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
50
-1
1/10
PC
A
surf
ace
pink
glo
ssy
opaq
ue ~
conv
ex
774
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-51
-2
1/
100
PCA
sur
face
lt p
k ~t
rans
glo
ssy
conv
ex
764
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
4-52
-2
N
A
surf
ace
pk/o
rang
e tra
ns g
loss
y co
nvex
98
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
164-
53
-2
NA
su
rfac
e or
ange
/tang
erin
e tra
ns g
loss
y co
nvex
68
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
165-
1 -1
M
A
surf
ace
glos
sy o
paqu
e lt
pk c
onve
x 77
2 G
eode
rmat
ophi
lus s
p. B
C51
8
0.99
A
J296
064
AT0
4-16
5-2
-1
MA
su
rfac
e gl
ossy
opa
que
lt ye
llow
ish
crea
m c
onve
x 77
1 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
165-
3 -1
M
A
surf
ace
glos
sy o
paqu
e of
f-w
hite
~co
nvex
74
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
165-
4 -2
M
A
surf
ace
glos
sy c
onve
x lt
pk o
paqu
e 72
9 G
eode
rmat
ophi
lus s
p. B
C51
8
0.98
A
J296
064
AT0
4-16
5-6
-1
MA
su
rfac
e gl
ossy
~co
nvex
opa
que
lt pi
nk
616
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
5-7
-1
MA
su
rfac
e gl
ossy
~co
nvex
opa
que
crea
m
716
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
5-8
-2
MA
su
rfac
e gl
ossy
con
vex
opaq
ue p
inki
sh o
rang
e 64
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
165-
9 -2
M
A
surf
ace
glos
sy c
onve
x op
aque
off
-whi
te c
ream
55
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
165-
10
-1
MA
su
rfac
e gl
ossy
con
vex
opaq
ue lt
pk
709
Geo
derm
atop
hilu
s sp.
BC
518
0.
99
AJ2
9606
4 A
T04-
165-
11
-1
MA
su
rfac
e gl
ossy
opa
que
conv
ex o
ff-w
hite
79
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
165-
12
-2
MA
su
rfac
e gl
ossy
opa
que
conv
ex p
ink
798
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
5-13
-2
M
A
surf
ace
glos
sy o
paqu
e co
nvex
off
-whi
te
779
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
5-14
-2
M
A
surf
ace
glos
sy o
paqu
e co
nvex
lt y
ello
w
766
Cel
lulo
mon
as c
ella
sea
0.
96
X83
804
AT0
4-16
5-15
-2
1/
10 P
CA
su
rfac
e gl
ossy
opa
que
conv
ex lt
pk
784
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
5-16
-2
1/
10 P
CA
su
rfac
e gl
ossy
opa
que
conv
ex lt
pin
k 77
2 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
165-
17
-2
1/10
PC
A
surf
ace
glos
sy o
paqu
e co
nvex
off
-whi
te
773
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
5-18
-1
1/
10 P
CA
su
rfac
e gl
ossy
opa
que
conv
ex c
ream
ish
687
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
5-19
-2
1/
10 P
CA
su
rfac
e gl
ossy
opa
que
conv
ex lt
pin
kish
75
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
165-
20
-1
1/10
PC
A
surf
ace
glos
sy o
paqu
e ~c
onve
x lt
pink
ish
oran
ge
802
Geo
derm
atop
hilu
s sp.
BC
518
0.
98
AJ2
9606
4 A
T04-
165-
21
-1
1/10
PC
A
surf
ace
glos
sy o
paqu
e ~c
onve
x lt
pk
777
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
5-22
-1
1/
10 P
CA
su
rfac
e ~g
loss
y op
aque
con
vex
brow
n 65
3 M
odes
toba
cter
sp. E
llin1
64
0.97
A
F409
006
AT0
4-16
5-23
-1
PC
A
surf
ace
tang
erin
e op
aque
con
vex
glos
sy
958
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
141
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
165-
25
-1
PCA
su
rfac
e ta
nger
ine
opaq
ue c
onve
x gl
ossy
10
23
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
5-27
-2
N
A
surf
ace
tang
erin
e/ta
n op
aque
con
vex
glos
sy
1028
Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
165-
29
-1
NA
su
rfac
e lt
pk/ta
n op
aque
glo
ssy
conv
ex
882
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
5-31
-2
N
A
surf
ace
tan/
peac
h gl
ossy
con
vex
opaq
ue
963
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
5-32
-2
1/
100
PCA
sur
face
lt p
k gl
ossy
con
vex
~tra
ns
783
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-1
-2
MA
su
rfac
e lt
pk g
loss
y bi
g co
nvex
opa
que
751
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
2 -2
M
A
surf
ace
smal
l lt p
k gl
ossy
con
vex
~opa
que
708
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-3
-2
MA
su
rfac
e bi
g bu
bble
gum
pk
~irr
egul
ar g
loss
y ~c
onve
x ~o
paqu
e 73
6 K
ocur
ia sp
. 221
6.35
.31
0.
98
AB
0944
67
AT0
4-16
6-4
-2
MA
su
rfac
e ye
llow
irre
gula
r glo
ssy
med
~op
aque
~co
nvex
72
3 K
ocur
ia e
ryth
rom
yxa
0.
98
Y11
330
AT0
4-16
6-5
-2
MA
su
rfac
e w
hite
~tra
ns g
loss
y co
nvex
77
5 C
ellu
lom
onas
car
tae
MSD
201
06
0.96
X
7945
6 A
T04-
166-
6 -2
M
A
surf
ace
flat s
mal
l pk
glos
sy tr
ans d
arke
r cen
ter
773
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-7
-2
MA
su
rfac
e lt
pk v
ery
~irr
egul
ar ~
dull
rais
ed ~
rigid
opa
que
781
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-8
-2
MA
su
rfac
e pk
irre
gula
r rai
sed
rigid
bum
py ~
trans
~gl
ossy
76
8 G
eorg
enia
sp. 2
216.
35.2
8
0.94
A
B09
4466
A
T04-
166-
9 -2
M
A
surf
ace
lt pk
opa
que
~irr
egul
ar ~
glos
sy c
onve
x bu
t mor
e co
nvex
in c
ente
r 77
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
10
-2
MA
su
rfac
e w
hite
tran
s glo
ssy
conv
ex
775
Cel
lulo
mon
as sp
. CJ1
0597
0.
95
AF5
0020
8 A
T04-
166-
11
-2
MA
su
rfac
e da
rker
pk
glos
sy c
onve
x ~t
rans
73
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
13
-2
MA
su
rfac
e pk
~op
aque
glo
ssy
conv
ex
773
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-14
-2
M
A
surf
ace
lt pk
opa
que
glos
sy c
onve
x 87
5 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
166-
15
-2
MA
su
rfac
e gl
ossy
flat
opa
que
brig
ht y
ello
wis
h w
ith ta
n 85
8 K
ocur
ia e
ryth
rom
yxa
0.
97
Y11
330
AT0
4-16
6-17
-2
M
A
surf
ace
opaq
ue c
ream
ish
clum
py c
onve
x ~d
ull
862
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-19
-2
M
A
surf
ace
glos
sy c
onve
x op
aque
cre
amis
h 86
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
20
-2
MA
su
rfac
e or
ange
/tan
~dul
l ~cl
umpy
~tra
ns c
onve
x 86
1 G
eorg
enia
sp. 2
216.
35.2
8
0.93
A
B09
4466
A
T04-
166-
21
-2
MA
su
rfac
e fla
t whi
te g
loss
y op
aque
86
3 Ar
thro
bact
er sp
. R-2
3173
0.
99
AJ7
8682
1
AT0
4-16
6-22
-2
M
A
surf
ace
hot p
k co
nvex
glo
ssy
~tra
ns
739
Arth
roba
cter
sp. F
a21
0.
99
AY
1312
25
AT0
4-16
6-23
-2
M
A
surf
ace
whi
te ~
opaq
ue g
loss
y co
nvex
86
0 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
24
-2
MA
su
rfac
e lt
pk tr
ans i
rreg
ular
rig
id ra
ised
bum
py
863
Geo
rgen
ia sp
. 221
6.35
.28
0.
93
AB
0944
66
AT0
4-16
6-25
-1
M
A
surf
ace
dark
er p
k/pe
ach
glos
sy c
onve
x da
rker
~tra
ns c
ente
r mor
e tra
ns e
dges
75
8 Bl
asto
cocc
us sp
. BC
521
0.
98
AJ3
1657
3 A
T04-
166-
26
-1
MA
su
rfac
e gl
ossy
flat
but
~co
nvex
hot
salm
on (p
inki
sh) o
paqu
e 77
8 K
ocur
ia e
ryth
rom
yxa
0.
97
Y11
330
AT0
4-16
6-27
-1
M
A
surf
ace
glos
sy fl
at p
each
col
ored
opa
que
761
Koc
uria
ery
thro
myx
a
0.97
Y
1133
0 A
T04-
166-
28
-1
MA
su
rfac
e gl
ossy
~co
nvex
brig
ht y
ello
w o
paqu
e 84
5 K
ocur
ia e
ryth
rom
yxa
0.
97
Y11
330
AT0
4-16
6-31
-1
M
A
surf
ace
flat ~
rais
ed o
ff-w
hite
/cre
am g
loss
y op
aque
circ
le
782
Baci
llus f
irm
us
0.99
D
1626
8 A
T04-
166-
32
-1
MA
su
rfac
e fla
t off
-whi
te/c
ream
glo
ssy
irreg
ular
73
7 B
acill
us fi
rmus
0.
99
D16
268
AT0
4-16
6-33
-1
M
A
surf
ace
flat g
loss
y op
aque
lt o
rang
e go
ld c
olor
77
6 K
ocur
ia e
ryth
rom
yxa
0.
98
Y11
330
AT0
4-16
6-34
-1
M
A
surf
ace
brig
ht sa
lmon
pin
kish
glo
ssy
opaq
ue fl
at c
ircle
s 80
5 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-35
-1
M
A
surf
ace
peac
h co
lore
d gl
ossy
flat
opa
que
circ
les
729
Koc
uria
sp. 2
216.
35.3
1 0.
99
AB
0944
67
AT0
4-16
6-36
-1
M
A
surf
ace
~glo
ssy
~con
vex
gold
ora
nge
blob
opa
que
790
Arth
roba
cter
sp. R
-231
73
0.99
A
J786
821
A
T04-
166-
37
-1
MA
su
rfac
e fla
t opa
que
glos
sy c
ream
col
ored
74
9 Ar
thro
bact
er sp
. R-2
3173
0.
99
AJ7
8682
1
AT0
4-16
6-38
-1
M
A
surf
ace
thin
lt o
rang
e ~g
loss
y la
yer o
ver e
ntire
pla
te
708
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-39
-1
M
A
surf
ace
flat o
paqu
e gl
ossy
brig
ht y
ello
w
404
Koc
uria
ery
thro
myx
a
0.97
Y
1133
0 A
T04-
166-
40
-1
MA
su
rfac
e du
ll co
nvex
opa
que
whi
te d
ots
805
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
41
-1
MA
su
rfac
e ~o
paqu
e ~c
lum
py c
ream
ish
conv
ex
786
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-42
-1
M
A
surf
ace
tiny
glos
sy ~
conv
ex lt
pin
k ci
rcle
s 77
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
43
-1
MA
su
rfac
e gl
ossy
flat
and
rais
ed p
each
col
ored
opa
que
770
Koc
uria
sp. 2
216.
35.3
1
0.98
A
B09
4467
A
T04-
166-
44
-1
MA
su
rfac
e gl
ossy
flat
and
rais
ed b
right
salm
on p
ink
opaq
ue
761
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
45
-1
MA
su
rfac
e du
ll w
hite
opa
que
conv
ex
771
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-46
-1
M
A
surf
ace
flat g
loss
y op
aque
brig
ht y
ello
w
532
Koc
uria
ery
thro
myx
a
0.97
Y
1133
0 A
T04-
166-
47
-1
MA
su
rfac
e br
owni
sh ta
n th
in la
yer
801
Koc
uria
sp. 2
216.
35.3
1
0.98
A
B09
4467
142
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
166-
48
-1
MA
su
rfac
e sm
all g
loss
y ~o
paqu
e co
nvex
lt p
inki
sh
749
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-49
-2
M
A
surf
ace
brig
ht p
ink
glos
sy fl
at o
paqu
e 76
4 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-50
-2
M
A
surf
ace
brig
ht y
ello
w fl
at g
loss
y op
aque
83
8 K
ocur
ia e
ryth
rom
yxa
0.
98
Y11
330
AT0
4-16
6-51
-2
M
A
surf
ace
smal
l cre
amis
h lt
pink
circ
les ~
opaq
ue g
loss
y co
nvex
81
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
52
-1
1/10
PC
A
surf
ace
brig
ht p
ink
glos
sy o
paqu
e ~c
onve
x/fla
t 80
6 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-53
-1
1/
10 P
CA
su
rfac
e lt
peac
h gl
ossy
~op
aque
/flat
opa
que
770
Koc
uria
ery
thro
myx
a
0.97
Y
1133
0 A
T04-
166-
54
-1
1/10
PC
A
surf
ace
dull
~clu
mpy
opa
que
conv
ex w
hite
74
8 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-55
-1
1/
10 P
CA
su
rfac
e or
ange
thin
laye
r cov
erin
g 1/
2 of
pla
te
756
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-56
-1
1/
10 P
CA
su
rfac
e gl
ossy
flat
yel
low
opa
que
772
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
57
-1
1/10
PC
A
surf
ace
dull
~fuz
zy o
paqu
e co
nvex
whi
te
801
Koc
uria
sp. 2
216.
35.3
1
0.98
A
B09
4467
A
T04-
166-
59
-1
1/10
PC
A
surf
ace
glos
sy ~
conv
ex/fl
at p
each
opa
que
753
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
60
-1
1/10
PC
A
surf
ace
glos
sy c
onve
x op
aque
cre
am/o
ffw
hite
76
7 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-61
-1
1/
10 P
CA
su
rfac
e gl
ossy
flat
/~co
nvex
brig
ht p
ink
opaq
ue
740
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
62
-2
1/10
PC
A
surf
ace
glos
sy c
onve
x op
aque
brig
ht p
ink
753
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
63
-2
1/10
PC
A
surf
ace
glos
sy c
onve
x ~t
rans
lt p
ink
76
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
64
-2
1/10
PC
A
surf
ace
glos
sy c
onve
x op
aque
yel
low
74
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
65
-2
1/10
PC
A
surf
ace
glos
sy c
onve
x br
ight
pin
k op
aque
75
6 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-66
-2
1/
10 P
CA
su
rfac
e gl
ossy
con
vex
peac
h op
aque
81
4 K
ocur
ia e
ryth
rom
yxa
0.
98
Y11
330
AT0
4-16
6-67
-2
1/
10 P
CA
su
rfac
e lt
pink
con
vex
~opa
que
glos
sy
761
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-68
-2
1/
10 P
CA
su
rfac
e lt
pink
con
vex
~opa
que
glos
sy
749
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-69
-2
1/
10 P
CA
su
rfac
e gl
ossy
opa
que
conv
ex b
right
yel
low
52
3 K
ocur
ia e
ryth
rom
yxa
0.
97
Y11
330
AT0
4-16
6-70
-1
1/
10 P
CA
su
rfac
e du
ll w
hite
con
vex
opaq
ue
746
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-71
-1
1/
10 P
CA
su
rfac
e br
ight
pin
k gl
ossy
flat
~co
nvex
opa
que
770
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
72
-1
1/10
PC
A
surf
ace
peac
h gl
ossy
~co
nvex
/flat
opa
que
77
8 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-73
-1
1/
10 P
CA
su
rfac
e lt
oran
ge th
in la
yer c
over
ing
plat
e
771
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-74
-2
1/
10 P
CA
su
rfac
e sa
lmon
opa
que
~fla
t glo
ssy
769
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
75
-2
1/10
PC
A
surf
ace
pink
opa
que
~con
vex
glos
sy
991
Arth
roba
cter
agi
lis
0.99
A
J577
725
AT0
4-16
6-77
-2
1/
10 P
CA
su
rfac
e lt
pink
opa
que
conv
ex g
loss
y 75
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
78
-2
1/10
PC
A
surf
ace
brig
ht y
ello
w o
paqu
e co
nvex
glo
ssy
970
Koc
uria
sp. S
26-8
0.
98
DQ
0603
77
AT0
4-16
6-79
-4
1/
10 P
CA
su
rfac
e lig
ht p
ink
glos
sy o
paqu
e ~c
onve
x 76
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
80
-4
1/10
PC
A
surf
ace
light
pin
k gl
ossy
opa
que
~con
vex
776
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-81
-4
1/
10 P
CA
su
rfac
e lig
ht p
ink
glos
sy o
paqu
e ~c
onve
x 72
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
82
-2
1/10
PC
A
surf
ace
pink
glo
ssy
opaq
ue c
onve
x 62
2 B
acte
rium
K2-
25
0.99
A
Y34
5428
A
T04-
166-
83
-2
1/10
PC
A
surf
ace
yello
w g
loss
y op
aque
con
vex
776
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
84
-1
1/10
PC
A
surf
ace
pink
glo
ssy
opaq
ue ~
conv
ex
818
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
85
-1
1/10
PC
A
surf
ace
gold
glo
ssy
opaq
ue ~
conv
ex
863
Koc
uria
aeg
yptia
stra
in Y
IM 7
0003
0.
96
DQ
0596
17
AT0
4-16
6-87
-3
1/
10 P
CA
su
rfac
e lig
ht p
ink
glos
sy o
paqu
e ~c
onve
x 61
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
88
-3
1/10
PC
A
surf
ace
yello
w o
paqu
e gl
ossy
~co
nvex
78
7 N
ocar
dioi
des k
ribb
ensi
s stra
in K
SL-6
0.
93
AY
8359
26
AT0
4-16
6-89
-3
1/
10 P
CA
su
rfac
e lig
ht p
ink
glos
sy o
paqu
e ~c
onve
x 51
0 Bl
asto
cocc
us sp
. BC
521
0.
84
AJ3
1657
3 A
T04-
166-
90
-3
1/10
PC
A
surf
ace
light
pin
k gl
ossy
opa
que
~con
vex
764
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-91
-3
1/
10 P
CA
su
rfac
e lig
ht y
ello
w ~
conv
ex o
paqu
e gl
ossy
61
1 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
166-
92
-1
1/10
PC
A
surf
ace
peac
h gl
ossy
~co
nvex
opa
que
739
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
93
-1
1/10
PC
A
surf
ace
pink
glo
ssy
~con
vex
opaq
ue
702
Arth
roba
cter
sp. M
uzt-C
11
0.99
A
Y52
6639
A
T04-
166-
95
-1
1/10
PC
A
surf
ace
pink
glo
ssy
~con
vex
opaq
ue
823
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
96
-1
1/10
PC
A
surf
ace
oran
ge ~
dull
flat
773
Geo
derm
atop
hilu
s sp.
BC
509
0.
99
AJ2
9606
3 A
T04-
166-
97
-1
1/10
PC
A
surf
ace
gold
glo
ssy
opaq
ue ~
conv
ex
980
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
98
-3
MA
su
rfac
e br
ight
pin
k gl
ossy
opa
que
conv
ex
773
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
143
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
166-
99
-3
MA
su
rfac
e lig
ht p
each
/pin
k op
aque
con
vex
glos
sy
885
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-10
0 -3
M
A
surf
ace
flesh
col
ored
~cl
umpy
con
vex
opaq
ue g
loss
y 97
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
101
-3
MA
su
rfac
e lig
ht p
ink
conv
ex o
paqu
e gl
ossy
75
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
102
-3
MA
su
rfac
e br
ight
yel
low
con
vex
opaq
ue g
loss
y 73
9 C
ellu
lom
onas
car
tae
MSD
201
06
0.
96
X79
456
AT0
4-16
6-10
3 -3
M
A
surf
ace
brig
ht p
ink
conv
ex o
paqu
e gl
ossy
78
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
104
-3
MA
su
rfac
e fle
sh c
lum
py o
paqu
e co
nvex
glo
ssy
766
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-10
5 -4
M
A
surf
ace
pink
/ora
nge
conv
ex o
paqu
e ~g
loss
y 76
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
107
-4
MA
su
rfac
e pe
ach
conv
ex o
paqu
e gl
ossy
76
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
108
-4
MA
su
rfac
e of
f whi
te c
onve
x op
aque
glo
ssy
755
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-10
9 -4
M
A
surf
ace
off w
hite
con
vex
opaq
ue d
ull ~
clum
py
979
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-11
0 -2
M
A
surf
ace
flat b
right
pin
k gl
ossy
opa
que
731
Koc
uria
sp. 2
216.
35.3
1
0.98
A
B09
4467
A
T04-
166-
111
-2
MA
su
rfac
e fla
t brig
ht y
ello
w g
loss
y op
aque
98
4 K
ocur
ia e
ryth
rom
yxa
0.
98
Y11
330
AT0
4-16
6-11
2 -2
M
A
surf
ace
off w
hite
flat
glo
ssy
opaq
ue
749
Arth
roba
cter
sp. R
-231
73
0.99
A
J786
821
AT0
4-16
6-11
3 -2
M
A
surf
ace
peac
h fla
t glo
ssy
opaq
ue
707
Koc
uria
sp. 2
216.
35.3
1
0.98
A
B09
4467
A
T04-
166-
114
-2
MA
su
rfac
e w
hite
opa
que
dull
conv
ex
707
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
6-11
6 -2
M
A
surf
ace
brig
ht p
ink
flat o
paqu
e gl
ossy
73
5 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-11
7 -2
M
A
surf
ace
off w
hite
flat
opa
que
glos
sy
577
Arth
roba
cter
sp. R
-231
73
0.96
A
J786
821
A
T04-
166-
118
-2
MA
su
rfac
e ye
llow
flat
opa
que
glos
sy
760
Koc
uria
ery
thro
myx
a
0.98
Y
1133
0 A
T04-
166-
119
-2
MA
su
rfac
e lig
ht p
ink
glos
sy c
onve
x op
aque
76
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
120
-2
MA
su
rfac
e lig
ht p
ink
glos
sy c
onve
x op
aque
79
9 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
121
-2
MA
su
rfac
e w
hite
dul
l con
vex
opaq
ue
734
Exig
uoba
cter
ium
sp. B
TAH
1
0.99
A
Y20
5564
A
T04-
166-
122
-2
MA
su
rfac
e br
ight
pin
k fla
t glo
ssy
753
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
123
-2
MA
su
rfac
e pe
ach
flat g
loss
y 80
6 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-12
4 -2
M
A
surf
ace
off w
hite
flat
glo
ssy
933
Arth
roba
cter
sp. R
-231
73
0.99
A
J786
821
AT0
4-16
6-12
5 -1
M
A
surf
ace
brig
ht p
ink
glos
sy o
paqu
e ~c
onve
x 87
0 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-12
6 -1
M
A
surf
ace
brig
ht y
ello
w g
loss
y op
aque
~co
nvex
78
9 K
ocur
ia e
ryth
rom
yxa
0.
98
Y11
330
AT0
4-16
6-12
7 -1
M
A
surf
ace
light
pin
k gl
ossy
opa
que
~con
vex
986
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
128
-1
MA
su
rfac
e of
f whi
te g
loss
y op
aque
con
vex
910
Arth
roba
cter
sp. R
-231
73
0.99
A
J786
821
AT0
4-16
6-12
9 -1
M
A
surf
ace
oran
ge ~
glos
sy o
paqu
e ~c
onve
x 75
3 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-13
0 -3
M
A
surf
ace
pink
con
vex
opaq
ue g
loss
y 77
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
132
-3
MA
su
rfac
e or
ange
pin
k co
nvex
opa
que
glos
sy
790
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-13
3 -3
M
A
surf
ace
flesh
con
vex
clum
py o
paqu
e ~g
loss
y 71
6 Bl
asto
cocc
us sp
. BC
448
0.
95
AJ3
1657
1 A
T04-
166-
134
-3
MA
su
rfac
e lig
ht y
ello
w c
onve
x cl
umpy
~tra
ns ~
dull
800
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
6-13
5 -4
M
A
surf
ace
light
pin
k co
nvex
opa
que
glos
sy
765
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-13
6 -4
M
A
surf
ace
pink
con
vex
opaq
ue g
loss
y 80
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
137
-4
MA
su
rfac
e cr
eam
dul
l con
vex
opaq
ue
603
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-13
8 -4
M
A
surf
ace
light
yel
low
opa
que
conv
ex g
loss
y 77
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
139
-1
MA
su
rfac
e pi
nk fl
at g
loss
y op
aque
98
2 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-14
1 -1
M
A
surf
ace
peac
h fla
t glo
ssy
opaq
ue
803
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
142
-1
MA
su
rfac
e tin
y lig
ht p
ink
~con
vex
~glo
ssy
753
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
144
-4
MA
su
rfac
e lig
ht p
ink
conv
ex g
loss
y op
aque
10
09
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-14
5 -1
M
A
surf
ace
yello
w fl
at g
loss
y op
aque
76
3 K
ocur
ia e
ryth
rom
yxa
0.
97
Y11
330
AT0
4-16
6-14
6 -1
M
A
surf
ace
pink
flat
glo
ssy
opaq
ue
774
Koc
uria
sp. 2
216.
35.3
1
0.99
A
B09
4467
A
T04-
166-
147
-1
MA
su
rfac
e pe
ach
flat g
loss
y op
aque
75
4 K
ocur
ia sp
. 221
6.35
.31
0.
99
AB
0944
67
AT0
4-16
6-14
8 -4
N
A
surf
ace
peac
h op
aque
glo
ssy
conv
ex
774
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-14
9 -4
N
A
surf
ace
pk/p
each
opa
que
glos
sy c
onve
x 78
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
150
-3
NA
su
rfac
e tra
ns p
each
/bro
wn
glos
sy ~
conv
ex
732
Blas
toco
ccus
sp. B
C52
1
0.97
A
J316
573
144
(Tab
le c
ontin
ued:
Sur
face
isol
ates
) Su
rfac
e Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
166-
151
-3
NA
su
rfac
e pe
ach
dark
er c
ente
r opa
que
~glo
ssy
conv
ex li
ghte
r edg
es
787
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-15
2 -3
N
A
surf
ace
trans
glo
ssy
conv
ex
496
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
4-16
6-15
3 -3
N
A
surf
ace
crea
m o
ffw
hite
~tra
ns g
loss
y co
nvex
83
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
154
-3
NA
su
rfac
e or
ange
~op
aque
tiny
~gl
ossy
~co
ncav
e 69
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
156
-3
NA
su
rfac
e pe
ach/
pk ~
opaq
ue ~
glos
sy c
onve
x 77
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
166-
157
-3
NA
su
rfac
e pe
ach
~opa
que
~glo
ssy
conv
ex
704
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
6-15
8 -3
N
A
surf
ace
oran
ge ~
opaq
ue ~
glos
sy ~
conc
ave
765
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-15
9 -3
N
A
surf
ace
oran
ge g
loss
y co
nvex
~tra
ns
842
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-16
0 -3
N
A
surf
ace
lt pk
tran
s glo
ssy
conv
ex
787
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-16
2 -4
1/
100
PCA
sur
face
pk
opaq
ue ~
conv
ex ~
glos
sy
488
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-16
6-16
3 -4
1/
100
PCA
sur
face
lt p
k tra
ns g
loss
y co
nvex
(dar
ker i
n ce
nter
tran
s edg
es)
588
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-16
4 -4
1/
100
PCA
sur
face
tra
ns g
loss
y ~l
t pk/
offw
hite
con
vex
641
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-16
5 -4
1/
100
PCA
sur
face
pk
opaq
ue ~
conv
ex ~
glos
sy
819
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-16
6 -4
1/
100
PCA
sur
face
tra
ns g
loss
y ~l
t pk/
offw
hite
con
vex
848
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-16
6-16
7 -3
1/
10 P
CA
su
rfac
e ta
n/or
ange
~op
aque
~gl
ossy
con
vex
roun
d 94
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
168
-3
1/10
PC
A
surf
ace
trans
~fla
t glo
ssy
955
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-16
9 -4
1/
10 P
CA
su
rfac
e bi
g br
ight
ora
nge
glos
sy o
paqu
e !~
irreg
ular
~co
nvex
94
0 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
166-
170
-4
1/10
PC
A
surf
ace
trans
bum
py ro
ugh
rigid
irre
gula
r glo
ssy
952
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-17
1 -3
1/
100
PCA
sur
face
sm
all o
rang
e/pk
tran
s glo
ssy
conv
ex
957
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-16
6-18
5 -1
N
A
surf
ace
hot o
rang
e ~c
onve
x !g
loss
y bi
g op
aque
92
3 K
ocur
ia sp
. 221
6.35
.31
0.
98
AB
0944
67
AT0
4-16
7-1
-1
NA
su
rfac
e irr
egul
ar fl
at ~
trans
cre
amy
offw
hite
glo
ssy
(may
be
cont
amin
atio
n)
1020
Ac
inet
obac
ter r
adio
resi
sten
s 0.
99
X81
666
145
(Tab
le c
ontin
ued:
Pit
isol
ates
) P
it Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
150-
1 -1
M
A
50 c
m
whi
te c
halk
y su
rfac
e op
aque
non
glos
sy ~
irreg
ular
rais
ed ~
bum
py
804
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
0-2
-1
MA
50
cm
w
hite
cha
lky
surf
ace
opaq
ue n
ongl
ossy
~irr
egul
ar ra
ised
~bu
mpy
80
4 Ps
eudo
noca
rdia
satu
rnea
0.
97
AJ2
5282
9 A
T04-
150-
3 -1
M
A
20 c
m
whi
te c
halk
y su
rfac
e op
aque
non
glos
sy ~
irreg
ular
rais
ed ~
bum
py
828
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-4
-1
MA
50
cm
w
hite
cha
lky
surf
ace
opaq
ue n
ongl
ossy
~irr
egul
ar ra
ised
~bu
mpy
99
5 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
150-
5 -1
M
A
40 c
m
whi
te c
halk
y su
rfac
e op
aque
non
glos
sy ~
irreg
ular
rais
ed ~
bum
py
772
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-6
-1
MA
40
cm
of
fwhi
te/ta
n ~g
loss
y ra
ised
~op
aque
bum
py ri
gid
hard
94
1 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
150-
7 -1
M
A
20 c
m
whi
te c
halk
y su
rfac
e op
aque
non
glos
sy ~
irreg
ular
rais
ed ~
bum
py
736
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-8
-1
MA
40
cm
br
ight
yel
low
opa
que
glos
sy ~
conv
ex
1020
M
icro
cocc
us lu
teus
0.
99
AJ4
0909
6 A
T04-
150-
9 -1
M
A
30 c
m
whi
te c
halk
y op
aque
dul
l irr
egul
ar ra
ised
bum
py ri
gid
tan
base
gro
ws i
nto
agar
88
3 Ps
eudo
noca
rdia
satu
rnea
0.
97
AJ2
5282
9 A
T04-
150-
10
-1
MA
30
cm
br
ight
mus
tard
yel
low
with
bro
wni
sh c
ente
r glo
ssy
conv
ex o
paqu
e 69
9 N
ocar
dioi
des s
p. G
WS-
BW
-H31
1M
0.98
A
Y33
2121
A
T04-
150-
11
-1
MA
30
cm
w
hite
opa
que
conv
ex g
loss
y 86
1 N
ocar
dioi
des s
p. 4
P1-A
0.
96
AY
0275
87
AT0
4-15
0-12
-1
M
A
30 c
m
oran
ge o
paqu
e ~g
loss
y co
nvex
but
cen
ter o
f col
ony
is !c
onve
x (li
ke d
ome)
77
9 M
ycob
acte
rium
sp. P
X3.
15
0.99
A
Y33
7605
A
T04-
150-
13
-1
MA
30
cm
w
hite
opa
que
conv
ex g
loss
y 81
7 N
ocar
dioi
dace
ae st
r. El
lin10
1 0.
96
AF4
0894
3 A
T04-
150-
14
-1
MA
30
cm
br
ight
yel
low
glo
ssy
conv
ex o
paqu
e 80
8 N
osto
coid
a ar
omat
ivor
a 0.
96
AY
6823
81
AT0
4-15
0-15
-2
M
A
40 c
m
whi
te c
halk
y du
ll irr
egul
ar ra
ised
opa
que
bum
py ri
gid
tan
base
in a
gar
855
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-16
-1
M
A
40 c
m
whi
te c
halk
y du
ll irr
egul
ar ra
ised
opa
que
bum
py ri
gid
tan
base
in a
gar
997
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
0-17
-1
M
A
40 c
m
offw
hite
/tan
~glo
ssy
rais
ed ~
opaq
ue b
umpy
rigi
d ha
rd b
ase
in a
gar
890
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-18
-2
M
A
40 c
m
tan
dull
rais
ed ~
opaq
ue w
hite
cha
lky
spot
s on
surf
ace
bum
py h
ard
in a
gar
877
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
0-19
-1
M
A
30 c
m
whi
te c
halk
y su
rfac
e du
ll irr
egul
ar ra
ised
opa
que
rigid
tan
base
in a
gar
882
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-21
-1
M
A
10 c
m
whi
te c
halk
y su
rfac
e du
ll irr
egul
ar ra
ised
opa
que
rigid
tan
base
in a
gar
966
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-24
-2
1/
10 P
CA
30
cm
pk
/ora
nge
~tra
ns g
loss
y co
nvex
87
9 G
eode
rmat
ophi
lus s
p.
0.98
X
9235
8 A
T04-
150-
25
-2
1/10
PC
A
30 c
m
oran
ge v
ery
conv
ex g
loss
y co
nvex
87
8 U
ncul
t soi
l bac
teriu
m c
lone
0.
96
AF4
2327
7 A
T04-
150-
27
-1
PCA
80
cm
pk
con
vex
~glo
ssy
roug
h sm
all b
umps
on
colo
ny ~
opaq
ue
873
Cur
toba
cter
ium
flac
cum
faci
ens
0.99
A
Y27
3210
A
T04-
150-
28
-1
PCA
80
cm
br
ight
mus
tard
yel
low
glo
ssy
~opa
que
flat i
rreg
ular
88
1 C
urto
bact
eriu
m fl
accu
mfa
cien
s 0.
98
AY
2732
10
AT0
4-15
0-29
-1
PC
A
70 c
m
brig
ht su
nshi
ne y
ello
w ~
dull
opaq
ue v
ery
conv
ex
869
Mic
roco
ccus
sede
ntar
ius
0.99
X
8775
5 A
T04-
150-
30
-1
PCA
70
cm
ta
n ~i
rreg
ular
opa
que
edge
s rin
g ar
ound
tran
s fla
t glo
ssy
cent
er w
ith sp
ots
414
Stap
hylo
cocc
us sp
. Tf 2
54
0.97
A
Y46
1700
A
T04-
150-
31
-1
PCA
70
cm
ta
n ~i
rreg
ular
opa
que
edge
s rin
g ar
ound
tran
s fla
t glo
ssy
cent
er w
ith sp
ots
727
Stap
hylo
cocc
us sp
. 16b
-5A
0.
99
AY
5615
56
AT0
4-15
0-32
-1
PC
A
70 c
m
tan
~irr
egul
ar o
paqu
e ed
ges r
ing
arou
nd tr
ans f
lat g
loss
y ce
nter
with
spot
s 73
4 M
ethy
loba
cter
ium
mes
ophi
licum
0.
98
AJ4
0091
9 A
T04-
150-
33
-1
PCA
40
cm
lt
pk ~
trans
glo
ssy
smal
l bum
ps o
n co
lony
~co
nvex
89
8 C
hela
toco
ccus
asa
ccha
rovo
rans
0.
98
AY
1678
39
AT0
4-15
0-34
-1
PC
A
40 c
m
lt pk
~tra
ns g
loss
y sm
all b
umps
on
colo
ny ~
conv
ex
900
Che
lato
cocc
us a
sacc
haro
vora
ns
0.97
A
Y16
7839
A
T04-
150-
37
-1
NA
10
cm
lt
pk/ta
n ve
ry g
loss
y co
nvex
~op
aque
91
0 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
150-
38
-2
NA
10
cm
bi
g irr
egul
ar ri
gid
~opa
que
glos
sy ~
flat y
ello
wis
h irr
egul
ar c
ente
r ~ta
n ed
ges
889
Baci
llus s
p. F
a29
16S
0.
98
AY
1312
22
AT0
4-15
2-1
-1
MA
10
cm
of
fwhi
te/ta
n ra
ised
~gl
ossy
~op
aque
~bu
mpy
~irr
egul
ar
895
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-2
-1
MA
10
cm
of
fwhi
te ~
trans
flat
dul
l edg
es p
urpl
e/br
own
near
ripp
led
whi
te fu
zzy
cent
er
785
Stre
ptom
yces
sp. I
M-7
082
0.
97
AF1
3154
9 A
T04-
152-
3 -1
M
A
10 c
m
chal
ky/w
hite
opa
que
dull
rais
ed ri
gid
surf
ace
offw
hite
bas
e gr
ows i
n ag
ar
792
Stre
ptom
ycet
acea
e
0.97
X
8732
0 A
T04-
152-
4 -1
M
A
10 c
m
whi
te c
halk
y su
rfac
e op
aque
~bu
mpy
rais
ed d
ull o
ffw
hite
bas
e gr
ows i
n ag
ar
777
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
2-5
-1
MA
10
cm
of
fwhi
te ~
trans
flat
dul
l edg
es p
urpl
e/br
own
near
ripp
led
whi
te fu
zzy
cent
er
800
Stre
ptom
yces
sp. I
M-7
082
0.97
A
F131
549
AT0
4-15
2-6
-1
MA
10
cm
of
f whi
te/ta
n ra
ised
~gl
ossy
~op
aque
~bu
mpy
~irr
egul
ar
889
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
2-7
-1
MA
10
cm
ch
alky
/whi
te o
paqu
e bu
mpy
dul
l rai
sed
cent
er ta
n op
aque
dul
l fla
t bas
e in
aga
r 79
0 St
rept
omyc
etac
eae
0.
95
X87
320
AT0
4-15
2-8
-1
MA
30
cm
ch
alky
/whi
te ~
irreg
ular
dul
l opa
que
bum
py o
ffw
hite
/tan
base
gro
ws i
n ag
ar
797
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-9
-1
MA
30
cm
of
f whi
te/ta
n ra
ised
~gl
ossy
~op
aque
smal
l. ~i
rreg
ular
88
0 Ps
eudo
noca
rdia
satu
rnea
0.
97
AJ2
5282
9 A
T04-
152-
10
-3
MA
10
cm
ch
alky
/whi
te d
ull o
paqu
e ~c
onve
x ~b
umpy
cen
ter t
an e
dges
bas
e in
aga
r 52
7 St
rept
omyc
es sp
. IM
-703
4
0.89
A
F131
546
AT0
4-15
2-11
-3
M
A
10 c
m
81
0 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-12
-3
M
A
10 c
m
~cha
lky/
whi
te d
ull ~
opaq
ue ra
ised
~irr
egul
ar ri
gid
tan
~dul
l bas
e in
aga
r 79
6 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
13
-3
MA
10
cm
of
fwhi
te/ta
n ra
ised
dul
l ~op
aque
~bu
mpy
~irr
egul
ar w
ith w
hite
cha
lky
in c
ente
r 79
9 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
14
-3
MA
10
cm
of
f whi
te/ta
n ra
ised
~gl
ossy
~op
aque
~bu
mpy
~irr
egul
ar
794
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-15
-1
M
A
20 c
m
chal
ky/w
hite
dul
l opa
que
surf
ace
rais
ed ~
irreg
ular
bum
py ta
n ba
se in
aga
r 77
9 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9
146
(Tab
le c
ontin
ued:
Pit
isol
ates
) P
it Is
olat
e
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
152-
16
-1
MA
20
cm
ch
alky
/whi
te d
ull o
paqu
e su
rfac
e ra
ised
~irr
egul
ar b
umpy
tan
base
in a
gar
785
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-17
-2
M
A
10 c
m
offw
hite
~tra
ns fl
at e
dges
pur
ple/
brow
n ne
ar ri
pple
d du
ll ~f
uzzy
rais
ed c
ente
r 77
7 St
rept
omyc
es sp
. IM
-708
2
0.97
A
F131
549
AT0
4-15
2-18
-2
M
A
10 c
m
chal
ky/w
hite
dul
l opa
que
~rai
sed
rigid
surfa
ce &
tan
edge
s bas
e in
aga
r 77
7 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-19
-1
M
A
10 c
m
tan
~tra
ns fl
at e
dges
pur
ple/
brow
n ne
ar ri
pple
d du
ll w
hite
fuzz
y ra
ised
cen
ter
767
Stre
ptom
yces
sp. I
M-7
082
0.
97
AF1
3154
9 A
T04-
152-
21
-1
MA
10
cm
w
hite
/cha
lky
~rai
sed
dull
opaq
ue ro
ugh
cent
er &
tan
~dul
l edg
es in
aga
r 75
0 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-22
-1
M
A
10 c
m
tan
~dul
l opa
que
irreg
ular
rais
ed (s
ome
rippl
es c
halk
y w
hite
opa
que
dull)
76
3 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-23
-1
M
A
10 c
m
offw
hite
/tan
conv
ex ~
glos
sy ~
trans
~bu
mpy
~irr
egul
ar
1004
Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
24
-2
MA
10
cm
ta
n ra
ised
edg
es c
onca
ve c
ente
r ~gl
ossy
~tra
ns ~
bum
py e
dges
~irr
egul
ar
762
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-25
-2
M
A
10 c
m
whi
te/c
halk
y op
aque
dul
l bum
py e
dges
con
cave
irre
gula
r tan
bas
e in
aga
r 76
6 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
26
-2
MA
10
cm
ta
n ~t
rans
flat
dul
l edg
e pu
rple
/bro
wn
near
opa
que
rippl
ed w
hite
cha
lky
cent
er
748
Stre
ptom
yces
late
ritiu
s 0.
97
AF4
5476
4 A
T04-
152-
27
-2
MA
10
cm
ta
n ba
se ~
irreg
ular
~op
aque
dul
l in
agar
with
cha
lky
whi
te o
n so
me
of su
rfac
e
753
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-28
-2
M
A
10 c
m
offw
hite
/tan
rais
ed ~
conv
ex ~
glos
sy ~
opaq
ue ~
bum
py ~
irreg
ular
75
3 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
29
-2
MA
10
cm
w
hite
/cha
lky
opaq
ue d
ull r
ippl
ed ra
ised
irre
gula
r rou
gh ta
n ~t
rans
bas
e in
aga
r 10
35
Stre
ptom
yces
sp. 8
0134
0.
98
AY
9968
29
AT0
4-15
2-30
-1
M
A
60 c
m
oran
ge ~
trans
glo
ssy
conv
ex
878
Myc
obac
teri
um sa
crum
BN
315
1
0.97
A
Y23
5429
A
T04-
152-
31
-1
MA
60
cm
ye
llow
~op
aque
glo
ssy
conv
ex
762
Mic
roco
ccus
sp. E
llin1
49
0.99
A
F408
991
AT0
4-15
2-32
-2
M
A
70 c
m
crea
m ta
n/br
own
~con
vex
glos
sy ~
opaq
ue d
arke
r in
cent
er
875
Noc
ardi
oide
s OS4
0.
97
U61
298
AT0
4-15
2-33
-1
M
A
20 c
m
whi
te/c
halk
y op
aque
irre
gula
r dul
l bum
py ra
ised
gre
en/b
row
n ba
se in
aga
r 88
6 Ps
eudo
noca
rdia
satu
rnea
0.
96
AJ2
5282
9 A
T04-
152-
34
-1
MA
30
cm
w
hite
/cha
lky
opaq
ue ir
regu
lar d
ull b
umpy
rais
ed e
dges
con
cave
bas
e in
aga
r 89
6 Ps
eudo
noca
rdia
satu
rnea
0.
97
AJ2
5282
9 A
T04-
152-
35
-1
MA
30
cm
w
hite
/cha
lky
opaq
ue ~
irreg
ular
roug
h du
ll bu
mpy
rais
ed w
ith ta
n ba
se in
aga
r 88
3 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
36
-1
MA
20
cm
w
hite
opa
que
chal
ky ir
regu
lar r
igid
dul
l rai
sed
edge
s con
cave
tan
base
in a
gar
885
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
2-37
-1
M
A
20 c
m
tan
base
in a
gar f
uzzy
whi
te o
paqu
e irr
egul
ar d
ull r
igid
gre
en/b
row
n be
low
74
6 St
rept
omyc
es sp
. AS
4.11
82
0.98
A
Y11
4179
A
T04-
152-
38
-1
MA
20
cm
w
hite
opa
que
chal
ky ~
irreg
ular
dul
l bum
py ra
ised
with
tan
base
in a
gar
879
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
2-39
-1
M
A
40 c
m
tan/
offw
hite
~gl
ossy
rais
ed ~
opaq
ue b
umpy
rigi
d ha
rd ir
regu
lar b
ase
in a
gar
877
Amyc
olat
opsi
s sp.
1B
dz
0.97
A
F479
268
AT0
4-15
2-41
-2
1/
10 P
CA
10
cm
ch
alky
~irr
egul
ar ri
ngs t
owar
d ce
nter
~fla
t opa
que
dull
trans
whi
te b
ase
in a
gar
897
Stre
ptom
ycet
acea
e
0.96
X
8732
0 A
T04-
152-
42
-2
1/10
PC
A
10 c
m
chal
ky ~
irreg
ular
ring
s tow
ard
cent
er ~
flat o
paqu
e du
ll tra
ns w
hite
bas
e in
aga
r 91
3 St
rept
omyc
etac
eae
0.
96
X87
320
AT0
4-15
2-43
-1
1/
10 P
CA
10
cm
ch
alky
~irr
egul
ar ri
ngs t
owar
d ce
nter
~fla
t opa
que
dull
trans
whi
te b
ase
in a
gar
901
Stre
ptom
ycet
acea
e
0.96
X
8732
0 A
T04-
152-
44
-1
1/10
PC
A
10 c
m
tan
trans
irre
gula
r edg
e in
aga
r gre
en/b
row
n ce
nter
cha
lky
whi
te v
eins
cen
ter
901
Stre
ptom
yces
sp. L
K13
23.3
0.
96
AY
4653
12
AT0
4-15
2-45
-1
1/
10 P
CA
10
cm
of
fwhi
te tr
ans ~
irreg
ular
edg
es in
aga
r tan
cha
lky
cent
er o
paqu
e du
ll 83
3 A
ctin
obac
teriu
m R
G-5
1
0.97
A
Y56
1610
A
T04-
152-
46
-1
1/10
PC
A
10 c
m
offw
hite
/cre
am sm
all g
loss
y ~o
paqu
e co
nvex
89
9 St
rept
omyc
etac
eae
0.
96
X87
320
AT0
4-15
2-47
-1
1/
10 P
CA
10
cm
tra
ns o
ffw
hite
~irr
egul
ar e
dges
gro
wn
in a
gar w
hite
cha
lky
cent
er o
paqu
e du
ll 88
7 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-48
-1
1/
10 P
CA
10
cm
sm
all t
rans
off
whi
te ~
irreg
ular
edg
es in
aga
r whi
te c
halk
y ce
nter
opa
que
dull
90
7 A
ctin
obac
teriu
m R
G-5
1
0.97
A
Y56
1610
A
T04-
152-
49
-1
1/10
PC
A
10 c
m
tan
base
in a
gar d
ull i
rreg
ular
rais
ed ri
gid
opaq
ue w
hite
cha
lky
spot
at c
ente
r 87
9 Ps
eudo
noca
rdia
pet
role
ophi
la
0.96
A
J252
828
AT0
4-15
2-50
-1
1/
10 P
CA
10
cm
ch
alky
~irr
egul
ar ri
ngs t
owar
d ce
nter
flat
opa
que
dull
trans
whi
te b
ase
in a
gar
876
Stre
ptom
ycet
acea
e
0.96
X
8732
0 A
T04-
152-
51
-1
1/10
PC
A
10 c
m
lt pk
/pea
ch ~
opaq
ue g
loss
y co
nvex
84
6 Bl
asto
cocc
us sp
. BC
448
0.
92
AJ3
1657
1 A
T04-
152-
52
-1
1/10
PC
A
10 c
m
offw
hite
smal
l ~op
aque
glo
ssy
conv
ex
876
Blas
toco
ccus
sp. B
C44
8
0.86
A
J316
571
AT0
4-15
2-53
-1
1/
10 P
CA
10
cm
sm
all w
hite
cha
lky
opaq
ue d
ull s
urfa
ce ~
irreg
ular
col
onie
s tan
bas
e in
aga
r 88
1 Ps
eudo
noca
rdia
pet
role
ophi
la
0.97
A
J252
828
AT0
4-15
2-54
-1
1/
10 P
CA
10
cm
bi
g tra
ns e
dges
in a
gar w
ith lt
bro
wn
~tra
ns c
ente
r (ce
nter
in a
gar a
lso)
84
0 A
ctin
obac
teriu
m R
G-5
1
0.95
A
Y56
1610
A
T04-
152-
55
-1
1/10
PC
A
40 c
m
brig
ht d
ark
oran
ge ~
opaq
ue ~
irreg
ular
glo
ssy
conv
ex ~
bum
py
877
Sphi
ngom
onas
sp. S
AFR
-028
0.
98
AY
1678
33
AT0
4-15
2-56
-1
1/
10 P
CA
40
cm
br
ight
dar
k or
ange
~op
aque
~irr
egul
ar g
loss
y co
nvex
~bu
mpy
88
8 Sp
hing
omon
as sp
. SA
FR-0
28
0.97
A
Y16
7833
A
T04-
152-
57
-1
1/10
PC
A
40 c
m
brig
ht d
ark
oran
ge ~
opaq
ue ~
irreg
ular
glo
ssy
conv
ex ~
bum
py
899
Sphi
ngom
onas
sp. S
AFR
-028
0.
98
AY
1678
33
AT0
4-15
2-58
-1
1/
10 P
CA
10
cm
w
hite
/cha
lky
~irr
egul
ar ri
ngs t
owar
d ce
nter
flat
opa
que
dull
~tra
ns b
ase
in a
gar
910
Stre
ptom
ycet
acea
e
0.96
X
8732
0 A
T04-
152-
59
-1
1/10
PC
A
10 c
m
whi
te c
halk
y ce
nter
rais
ed ~
trans
bas
e in
aga
r 86
2 St
rept
omyc
etac
eae
0.
96
X87
320
AT0
4-15
2-60
-1
1/
10 P
CA
10
cm
ta
n sm
all ~
irreg
ular
rais
ed ~
dull
~opa
que
782
Actin
obis
pora
xin
jiang
ensi
s 0.
98
AF3
2572
8 A
T04-
152-
62
-1
PCA
10
cm
of
fwhi
te ~
opaq
ue v
einy
~du
ll irr
egul
ar b
ase
in a
gar &
"vie
ns"
abov
e su
rfac
e 89
8 A
ctin
obac
teriu
m R
G-5
1
0.96
A
Y56
1610
A
T04-
152-
69
-1
PCA
10
cm
tra
ns ~
dull
in a
gar c
rack
ed in
x e
dges
off
whi
te d
ull o
paqu
e bu
mps
aro
und
899
Stre
ptom
ycet
acea
e
0.95
X
8732
0 A
T04-
152-
70
-1
PCA
10
cm
tra
ns ~
glos
sy in
aga
r cra
cked
in x
edg
es o
ffwhi
te ~
dull
opaq
ue b
umps
aro
und
854
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
2-72
-1
PC
A
10 c
m
big
irreg
ular
tran
s tan
rigi
d ~g
loss
y co
ncav
e an
d ba
se in
aga
r 89
5 A
ctin
obac
teriu
m R
G-5
1
0.97
A
Y56
1610
A
T04-
152-
73
-2
PCA
10
cm
of
fwhi
te ~
trans
~du
ll bu
mpy
edg
e ce
nter
whi
te c
halk
y op
aque
dul
l rai
sed
ridge
s 76
8 St
rept
omyc
etac
eae
0.
96
X87
320
147
(Tab
le c
ontin
ued:
Pit
isol
ates
) Pi
t Iso
late
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
152-
74
-1
PCA
10
cm
tra
ns ~
glos
sy c
rack
ed in
x e
dges
off
whi
te ~
dull
opaq
ue b
umps
in a
gar
909
Stre
ptom
ycet
acea
e
0.96
X
8732
0 A
T04-
152-
75
-1
PCA
10
cm
bi
g irr
egul
ar ~
opaq
ue ta
n rig
id ~
dull
in a
gar r
aise
d on
ridg
es o
r "ve
ins"
86
8 A
ctin
obac
teriu
m R
G-5
1
0.97
A
Y56
1610
A
T04-
152-
76
-2
PCA
10
cm
tra
ns ~
irreg
ular
rigi
d du
ll ed
ges r
aise
d w
hite
cha
lky
vein
y op
aque
cen
ter
888
Act
inob
acte
rium
RG
-51
0.
96
AY
5616
10
AT0
4-15
2-77
-2
PC
A
10 c
m
trans
~irr
egul
ar ri
gid
dull
edge
s rai
sed
whi
te c
halk
y ve
iny
opaq
ue c
ente
r 89
3 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-78
-2
PC
A
10 c
m
brow
n ve
ins ~
dull
rais
ed o
paqu
e bu
mps
on
surf
ace
~tra
ns ~
irreg
ular
in a
gar
817
Act
inob
acte
rium
RG
-51
0.
98
AY
5616
10
AT0
4-15
2-79
-2
PC
A
10 c
m
offw
hite
vei
ns (m
ore
in c
ente
r) ~
dull
rais
ed o
paqu
e gr
ows i
n ag
ar ~
irreg
ular
83
4 A
ctin
obac
teriu
m R
G-5
1
0.98
A
Y56
1610
A
T04-
152-
80
-2
PCA
10
cm
tra
ns c
onca
ve ~
dull
rigid
irre
gula
r cha
lky
yello
w v
eins
opa
que
bum
ps a
roun
d 79
5 St
rept
omyc
etac
eae
0.
96
X87
320
AT0
4-15
2-81
-2
PC
A
30 c
m
flat t
rans
off
-whi
te ir
regu
lar g
loss
y 86
6 St
aphy
loco
ccus
epi
derm
idis
0.
98
AY
0303
42
AT0
4-15
2-82
-2
PC
A
surf
ace
brig
ht y
ello
w b
ig g
loss
y co
nvex
~op
aque
87
9 U
ncul
t act
inob
acte
rium
clo
ne
0.98
A
Y36
0679
A
T04-
152-
83
-1
PCA
10
cm
of
fwhi
te o
paqu
e rip
pled
(vei
ny) ~
dull
irreg
ular
bas
e in
aga
r "ve
ins"
on
surf
ace
901
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
2-85
-1
PC
A
10 c
m
offw
hite
tran
s ~gl
ossy
dig
s all
the
way
into
aga
r cen
ter i
s cra
cked
in a
n x
902
Stre
ptom
ycet
acea
e
0.97
X
8732
0 A
T04-
152-
88
-2
1/10
0 PC
A 4
0 cm
or
ange
/yel
low
irre
gula
r glo
ssy
conv
ex ~
trans
& lt
ora
nge
!irre
gula
r edg
es fl
at
881
Pseu
dom
onas
stut
zeri
0.
99
X98
607
AT0
4-15
2-89
-2
1/
100
PCA
40
cm
lt ye
llow
opa
que
conv
ex d
ull m
ed
884
Mic
roco
ccus
sp. E
llin1
49
0.99
A
F408
991
AT0
4-15
2-90
-2
1/
100
PCA
10
cm
whi
te tr
ans i
n ag
ar w
hite
cha
lky/
fuzz
y op
aque
dul
l spo
kes r
adia
te fr
om c
ente
r 83
9 A
ctin
omyc
etal
es b
acte
rium
HPA
66
0.97
D
Q14
4230
A
T04-
152-
91
-2
1/10
0 PC
A 1
0 cm
w
hite
tran
s in
agar
with
few
spot
s of c
halk
y/fu
zzy
opaq
ue d
ull o
n su
rfac
e
787
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
2-93
-1
1/
100
PCA
20
cm
whi
te tr
ans i
n ag
ar w
ith o
ffw
hite
cha
lky
in c
ente
r of c
olon
y in
shap
e of
a st
ar
586
Stre
ptom
yces
sp. A
S 4.
1182
0.
97
AY
1141
79
AT0
4-15
2-10
0 -1
1/
100
PCA
10
cm
whi
te tr
ans i
n ag
ar w
hite
cha
lky/
fuzz
y op
aque
dul
l spo
kes r
adia
te fr
om c
ente
r 81
3 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-10
1 -1
1/
100
PCA
10
cm
whi
te tr
ans i
n ag
ar w
ith w
hite
cha
lky
opaq
ue d
ull r
ing
arou
nd c
ente
r on
agar
60
0 St
rept
omyc
es sp
. 337
F08
0.
98
AB
1243
65
AT0
4-15
2-10
2 -1
1/
100
PCA
10
cm
offw
hite
/tan
base
gro
wn
into
aga
r sm
all w
hite
opa
que
chal
ky/fu
zzy
surf
ace
720
Pseu
dono
card
ia p
etro
leop
hila
0.
97
AJ2
5282
8 A
T04-
152-
103
-1
1/10
0 PC
A 1
0 cm
w
hite
tran
s w
ith sp
ots o
f fuz
zy g
reen
/off
whi
te o
paqu
e du
ll on
surf
ace
806
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
2-10
4 -1
1/
100
PCA
10
cm
whi
te tr
ans i
n ag
ar c
halk
y op
aque
dul
l rin
gs &
spok
es ra
diat
e fro
m c
ente
r 78
8 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-10
5 -1
1/
100
PCA
10
cm
whi
te tr
ans g
row
n in
aga
r sur
face
has
spot
s of w
hite
fuzz
y op
aque
dul
l 90
2 A
ctin
obac
teriu
m R
G-5
1
0.97
A
Y56
1610
A
T04-
152-
106
-1
1/10
0 PC
A 1
0 cm
in
aga
r sur
face
nea
r cen
ter i
rreg
ular
off
whi
te fu
zzy/
chal
ky o
paqu
e du
ll 85
7 A
ctin
obac
teriu
m R
G-5
1
0.97
A
Y56
1610
A
T04-
152-
107
-1
1/10
0 PC
A 1
0 cm
w
hite
cha
lky
opaq
ue d
ull r
ing
arou
nd c
ente
r & w
hite
cha
lky
outs
ide
ring
89
2 St
rept
omyc
etac
eae
0.
96
X87
320
AT0
4-15
2-10
8 -2
1/
100
PCA
10
cm
~tra
ns in
aga
r whi
te c
halk
y sp
ecks
opa
que
dull
(con
vex
big
spec
k in
cen
ter)
75
8 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-10
9 -2
N
A
surf
ace
whi
te ~
opaq
ue g
loss
y fla
t ~irr
egul
ar
794
Stap
hylo
cocc
us e
pide
rmid
is
0.99
A
Y03
0342
A
T04-
152-
111
-1
NA
20
cm
du
ll ra
ised
bum
py w
hite
cha
lky
opaq
ue e
dges
bro
wn
opaq
ue c
ente
r in
agar
75
7 St
rept
omyc
es sp
. AS
4.11
82
0.97
A
Y11
4179
A
T04-
152-
112
-2
NA
10
cm
bi
g ta
n tra
ns ir
regu
lar ~
flat ~
dull
digs
into
aga
r bro
wn
in c
ente
r in
shap
e of
x
739
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
2-11
3 -2
N
A
10 c
m
rais
ed ~
trans
roug
h rig
id b
umpy
off
whi
te ~
dull
~irr
egul
ar
826
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-11
4 -2
N
A
10 c
m
tan
irreg
ular
opa
que
ring
arou
nd c
onca
ve c
ente
r out
side
ring
cha
lky
dull
895
Stre
ptom
yces
sp. A
S 4.
1182
0.
97
AY
1141
79
AT0
4-15
2-11
7 -1
N
A
10 c
m
~irr
egul
ar tr
ans f
lat ~
glos
sy e
dges
whi
te c
halk
y irr
egul
ar ~
rais
ed o
paqu
e du
ll 89
3 St
rept
omyc
etac
eae
0.
96
X87
320
AT0
4-15
2-11
8 -1
N
A
10 c
m
trans
tan
irreg
ular
flat
roug
h rig
id g
loss
y 90
8 A
ctin
obac
teriu
m R
G-5
1
0.96
A
Y56
1610
A
T04-
152-
120
-2
NA
10
cm
irr
egul
ar fl
at o
ffw
hite
spot
s in
agar
& o
n co
lony
cha
lky
opaq
ue d
ull c
ente
r 83
2 St
rept
omyc
es sp
. MS-
037
0.
95
AY
6459
01
AT0
4-15
2-12
1 -2
N
A
10 c
m
irreg
ular
flat
off
whi
te sp
ots i
n ag
ar &
on
colo
ny c
halk
y op
aque
dul
l cen
ter
874
Stre
ptom
ycet
acea
e
0.96
X
8732
0 A
T04-
152-
122
-2
NA
10
cm
br
own
vein
y rip
pled
in x
glo
ssy
opaq
ue in
aga
r irr
egul
ar w
hite
bum
py e
dges
88
5 A
ctin
obac
teriu
m R
G-5
1
0.96
A
Y56
1610
A
T04-
152-
123
-1
NA
10
cm
~o
paqu
e ta
n ~g
loss
y rig
id c
ente
r edg
es v
einy
& ri
pple
d ~f
lat d
igs i
n ag
ar
870
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
2-12
4 -1
N
A
10 c
m
trans
lt b
row
n/ta
n di
g in
to a
gar ~
glos
sy c
rack
ed in
cen
ter
877
Stre
ptom
yces
sp. L
K12
24.4
0.
96
AY
4652
68
AT0
4-15
2-12
5 -1
N
A
10 c
m
lt ye
llow
tran
s cra
cked
& c
aved
in c
ente
r dig
s in
agar
~gl
ossy
88
4 St
rept
omyc
etac
eae
0.
96
X87
320
AT0
4-15
2-12
6 -1
N
A
10 c
m
~opa
que
tan
~glo
ssy
bum
py ri
gid
digs
in a
gar
872
Act
inob
acte
rium
RG
-51
0.
96
AY
5616
10
AT0
4-15
2-12
7 -1
N
A
10 c
m
whi
te c
halk
y op
aque
dul
l rig
id ~
rais
ed c
ente
r tra
ns e
dges
dig
s in
agar
87
1 St
rept
omyc
etac
eae
0.
96
X87
320
AT0
4-15
2-12
9 -1
N
A
10 c
m
brow
n/ta
n tra
ns ~
irreg
ular
rigi
d gl
ossy
~fla
t dig
s in
agar
but
is e
asy
to g
et u
p 88
8 A
ctin
obac
teriu
m R
G-5
1
0.97
A
Y56
1610
A
T04-
153-
1 -2
N
A
10 c
m
flat y
ello
w g
loss
y op
aque
74
9 Br
evib
acill
us a
gri
0.
99
AJ5
8638
8 A
T04-
153-
2 -1
N
A
surf
ace
crea
m c
onve
x gl
ossy
tran
s 76
8 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
3 -1
N
A
surf
ace
light
pin
k co
nvex
glo
ssy
~tra
ns
798
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
3-4
-1
NA
70
cm
pe
ach
conv
ex g
loss
y op
aque
79
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
5 -1
PC
A
20 c
m
dark
pin
k w
rinkl
y co
nvex
opa
que
glos
sy
643
Unc
ult M
ethy
loba
cter
iace
ae b
acte
rium
clo
ne 1
0-3B
a02
0.97
A
Y36
0526
A
T04-
153-
6 -1
PC
A
20 c
m
oran
ge c
onve
x op
aque
glo
ssy
814
Unc
ult A
ctin
omyc
etal
es b
acte
rium
clo
ne E
B10
74
0.96
A
Y39
5393
148
(Tab
le c
ontin
ued:
Pit
isol
ates
) Pi
t Iso
late
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
153-
7 -1
PC
A
20 c
m
brig
ht y
ello
w fl
at g
loss
y op
aque
47
6 C
urto
bact
eriu
m fl
accu
mfa
cien
s pv.
bas
ella
e
0.99
A
Y27
3210
A
T04-
153-
8 -1
PC
A
20 c
m
oran
ge o
paqu
e co
nvex
glo
ssy
~clu
mpy
65
9 Fr
anki
a sp
. (st
rain
AV
N17
s)
0.94
L4
0613
A
T04-
153-
9 -1
PC
A
80 c
m
light
yel
low
opa
que
conv
ex g
loss
y 85
1 M
icro
cocc
us lu
teus
0.
98
AJ4
0909
6 A
T04-
153-
10
-1
1/10
PC
A
surf
ace
whi
te/c
ream
~tra
ns c
lum
py c
onve
x 84
7 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
153-
11
-1
1/10
PC
A
surf
ace
tiny
light
pin
k co
nvex
glo
ssy
772
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
3-12
-1
1/
10 P
CA
su
rfac
e lig
ht p
ink
~tra
ns ~
conv
ex g
loss
y 66
8 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
153-
14
-1
1/10
PC
A
80 c
m
flat l
ight
pin
k tra
ns g
loss
y 67
5 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
153-
15
-1
1/10
PC
A
70 c
m
light
pin
k ~c
onve
x gl
ossy
~tra
ns
763
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
4-15
3-16
-2
1/
10 P
CA
su
rfac
e lig
ht p
ink
conv
ex g
loss
y ~t
rans
79
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
17
-1
1/10
PC
A
surf
ace
light
pin
k co
nvex
glo
ssy
~tra
ns
766
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
3-20
-2
M
A
10 c
m
light
yel
low
~co
nvex
glo
ssy
opaq
ue
814
Unc
ult A
ctin
omyc
etal
es b
acte
rium
clo
ne E
B10
74
0.96
A
Y39
5393
A
T04-
153-
21
-1
MA
su
rfac
e cr
eam
~gl
ossy
con
vex
opaq
ue
789
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-15
3-22
-1
M
A
70 c
m
off w
hite
con
vex
glos
sy ~
trans
77
2 C
ellu
lom
onas
car
tae
MSD
201
06
0.93
X
7945
6 A
T04-
153-
23
-1
MA
70
cm
lig
ht y
ello
w ~
conv
ex g
loss
y op
aque
57
3 M
icro
cocc
us lu
teus
0.
98
AJ4
0909
6 A
T04-
153-
24
-1
MA
80
cm
pe
ach
conv
ex g
loss
y op
aque
84
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
25
-1
MA
20
cm
lig
ht p
ink
conv
ex g
loss
y op
aque
78
7 G
eode
rmat
ophi
lus o
bscu
rus d
icty
ospo
rus
0.98
L4
0621
A
T04-
153-
26
-2
MA
su
rfac
e lig
ht p
ink
trans
clu
mpy
con
vex
~dul
l 79
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
27
-1
MA
su
rfac
e ye
llow
clu
mpy
con
vex
trans
~du
ll 80
7 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
153-
28
-1
MA
su
rfac
e lig
ht p
ink
conv
ex g
loss
y op
aque
82
5 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
153-
29
-1
MA
su
rfac
e w
hite
con
vex
glos
sy o
paqu
e 78
7 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
30
-1
MA
su
rfac
e or
ange
con
vex
glos
sy o
paqu
e 78
6 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
31
-1
MA
su
rfac
e pe
ach
~dul
l clu
mpy
con
vex
trans
83
8 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
32
-2
MA
30
cm
lig
ht p
ink
conv
ex g
loss
y op
aque
98
1 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
159-
1 -3
1/
10 P
CA
su
rfac
e pe
ach/
pk o
paqu
e co
nvex
~du
ll 74
2 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
159-
3 -3
1/
10 P
CA
su
rfac
e da
rker
gre
en/b
row
n ce
nter
opa
que
glos
sy c
onve
x ed
ges ~
trans
whi
te
786
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
98
L406
20
AT0
4-15
9-4
-3
1/10
PC
A
surf
ace
trans
pk/
purp
le g
loss
y co
nvex
58
2 H
ymen
obac
ter s
p. 2
9F
0.96
A
Y64
7897
A
T04-
159-
5 -3
1/
10 P
CA
su
rfac
e tra
ns p
k/re
d gl
ossy
~co
nvex
65
3 H
ymen
obac
ter s
p. 2
9F
0.9
AY
6478
97
AT0
4-15
9-7
-3
1/10
PC
A
surf
ace
lt pk
opa
que
~glo
ssy
conv
ex
750
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
4-15
9-8
-3
1/10
PC
A
surf
ace
smal
l yel
low
opa
que
~glo
ssy
conv
ex
784
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
98
L406
20
AT0
4-15
9-9
-3
1/10
PC
A
surf
ace
brig
ht re
d/or
ange
ver
y co
nvex
cen
ter o
paqu
e ce
nter
~tra
ns e
dges
glo
ssy
555
Che
lato
cocc
us a
sacc
haro
vora
ns st
rain
SA
FR-0
17
0.97
A
Y16
7839
A
T04-
159-
10
-3
1/10
PC
A
surf
ace
lt pk
/pea
ch o
paqu
e gl
ossy
con
vex
749
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
4-15
9-11
-3
1/
10 P
CA
su
rfac
e br
own
glos
sy o
paqu
e co
nvex
64
9 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.96
L4
0620
A
T04-
159-
12
-3
1/10
PC
A
surf
ace
yello
w w
ith c
halk
y du
ll w
hite
con
cave
cen
ter o
paqu
e du
ll 66
6 Ac
tinob
ispo
ra x
injia
ngen
sis
0.97
A
F325
728
AT0
4-15
9-13
-3
1/
10 P
CA
su
rfac
e pa
le p
ink
glos
sy c
onve
x op
aque
74
1 Bl
asto
cocc
us a
ggre
gatu
s 0.
96
AJ4
3019
3 A
T04-
159-
14
-3
1/10
PC
A
surf
ace
brig
ht p
ink
glos
sy ~
trans
flat
49
8 Ar
thro
bact
er sp
. Fa2
1
0.99
A
Y13
1225
A
T04-
159-
15
-3
1/10
PC
A
surf
ace
oran
ge c
onve
x op
aque
glo
ssy
793
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
4-15
9-16
-3
1/
10 P
CA
su
rfac
e cr
eam
/lt y
ello
w ~
glos
sy o
paqu
e co
nvex
78
5 Ar
thro
bact
er sp
. Fa2
1
0.99
A
Y13
1225
A
T04-
159-
17
-3
1/10
PC
A
surf
ace
trans
brig
ht y
ello
w g
loss
y ~c
onve
x 80
6 Ar
thro
bact
er sp
. Fa2
1
0.99
A
Y13
1225
A
T04-
159-
18
-3
1/10
PC
A
surf
ace
crea
m tr
ans g
loss
y co
nvex
91
8 C
ellu
lom
onas
cel
lase
a
0.97
X
8380
4 A
T04-
159-
19
-5
1/10
PC
A
20 c
m
crea
m ~
dull
opaq
ue w
hite
cha
lky
cent
er
631
Unc
ult S
phin
gom
onas
sp. c
lone
BL0
04B
95
0.97
A
Y80
6057
A
T04-
159-
20
-5
1/10
PC
A
20 c
m
lt or
ange
glo
ssy
flat o
paqu
e 89
4 Ps
eudo
noca
rdia
zijin
gens
is
0.97
A
F325
725
AT0
4-15
9-21
-4
1/
10 P
CA
su
rfac
e cr
eam
/lt p
each
opa
que
glos
sy fl
at
770
Blas
toco
ccus
agg
rega
tus
0.98
A
J430
193
AT0
4-15
9-22
-4
1/
10 P
CA
su
rfac
e gl
ossy
dar
k or
ange
opa
que
~con
vex
661
Mod
esto
bact
er sp
. Elli
n165
0.
99
AF4
0900
7 A
T04-
159-
23
-4
1/10
PC
A
surf
ace
lt or
ange
glo
ssy
opaq
ue c
onve
x 67
8 Bl
asto
cocc
us a
ggre
gatu
s 0.
96
AJ4
3019
3 A
T04-
159-
24
-4
1/10
PC
A
surf
ace
lt pk
opa
que
glos
sy c
onve
x 60
3 G
eode
rmat
ophi
lus s
p.
0.97
X
9235
8 A
T04-
159-
25
-4
1/10
PC
A
surf
ace
oran
ge o
paqu
e gl
ossy
flat
48
5 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T04-
159-
26
-4
1/10
PC
A
surf
ace
glos
sy c
ream
opa
que
conv
ex w
ith b
lack
dot
in c
ente
r 76
8 M
odes
toba
cter
sp. E
llin1
65
0.99
A
F409
007
149
(Tab
le c
ontin
ued:
Pit
isol
ates
) Pi
t Iso
late
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
159-
27
-4
1/10
PC
A
surf
ace
lt or
ange
rais
ed b
ut fl
at g
loss
y op
aque
77
8 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T04-
159-
28
-4
1/10
PC
A
surf
ace
crea
m o
paqu
e co
nvex
glo
ssy
540
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
96
L406
20
AT0
4-15
9-29
-4
1/
10 P
CA
su
rfac
e lt
oran
ge o
paqu
e co
nvex
glo
ssy
557
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
4-15
9-30
-4
1/
10 P
CA
su
rfac
e pi
nk g
loss
y fla
t tra
ns
596
Hym
enob
acte
r sp.
29F
0.
96
AY
6478
97
AT0
4-15
9-31
-5
1/
10 P
CA
10
cm
go
ld ~
trans
glo
ssy
opaq
ue
1055
Sp
hing
omon
as sp
. Alp
ha4-
5
0.93
A
Y77
1798
A
T04-
159-
32
-4
1/10
PC
A
surf
ace
crea
m/lt
yel
low
~du
ll op
aque
~co
nvex
/flat
74
8 Bl
asto
cocc
us sp
. BC
421
0.
98
AJ3
1657
4 A
T04-
159-
33
-4
1/10
PC
A
surf
ace
lt pk
~du
ll/~g
loss
y op
aque
~co
nvex
/flat
75
0 M
odes
toba
cter
mul
tisep
tatu
s 0.
98
AJ8
7130
4 A
T04-
159-
34
-4
1/10
PC
A
surf
ace
clum
py g
loss
y tra
ns &
opa
que
yello
w c
onve
x 66
3 Bl
asto
cocc
us sp
. BC
521
0.
97
AJ3
1657
3 A
T04-
159-
35
-4
1/10
PC
A
surf
ace
glos
sy fl
at o
paqu
e cr
eam
77
6 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.98
L4
0620
A
T04-
159-
36
-4
1/10
PC
A
surf
ace
trans
cre
am g
loss
y fla
t 75
3 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.98
L4
0620
A
T04-
159-
37
-4
1/10
PC
A
surf
ace
lt or
ange
glo
ssy
flat o
paqu
e 64
4 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
159-
38
-4
1/10
PC
A
surf
ace
lt pk
~op
aque
glo
ssy
conv
ex
635
Geo
derm
atop
hilu
s sp.
0.
98
X92
358
AT0
4-15
9-39
-5
1/
10 P
CA
su
rfac
e or
ange
flat
opa
que
glos
sy
656
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
4-15
9-40
-5
1/
10 P
CA
su
rfac
e gl
ossy
tran
s with
whi
te c
ente
r ins
ide
flat
713
Paen
ibac
illus
sp. D
SM 1
352
0.95
A
J345
017
AT0
4-15
9-41
-4
1/
10 P
CA
20
cm
of
fwhi
te ~
trans
glo
ssy
cent
er !c
onve
x 59
2 M
esor
hizo
bium
sp. H
B5A
4
0.98
A
J007
857
AT0
4-15
9-42
-5
1/
10 P
CA
10
cm
w
hite
cha
lky
irreg
ular
opa
que
rais
ed d
ull
497
Hon
gia
sp. 3
84G
08
0.98
A
B12
2843
A
T04-
159-
43
-4
1/10
PC
A
20 c
m
gree
n ch
alky
surf
ace
opaq
ue ra
ised
bas
e in
aga
r irr
egul
ar
787
Stre
ptom
yces
sp. N
RR
L 57
99
0.98
A
J391
814
AT0
4-15
9-44
-5
1/
10 P
CA
40
cm
in
aga
r off
whi
te ir
regu
lar ~
opaq
ue
808
Stre
ptom
yces
sp. I
M-7
140
0.
96
AF1
3155
4 A
T04-
159-
45
-4
1/10
PC
A
20 c
m
in a
gar w
hite
cha
lky
opaq
ue d
ull s
urfa
ce ~
rais
ed in
cen
ter
804
Stre
ptom
yces
ple
mor
phus
0.
98
AY
2223
23
AT0
4-15
9-46
-4
1/
10 P
CA
10
cm
as
hy b
row
nish
whi
te ir
regu
lar r
aise
d bu
mpy
roug
h op
aque
dul
l 59
6 A
ctin
obac
teriu
m R
G-5
1
0.98
A
Y56
1610
A
T04-
159-
47
-5
1/10
PC
A
10 c
m
offw
hite
cha
lky
irreg
ular
opa
que
rais
ed d
ull
561
Stre
ptom
yces
alb
idoc
hrom
ogen
es st
rain
AS
4.18
63
0.99
A
Y99
9884
A
T04-
159-
48
-3
1/10
PC
A
40 c
m
gree
n ch
alky
surf
ace
opaq
ue ra
ised
bas
e in
aga
r 85
0 St
rept
omyc
es sp
. NR
RL
5799
0.
98
AJ3
9181
4 A
T04-
159-
49
-4
1/10
PC
A
10 c
m
whi
te c
halk
y su
rfac
e co
nvex
opa
que
dull
base
in a
gar
852
Actin
obis
pora
yun
nane
nsis
0.
97
D85
472
AT0
4-15
9-51
-5
1/
10 P
CA
30
cm
in
aga
r tan
irre
gula
r gre
en c
halk
y/fu
zzy
surf
ace
rais
ed o
paqu
e du
ll 84
8 St
rept
omyc
es sp
. NR
RL
5799
0.
98
AJ3
9181
4 A
T04-
159-
52
-5
1/10
PC
A
30 c
m
in a
gar ~
opaq
ue ir
regu
lar o
ffw
hite
80
8 St
rept
omyc
es ru
tger
sens
is (D
SM 4
0077
T)
0.96
Z7
6688
A
T04-
159-
53
-5
1/10
PC
A
40 c
m
in a
gar ~
opaq
ue ir
regu
lar o
ffw
hite
80
6 St
rept
omyc
es sp
. IM
-714
0
0.96
A
F131
554
AT0
4-15
9-54
-3
1/
10 P
CA
30
cm
gr
een
chal
ky su
rfac
e op
aque
rais
ed b
ase
in a
gar
825
Stre
ptom
yces
car
pine
nsis
stra
in N
RR
L B
-169
21
0.99
A
Y99
9908
A
T04-
159-
55
-3
1/10
PC
A
40 c
m
trans
off
whi
te g
loss
y co
nvex
93
2 U
ncul
t alp
ha p
rote
obac
teriu
m c
lone
AK
YG
1666
0.
95
AY
9217
86
AT0
4-15
9-56
-3
1/
10 P
CA
40
cm
tra
ns o
ffw
hite
glo
ssy
conv
ex
829
Unc
ult a
lpha
pro
teob
acte
rium
clo
ne A
KY
G16
66
0.95
A
Y92
1786
A
T04-
159-
57
-3
1/10
PC
A
20 c
m
gree
n ch
alky
opa
que
bum
py c
onve
x 51
2 St
rept
omyc
es sp
. ATT
508
0.
99
AY
7581
94
AT0
4-15
9-58
-3
1/
10 P
CA
20
cm
of
fwhi
te o
paqu
e gr
ows i
n ag
ar ti
ny p
art g
row
ing
on to
p of
aga
r ~co
nvex
81
8 Pr
omic
rom
onos
pora
aer
olat
a
0.98
A
J487
303
AT0
4-15
9-59
-3
1/
10 P
CA
20
cm
ye
llow
opa
que
conv
ex c
halk
y/~f
uzzy
68
6 M
ycob
acte
rium
cel
atum
0.
92
AJ5
3604
0 A
T04-
159-
60
-3
1/10
PC
A
20 c
m
whi
te o
paqu
e co
nvex
cha
lky
base
in a
gar
744
Stre
ptom
yces
sp. 1
413
0.
95
AY
8769
43
AT0
4-15
9-61
-3
1/
10 P
CA
20
cm
ta
n/ye
llow
flat
in a
gar t
rans
84
9 St
rept
omyc
es sp
. NR
RL
5799
0.
98
AJ3
9181
4 A
T04-
159-
62
-3
1/10
PC
A
20 c
m
crea
m tr
ans g
loss
y co
nvex
66
2 U
ncul
t alp
ha p
rote
obac
teriu
m
0.95
A
J318
115
AT0
4-15
9-63
-3
1/
10 P
CA
20
cm
cr
eam
bas
e in
aga
r with
whi
te o
paqu
e ch
alky
circ
le o
n to
p 61
4 C
ellu
lom
onas
sp. X
7
0.98
A
F060
791
AT0
4-15
9-64
-3
1/
10 P
CA
20
cm
gr
een
chal
ky o
paqu
e ~c
onve
x 82
6 St
rept
omyc
es c
arpi
nens
is st
rain
NR
RL
B-1
6921
0.
98
AY
9999
08
AT0
4-15
9-65
-3
1/
10 P
CA
20
cm
of
fwhi
te c
halk
y ho
les o
n to
p ~c
onve
x op
aque
66
1 A
ctin
obac
teriu
m R
G-5
1
0.98
A
Y56
1610
A
T04-
159-
66
-3
1/10
PC
A
20 c
m
crea
m ~
glos
sy tr
ans f
lat g
row
s in
agar
81
0 St
rept
omyc
es a
lbid
ochr
omog
enes
stra
in A
S 4.
1863
0.
99
AY
9998
84
AT0
4-15
9-67
-3
1/
10 P
CA
su
rfac
e lt
pk ~
opaq
ue g
loss
y co
nvex
84
5 M
odes
toba
cter
sp. E
llin1
65
0.99
A
F409
007
AT0
4-15
9-69
-4
1/
10 P
CA
20
cm
gr
ey ir
regu
lar l
umpy
con
vex
opaq
ue
544
Stre
ptom
yces
sp. A
TT50
8
0.98
A
Y75
8194
A
T04-
159-
71
-4
1/10
PC
A
20 c
m
smal
l whi
te o
paqu
e fla
t rou
nd
627
Stre
ptom
yces
sp. A
TT50
8
0.98
A
Y75
8194
A
T04-
159-
72
-3
1/10
PC
A
surf
ace
pink
~tra
ns g
loss
y co
nvex
roun
d 53
1 Ar
thro
bact
er sp
. Fa2
1
0.97
A
Y13
1225
A
T04-
159-
73
-3
1/10
PC
A
surf
ace
smal
l ora
nge
roun
d gl
ossy
con
vex
opaq
ue
423
Blas
toco
ccus
sp. B
C52
1
0.97
A
J316
573
AT0
4-15
9-74
-3
1/
10 P
CA
su
rfac
e la
rge
brow
n irr
egul
ar fl
at ri
dges
aro
und
edge
63
0 St
rept
omyc
es sp
. AS
4.11
82
0.96
A
Y11
4179
A
T04-
159-
75
-3
1/10
PC
A
surf
ace
tiny
whi
te tr
ans c
onve
x gl
ossy
roun
d 74
7 St
rept
omyc
es sp
. AS
4.11
82
0.97
A
Y11
4179
A
T04-
159-
77
-4
1/10
PC
A
40 c
m
smal
l rou
nd w
hite
tran
s fla
t 53
6 U
ncul
t alp
ha p
rote
obac
teriu
m
0.94
A
J318
115
150
(Tab
le c
ontin
ued:
Pit
isol
ates
) Pi
t Iso
late
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
159-
78
-4
1/10
PC
A
40 c
m
smal
l rou
nd w
hite
tran
s ~fla
t 93
6 U
ncul
t alp
ha p
rote
obac
teriu
m c
lone
AK
YG
1666
0.
95
AY
9217
86
AT0
4-15
9-79
-3
1/
10 P
CA
10
cm
w
hite
cha
lky
text
ure
like
surf
ace
of m
oon
opaq
ue ir
regu
lar r
aise
d du
ll 61
4 A
ctin
obac
teriu
m R
G-5
1
0.98
A
Y56
1610
A
T04-
159-
81
-5
1/10
PC
A
30 c
m
whi
te ~
opaq
ue v
ery
cent
er c
onve
x th
e re
st is
~fla
t ~irr
egul
ar ~
glos
sy
705
Stre
ptom
ycet
acea
e ba
cter
ium
CN
Q71
9
0.98
A
Y46
4544
A
T04-
159-
82
-4
1/10
PC
A
30 c
m
gree
n fu
zzy
opaq
ue d
ull r
aise
d ce
nter
flat
spar
ce e
dges
61
1 St
rept
omyc
es sp
. NR
RL
5799
0.
98
AJ3
9181
4 A
T04-
159-
83
-4
1/10
PC
A
40 c
m
in a
gar t
rans
~cr
eam
y gl
ossy
73
0 St
rept
omyc
es sp
. IM
-714
0
0.95
A
F131
554
AT0
4-15
9-84
-4
1/
10 P
CA
40
cm
gr
een
fuzz
y/ch
alky
opa
que
dull
rais
ed
611
Stre
ptom
yces
sp. N
RR
L 57
99
0.98
A
J391
814
AT0
4-15
9-85
-4
1/
10 P
CA
40
cm
tin
y tra
ns c
ream
y co
nvex
roun
d 84
8 U
ncul
t alp
ha p
rote
obac
teriu
m c
lone
AK
YG
1666
0.
94
AY
9217
86
AT0
4-15
9-86
-3
1/
10 P
CA
40
cm
gr
een
fuzz
y/ch
alky
opa
que
dull
rais
ed
658
Stre
ptom
yces
sp. N
RR
L 57
99
0.97
A
J391
814
AT0
4-15
9-87
-3
1/
10 P
CA
40
cm
sm
all t
rans
~cr
eam
y co
nvex
roun
d 64
7 St
rept
omyc
es sp
. NR
RL
5799
0.
98
AJ3
9181
4 A
T04-
159-
88
-4
1/10
PC
A
20 c
m
big
in a
gar r
ust c
ente
r is ~
rais
ed fr
om a
gar ~
trans
roun
d ~g
loss
y 65
4 Sa
ccha
roth
rix
sp. A
S 4.
1731
0.
96
AY
1356
93
AT0
4-15
9-89
-4
1/
10 P
CA
20
cm
of
fwhi
te in
aga
r mak
es a
gar c
onve
x op
aque
glo
ssy
big
806
Noc
ardi
opsi
s sp.
98-
08-2
90A
0.
97
AF3
2840
8 A
T04-
159-
90
-4
1/10
PC
A
20 c
m
offw
hite
smal
l har
d in
aga
r rou
nd c
onve
x op
aque
85
3 Ac
tinom
adur
a sp
. IM
-622
6
0.97
A
F131
308
AT0
4-15
9-92
-4
1/
10 P
CA
20
cm
of
fwhi
te b
ase
dull
in a
gar w
hite
surf
ace
roug
h rig
id b
umpy
73
4 N
ocar
dioi
dace
ae st
r. IM
-775
7
0.96
A
F131
634
AT0
4-15
9-92
B
-4
1/10
0 PC
A 2
0 cm
536
Unc
ult S
phin
gom
onas
sp. c
lone
BL0
04B
95
0.98
A
Y80
6057
A
T04-
159-
93
-3
1/10
PC
A
30 c
m
trans
glo
ssy
roun
d sm
all c
onve
x ce
nter
is w
hite
84
0 U
ncul
t alp
ha p
rote
obac
teriu
m c
lone
AK
YG
1666
0.
95
AY
9217
86
AT0
4-15
9-94
-4
1/
10 P
CA
20
cm
tra
ns ta
n gl
ossy
con
vex
1057
Sp
hing
omon
as sp
. Alp
ha4-
5
0.93
A
Y77
1798
A
T04-
159-
97
-3
1/10
PC
A
30 c
m
whi
te ti
ny c
halk
y rin
g ar
ound
cen
ter i
n ag
ar d
ull o
paqu
e ro
und
490
Stre
ptom
yces
sp. 4
31E1
1
0.98
A
B12
3477
A
T04-
159-
100
-3
1/10
PC
A
20 c
m
flat ~
trans
off
whi
te g
loss
y 56
3 Ba
cillu
s sp.
IDA
0663
0.
97
AJ5
6353
3 A
T04-
159-
102
-3
1/10
PC
A
20 c
m
med
off
whi
te ~
trans
glo
ssy
852
Rhiz
obiu
m sp
. H-4
0.
97
AF2
7988
9 A
T04-
159-
104
-3
1/10
PC
A
20 c
m
~irr
egul
ar d
ull c
ente
r is w
hite
cha
lky
~opa
que
712
Noc
ardi
oida
ceae
str.
IM-7
757
0.
97
AF1
3163
4 A
T04-
159-
106
-5
NA
su
rfac
e lt
pk/ta
n m
ed o
paqu
e co
nvex
glo
ssy
667
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
4-15
9-10
7 -5
N
A
20 c
m
offw
hite
rais
ed o
paqu
e ~i
rreg
ular
not
smoo
th ~
glos
sy
707
Actin
obis
pora
yun
nane
nsis
0.
95
D85
472
AT0
4-15
9-11
2 -4
N
A
surf
ace
whi
te ~
trans
glo
ssy
conv
ex c
ente
r is b
umpy
66
1 M
odes
toba
cter
sp. E
llin1
65
0.97
A
F409
007
AT0
4-15
9-11
3 -4
N
A
surf
ace
lt pk
/tan
~opa
que
glos
sy c
onve
x 88
0 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
159-
114
-4
NA
su
rfac
e bi
g ho
t pk/
hot p
each
opa
que
~con
vex
text
ured
bum
py &
rigi
d ~i
rreg
ular
68
6 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
159-
116
-4
NA
10
cm
!ir
regu
lar w
hite
cha
lky
surf
ace
base
in a
gar d
ull o
paqu
e 68
2 Ac
tinob
acte
rium
RG
-51
0.
98
AY
5616
10
AT0
4-15
9-11
8 -4
N
A
surf
ace
yello
w ~
opaq
ue g
loss
y !c
onve
x 64
7 C
ellu
losi
mic
robi
um fu
nkei
isol
ate
W6
0.
93
AY
7299
60
AT0
4-15
9-11
9 -4
N
A
surf
ace
hot p
k bi
g ~i
rreg
ular
glo
ssy
~con
vex
~opa
que
850
Arth
roba
cter
agi
lis
0.99
A
J577
725
AT0
4-15
9-12
0 -4
N
A
surf
ace
pk/p
each
~du
ll bu
mpy
rigi
d ro
ugh
~opa
que
conv
ex
610
Mod
esto
bact
er m
ultis
epta
tus
0.97
A
J871
304
AT0
4-15
9-12
1 -4
N
A
surf
ace
oran
ge ~
trans
glo
ssy
conv
ex
665
Blas
toco
ccus
agg
rega
tus
0.97
A
J430
193
AT0
4-15
9-12
2 -4
N
A
surf
ace
big
pk/p
each
~op
aque
glo
ssy
conv
ex
665
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
9-12
3 -4
N
A
surf
ace
peac
h tra
ns ri
dges
~op
aque
cen
ter g
loss
y co
nvex
44
3 Bl
asto
cocc
us sp
. BC
521
0.
97
AJ3
1657
3 A
T04-
159-
124
-4
NA
su
rfac
e w
hite
glo
ssy
conv
ex ~
trans
bum
py su
rfac
e in
cen
ter
697
Mod
esto
bact
er sp
. Elli
n165
0.
98
AF4
0900
7 A
T04-
159-
125
-4
NA
su
rfac
e ta
n/lt
pk g
loss
y co
nvex
~op
aque
67
0 Bl
asto
cocc
us a
ggre
gatu
s 0.
96
AJ4
3019
3 A
T04-
159-
126
-4
NA
su
rfac
e 67
7 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
159-
127
-4
NA
su
rfac
e tra
ns g
loss
y co
nvex
65
0 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.97
L4
0620
A
T04-
159-
128
-4
NA
su
rfac
e w
hite
rais
ed d
ull o
paqu
e bu
mpy
roug
h rig
id ~
irreg
ular
88
0 G
eode
rmat
ophi
lus s
p.
0.98
X
9235
8 A
T04-
159-
129
-4
NA
su
rfac
e tra
ns g
loss
y co
nvex
with
whi
te ~
irreg
ular
opa
que
bum
py ro
ugh
in c
ente
r 67
1 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
159-
132
-4
NA
10
cm
tra
ns b
row
n rip
pled
rigi
d ra
ised
in c
ente
r & o
n rip
ples
glo
ssy
540
Stre
ptom
yces
sp. 3
37F0
8
0.99
A
B12
4365
A
T04-
159-
133
-4
NA
10
cm
of
fwhi
te ~
trans
ripp
led
rigid
rais
ed o
n rip
ples
~gl
ossy
65
5 Sa
ccha
roth
rix
sp. A
S 4.
1731
0.
97
AY
1356
93
AT0
4-15
9-14
1 -4
N
A
20 c
m
whi
te g
loss
y fla
t mor
e op
aque
in c
ente
r ~tra
ns e
dges
68
5 Pa
enib
acill
us sp
. DSM
135
2
0.95
A
J345
017
AT0
4-15
9-14
8 -3
N
A
surf
ace
brow
nish
yel
low
rais
ed ro
ugh
rippl
ed ri
gid
opaq
ue ~
irreg
ular
74
8 Sa
ccha
roth
rix
sp. A
S 4.
1731
0.
98
AY
1356
93
AT0
4-15
9-14
9 -3
N
A
surf
ace
whi
te ~
irreg
ular
rais
ed ~
dull
roug
h bu
mpy
~op
aque
con
cave
cen
ter
801
Actin
obis
pora
ala
nini
phila
0.
98
AF3
2572
6 A
T04-
159-
150
-3
NA
10
cm
bi
g lt
yello
w tr
ans i
n ag
ar li
ke v
eins
~gl
ossy
ripp
led
rigid
rais
ed o
n rip
ples
81
7 St
rept
omyc
es a
lbid
ochr
omog
enes
stra
in A
S 4.
1863
0.
99
AY
9998
84
AT0
4-15
9-15
1 -3
N
A
10 c
m
yello
w/g
reen
opa
que
~dul
l rai
sed
rigid
pun
ctifo
rm tr
ans y
ello
w sp
ots a
roun
d
893
Unc
ult S
trep
tom
yces
sp. c
lone
clo
RD
L+29
0.
98
AY
8343
79
AT0
4-15
9-15
2 -5
M
A
surf
ace
smal
l pin
k op
aque
con
vex
dull
654
Blas
toco
ccus
sp. B
C41
2
0.95
A
J316
574
AT0
4-15
9-15
3 -3
M
A
10 c
m
larg
e irr
egul
ar fl
at c
ream
glo
ssy
trans
on
edge
s opa
que
in c
ente
r 65
0 Ba
cillu
s sp.
LM
G 2
0243
0.
9 A
J316
317
151
(Tab
le c
ontin
ued:
Pit
isol
ates
) Pi
t Iso
late
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
159-
154
-3
MA
10
cm
la
rge
crea
m fl
at w
hite
on
edge
s opa
que
glos
sy ir
regu
lar b
umps
on
edge
s 63
1 Ar
thro
bact
er sp
. 1c-
1
0.98
A
Y56
1525
A
T04-
159-
155
-3
MA
10
cm
w
hite
roun
d op
aque
glo
ssy
flat d
arke
r in
cent
er
615
Unc
ult B
acill
us sp
.
0.97
A
J640
178
AT0
4-15
9-15
6 -3
M
A
10 c
m
brow
n tra
ns fl
at ri
dges
on
edge
s dar
ker i
n ce
nter
cle
ar a
rea
arou
nd it
58
7 N
ocar
diop
sis s
p. 9
8-08
-290
A
0.94
A
F328
408
AT0
4-15
9-15
7 -3
M
A
10 c
m
whi
te ir
regu
lar o
paqu
e du
ll ro
und
in c
ente
r with
2 p
oint
s com
ing
out f
lat
564
Baci
llus s
p. V
AN
14
0.94
A
F286
482
AT0
4-15
9-15
8 -3
M
A
10 c
m
smal
l yel
low
con
vex
bum
py o
paqu
e 61
6 Ps
eudo
noca
rdia
pet
role
ophi
la
0.96
A
J252
828
AT0
4-15
9-16
0 -3
M
A
10 c
m
smal
l rou
nd w
hite
tran
s fla
t dul
l 59
8 Ba
cillu
s sp.
JL-3
4
0.95
A
Y74
5866
A
T04-
159-
161
-4
MA
su
rfac
e pi
nk ro
und
opaq
ue fl
at g
loss
y 54
7 M
odes
toba
cter
sp. E
llin1
65
0.99
A
F409
007
AT0
4-15
9-16
2 -4
M
A
surf
ace
pink
roun
d op
aque
con
vex
non-
glos
sy "d
onut
-like
" w
ith h
ole
in c
ente
r 60
8 Bl
asto
cocc
us sp
. BC
521
0.
95
AJ3
1657
3 A
T04-
159-
163
-4
MA
su
rfac
e ye
llow
irre
gula
r sha
pe c
onve
x op
aque
non
-glo
ssy
"bum
py"
642
Cel
lulo
mon
as c
ella
sea
0.
95
X83
804
AT0
4-15
9-16
4 -4
M
A
surf
ace
crea
m ro
und
conv
ex g
loss
y op
aque
66
1 C
ellu
losi
mic
robi
um fu
nkei
isol
ate
W6
0.
94
AY
7299
60
AT0
4-15
9-16
5 -4
M
A
surf
ace
smal
l pin
k ro
und
conv
ex d
ull o
paqu
e 98
2 G
eode
rmat
ophi
lus s
p.
0.98
X
9235
8 A
T04-
159-
170
-4
MA
30
cm
sm
all r
ound
cre
am ~
trans
con
vex
glos
sy
773
Stre
ptom
yces
sp. N
RR
L 57
99
0.98
A
J391
814
AT0
4-15
9-17
3 -5
M
A
surf
ace
yello
w ro
ugh
rigid
bum
py ir
regu
lar ~
glos
sy ra
ised
opa
que
802
Cel
lulo
mon
as c
ella
sea
0.
96
X83
804
AT0
4-15
9-17
5 -4
M
A
surf
ace
offw
hite
opa
que
glos
sy c
onve
x ~r
ound
81
9 Ba
cter
ium
K2-
12
0.94
A
Y34
5437
A
T04-
159-
176
-4
MA
su
rfac
e pi
nk o
paqu
e rig
id ro
ugh
bum
py ir
regu
lar ~
glos
sy
815
Blas
toco
ccus
sp. B
C44
8
0.95
A
J316
571
AT0
4-15
9-17
7 -4
M
A
surf
ace
yello
wis
h/ta
n gl
ossy
roun
d co
nvex
~tra
ns
838
Mic
roba
cter
iace
ae st
r. El
lin16
6
0.96
A
F409
008
AT0
4-15
9-17
8 -4
M
A
surf
ace
lt pk
opa
que
glos
sy c
onve
x ro
und
753
Bac
teriu
m E
llin6
023
0.
98
AY
2346
75
AT0
4-15
9-18
0 -4
M
A
10 c
m
brow
n in
aga
r opa
que
flat d
ull ~
roun
d 80
7 St
rept
omyc
es sp
. IM
-736
2
0.99
A
F131
591
A
T04-
159-
182
-5
MA
20
cm
cr
eam
/off
whi
te ir
regu
lar g
loss
y ra
ised
~op
aque
66
8 O
xalo
phag
us o
xalic
us
0.95
Y
1458
1 A
T04-
159-
183
-5
MA
40
cm
tra
ns o
ffw
hite
~irr
egul
ar g
loss
y ~c
onve
x 61
2 Ba
cillu
s kru
lwic
hiae
0.
96
AB
0868
97
AT0
4-15
9-18
4 -3
M
A
surf
ace
~irr
egul
ar !h
ot p
ink
~fla
t glo
ssy
opaq
ue m
ed
708
Arth
roba
cter
sp. F
a21
0.
97
AY
1312
25
AT0
4-15
9-18
5 -3
M
A
surf
ace
!hot
pin
k ~f
lat g
loss
y op
aque
med
roun
d 66
9 Ar
thro
bact
er sp
. Muz
t-F95
0.
94
AY
5266
77
AT0
4-15
9-18
6 -3
M
A
surf
ace
whi
te/c
ream
opa
que
glos
sy c
onve
x 61
7 C
ellu
lom
onas
sept
ica
stra
in W
7388
0.
94
AY
6557
33
AT0
4-15
9-18
7 -3
M
A
surf
ace
lt pk
glo
ssy
conv
ex o
paqu
e (li
ke c
ream
save
r)
846
Blas
toco
ccus
sp. B
C44
8
0.95
A
J316
571
AT0
4-15
9-18
8 -3
M
A
surf
ace
yello
w ~
glos
sy o
paqu
e irr
egul
ar ra
ised
but
in a
gar r
igid
fray
ed e
dges
81
7 C
ellu
losi
mic
robi
um fu
nkei
isol
ate
W6
0.
91
AY
7299
60
AT0
4-15
9-18
9 -3
M
A
surf
ace
lt ye
llow
~gl
ossy
opa
que
irreg
ular
rais
ed b
ut in
aga
r rig
id fr
ayed
edg
es
775
Cel
lulo
mon
as u
da
0.96
X
8380
1 A
T04-
159-
190
-3
MA
su
rfac
e pk
~op
aque
glo
ssy
rais
ed ro
und
807
Arth
roba
cter
sp. F
a21
0.
97
AY
1312
25
AT0
4-15
9-19
1 -3
M
A
surf
ace
smal
l ora
nge
~irr
egul
ar d
ull o
paqu
e ro
ugh
787
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
98
L406
20
AT0
4-15
9-19
2 -3
M
A
surf
ace
lt pk
~tra
ns ~
irreg
ular
flat
dul
l 60
2 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.98
L4
0620
A
T04-
159-
193
-3
MA
su
rfac
e tin
y ~t
rans
yel
low
edg
es b
row
n ce
nter
glo
ssy
conv
ex
171
Unc
ult C
ellu
lom
onad
acea
e ba
cter
ium
clo
ne m
M3
0.
97
AY
7314
68
A
T04-
159-
194
-3
MA
su
rfac
e ho
t yel
low
har
d irr
egul
ar d
ull o
paqu
e ra
ised
roug
h 80
0 Ac
tinob
ispo
ra a
lani
niph
ila
0.98
A
F325
726
AT0
4-15
9-19
5 -3
M
A
10 c
m
whi
te c
halk
y op
aque
cen
ter f
lat i
n ag
ar b
ig ro
und
trans
dul
l edg
es
815
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
9-19
7 -3
M
A
10 c
m
offw
hite
opa
que
fray
ed e
dges
glo
ssy
~rou
nd ~
conv
ex
863
Unc
ult B
acill
us sp
.
0.95
A
J640
178
AT0
4-15
9-19
8 -3
M
A
10 c
m
trans
off
whi
te ro
und
glos
sy fl
at
757
Subt
erco
la p
rate
nsis
0.
97
AJ3
1041
2 A
T04-
159-
200
-5
MA
30
cm
ta
n ed
ges b
row
n ce
nter
opa
que
smoo
th ro
und
glos
sy in
aga
r ~co
nvex
83
6 St
rept
omyc
es sp
. EF-
76
0.96
A
F112
173
AT0
4-15
9-20
1 -4
1/
100
PCA
sur
face
lt p
k ro
und
opaq
ue fl
at g
loss
y 81
7 M
odes
toba
cter
sp. E
llin1
65
0.99
A
F409
007
AT0
4-15
9-20
2 -4
1/
100
PCA
sur
face
ora
nge
irreg
ular
opa
que
flat d
ull
984
Mod
esto
bact
er sp
. Elli
n165
0.
99
AF4
0900
7 A
T04-
159-
203
-4
1/10
0 PC
A s
urfa
ce c
ream
roun
d fla
t dul
l ~tra
ns
658
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
96
L406
20
AT0
4-15
9-20
5 -5
1/
100
PCA
30
cm
very
larg
e cr
eam
dar
ker i
n ce
nter
roun
d ~t
rans
flat
dul
l 67
3 St
rept
omyc
es sp
. NR
RL
5799
0.
98
AJ3
9181
4 A
T04-
159-
207
-4
1/10
0 PC
A 4
0 cm
gr
ey ro
und
flat d
ull o
paqu
e 71
5 St
rept
omyc
es sp
. NR
RL
5799
0.
97
AJ3
9181
4 A
T04-
159-
209
-3
1/10
0 PC
A 3
0 cm
gr
ey ro
und
flat d
ull ~
trans
70
8 St
rept
omyc
es sp
. NR
RL
5799
0.
98
AJ3
9181
4 A
T04-
159-
214
-3
MA
20
cm
la
rge
beig
e ro
und
trans
glo
ssy
flat
797
Baci
llus s
p. L
MG
202
43
0.98
A
J316
317
AT0
4-15
9-21
5 -3
M
A
20 c
m
smal
l bro
wn
roun
d fla
t dul
l opa
que
794
Baci
llus s
p. L
MG
202
43
0.99
A
J316
317
AT0
4-15
9-21
7 -3
M
A
20 c
m
crea
m fl
at g
loss
y op
aque
roun
d 88
7 Ba
cillu
s lito
ralis
0.
98
AY
6086
05
AT0
4-15
9-21
8 -3
M
A
20 c
m
brow
n co
nvex
dul
l ~tra
ns ro
und
spot
ted
with
bro
wn
690
Noc
ardi
opsi
s sp.
98-
08-2
90A
0.
97
AF3
2840
8 A
T04-
159-
220
-3
MA
20
cm
op
aque
bei
ge c
ream
mar
gin
trans
out
side
flat
dul
l whi
te in
cen
ter i
rreg
ular
78
7 Ba
cillu
s sp.
VA
N14
0.
95
AF2
8648
2 A
T04-
159-
223A
-3
M
A
20 c
m
crea
m g
loss
y irr
egul
ar o
paqu
e fla
t 68
4 Ps
eudo
noca
rdia
zijin
gens
is
0.96
A
F325
725
152
(Tab
le c
ontin
ued:
Pit
isol
ates
) Pi
t Iso
late
D
ilutio
n M
edia
D
epth
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
Sim
ilari
ty A
cces
sion
no.
A
T04-
159-
223B
-3
M
A
20 c
m
71
4 Ba
cillu
s lito
ralis
0.
95
AY
6086
05
AT0
4-15
9-22
4 -3
M
A
20 c
m
crea
m d
ull f
lat r
ound
smal
l ~tra
ns
761
Baci
llus s
p. L
MG
202
43
0.99
A
J316
317
AT0
4-15
9-22
5 -4
1/
10 P
CA
10
cm
ta
n irr
egul
ar w
ith o
paqu
e da
rk ta
n sp
ots t
rans
on
1 si
de o
paqu
e ot
her f
lat d
ull
782
Baci
llus n
iaci
ni
0.99
A
F468
221
AT0
4-15
9-22
8 -3
1/
10 P
CA
30
cm
da
rk g
rey
roun
d op
aque
dul
l bum
py c
onve
x 74
0 St
rept
omyc
es sp
. NR
RL
5799
0.
97
AJ3
9181
4 A
T04-
159-
229
-3
1/10
PC
A
30 c
m
tan
roun
d op
aque
flat
dul
l 44
9 St
rept
omyc
es sp
. 434
F11
0.
97
AB
1235
50
153
(Tab
le c
ontin
ued:
Enr
ichm
ent i
sola
tes)
E
nric
hmen
t Iso
late
Dilu
tion
Med
ia
Dep
th
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
S
imila
rity
Acc
essi
on n
o.
AT0
4-15
0-B
6 -1
1/
100
PCA
50
cm
whi
te o
paqu
e ~c
onve
x 79
9 St
rept
omyc
es sp
. AS
4.11
82
0.98
A
Y11
4179
A
T04-
150-
B8
-1
1/10
0 PC
A 1
0 cm
tin
y w
hite
opa
que
82
2 Br
evib
acill
us a
gri
0.99
A
J586
388
AT0
4-15
0-B
9 -2
1/
100
PCA
40
cm
whi
te o
paqu
e ~f
uzzy
con
vex
yello
wis
h in
side
78
8 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
150-
B10
-2
PC
A
40 c
m
lt ye
llow
ish
clum
py c
onve
x ~t
rans
57
6 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
150-
B11
-1
PC
A
10 c
m
lt ye
llow
clu
mpy
con
vex
~tra
ns
816
uncu
lt al
pha
prot
eoba
cter
ium
0.
96
AJ3
1196
2 A
T04-
150-
B12
-1
PC
A
10 c
m
yello
w b
lob
glos
sy o
paqu
e fla
t 80
7 Br
evib
acill
us a
gri
0.
99
AJ5
8638
8 A
T04-
150-
B13
-1
PC
A
10 c
m
offw
hite
glo
ssy
opaq
ue ~
conv
ex
973
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
0-B
14
-1
PCA
40
cm
ye
llow
ish
clum
ps ir
regu
lar c
onve
x ~t
rans
69
7 St
rept
omyc
es sp
. AS
4.11
82
0.97
A
Y11
4179
A
T04-
150-
B15
-2
N
A
40 c
m
lt ye
llow
clu
mpy
con
vex
~tra
ns
805
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
0-B
19
-1
NA
10
cm
cr
eam
glo
ssy
opaq
ue c
onve
x cr
eam
ish
pk
812
Brev
ibac
illus
agr
i
0.99
A
J586
388
AT0
4-15
0-B
20
-1
NA
50
cm
w
hite
clu
mpy
opa
que
conv
ex y
ello
w in
side
98
3 Br
evib
acill
us a
gri
0.
99
AJ5
8638
8 A
T04-
150-
B21
-1
1/
10 P
CA
50
cm
w
hite
opa
que
conv
ex y
ello
w in
side
that
gro
ws i
n ag
ar
869
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
0-B
22
-2
1/10
PC
A
40 c
m
whi
te o
paqu
e cl
umpy
yel
low
insi
de c
onve
x 10
20
Her
basp
irill
um se
rope
dica
e st
rain
X8
0.
96
AF1
6406
5 A
T04-
150-
B23
-2
1/
10 P
CA
40
cm
gl
ossy
ora
nge
opaq
ue c
onve
x 84
2 Ps
eudo
noca
rdia
satu
rnea
0.
97
AJ2
5282
9 A
T04-
150-
B24
-2
1/
10 P
CA
50
cm
op
aque
whi
te c
onve
x cl
umpy
yel
low
insi
de
786
Brev
ibac
illus
agr
i
0.99
A
J586
388
AT0
4-15
0-B
25
-2
1/10
PC
A
surf
ace
lt p
k ~g
loss
y th
in la
yer c
over
ing
part
of p
late
83
9 St
rept
omyc
es c
alifo
rnic
us st
rain
DSM
400
58
0.98
A
Y99
9845
A
T04-
150-
B26
-1
1/
10 P
CA
10
cm
th
in lt
pk
laye
r cov
erin
g pl
ate
736
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-15
0-B
27
-2
1/10
PC
A
10 c
m
smal
l glo
ssy
lt pk
con
vex
opaq
ue
817
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-B
28
-1
1/10
PC
A
40 c
m
smal
l whi
te c
onve
x du
ll op
aque
yel
low
ish
cent
er
813
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-B
29
-1
1/10
PC
A
surf
ace
bro
wn
irreg
ular
gro
win
g in
aga
r with
lt p
k la
yer o
n to
p 46
7 Bl
asto
cocc
us sp
. BC
521
0.
98
AJ3
1657
3 A
T04-
150-
B30
-2
M
A
40 c
m
whi
te c
onve
x op
aque
~fu
zzy
base
gro
ws i
n ag
ar
799
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
0-B
31
-1
MA
30
cm
641
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
0-B
32
-1
MA
su
rfac
e lt
pk/
tan
glos
sy ~
conv
ex th
in la
yer c
over
s pla
te
838
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
0-B
33
-1
MA
20
cm
w
hite
/off
whi
te ~
conv
ex o
paqu
e 77
8 Ps
eudo
noca
rdia
satu
rnea
0.
97
AJ2
5282
9 A
T04-
150-
B34
-2
M
A
30 c
m
whi
te ~
fuzz
y op
aque
con
vex
yello
w c
ente
r 57
9 Ps
eudo
noca
rdia
satu
rnea
0.
97
AJ2
5282
9 A
T04-
150-
B35
-2
M
A
20 c
m
tiny
crea
m ~
opaq
ue ~
conv
ex g
loss
y 76
1 U
ncul
t firm
icut
e cl
one
smal
l1E0
7
0.94
A
F445
685
AT0
4-15
0-B
37
-2
MA
50
cm
w
hite
opa
que
~fuz
zy c
onve
x di
p ce
nter
top
834
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
0-B
38
-1
MA
10
cm
w
hite
opa
que
~fuz
zy y
ello
wis
h in
side
con
vex
853
Stre
ptom
yces
sp. A
S 4.
1182
0.
98
AY
1141
79
AT0
4-15
0-B
39
-1
MA
50
cm
w
hite
opa
que
conv
ex ~
fuzz
y 10
01
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
0-B
40
-1
MA
40
cm
w
hite
opa
que
conv
ex ~
fuzz
y 54
0 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
150-
B41
-1
PC
A
surf
ace
ora
nge/
pk g
loss
y op
aque
con
vex
854
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
0-B
42
-1
1/10
PC
A
30 c
m
whi
te o
paqu
e co
nvex
dul
l yel
low
insi
de g
row
s in
agar
83
0 Ps
eudo
noca
rdia
satu
rnea
0.
97
AJ2
5282
9 A
T04-
152-
B1
-1
1/10
0 PC
A 1
0 cm
w
hite
~irr
egul
ar ~
fuzz
y ~c
onve
x op
aque
93
4 St
rept
omyc
es c
alifo
rnic
us st
rain
DSM
400
58
0.98
A
Y99
9845
A
T04-
152-
B4
-1
1/10
0 PC
A 2
0 cm
w
hite
smal
l fuz
zy o
paqu
e 87
7 Br
evib
acill
us a
gri
0.
99
AJ5
8638
8 A
T04-
152-
B5
-2
1/10
0 PC
A 1
0 cm
sm
all ~
irreg
ular
~fu
zzy
opaq
ue w
hite
88
5 St
rept
omyc
es sp
. LK
1322
.1
0.96
A
Y46
5307
A
T04-
152-
B6
-2
PCA
10
cm
of
fwhi
te ~
irreg
ular
con
vex
~glo
ssy
opaq
ue
888
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
2-B
7 -2
PC
A
10 c
m
brow
n irr
egul
ar d
ull o
paqu
e ~c
onca
ve
736
Act
inob
acte
rium
RG
-51
0.
97
AY
5616
10
AT0
4-15
2-B
8 -1
PC
A
30 c
m
yello
w g
reen
ish
clum
ps ~
trans
con
vex
899
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
2-B
9 -1
PC
A
10 c
m
whi
te d
ull o
paqu
e ~c
onve
x br
owni
sh in
side
90
3 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
B10
-1
PC
A
20 c
m
whi
te ir
regu
lar ~
opaq
ue ~
conc
ave
grow
ing
in a
gar
916
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-15
2-B
11
-1
PCA
su
rfac
e pi
nkis
h or
ange
cov
ers p
art o
f pla
te o
paqu
e du
ll 74
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
152-
B12
-1
PC
A
40 c
m
crea
m d
ull g
row
s cov
erin
g en
tire
plat
e 84
5 Ba
cillu
s cer
eus s
train
SY
PA3-
2
1.00
A
Y96
8700
A
T04-
152-
B13
-1
N
A
20 c
m
whi
te d
ull ~
conv
ex o
paqu
e lit
tle d
ip in
cen
ter o
n to
p 86
3 Ps
eudo
noca
rdia
satu
rnea
0.
96
AJ2
5282
9 A
T04-
152-
B14
-1
N
A
20 c
m
crea
mis
h in
aga
r ~tra
ns c
onca
ve
805
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
2-B
15
-2
NA
20
cm
cr
eam
ish
with
som
e w
hite
spot
s ~tra
ns ~
conv
ex ~
in a
gar
638
Unc
ult f
irmic
ute
clon
e SM
1E07
0.
93
AF4
4568
5 A
T04-
152-
B16
-1
N
A
surf
ace
lt pi
nkis
h or
ange
thin
laye
r cov
erin
g en
tire
plat
e ~t
rans
89
1 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
152-
B17
-1
N
A
10 c
m
offw
hite
~gl
ossy
~co
nvex
but
gro
win
g in
aga
r ~tra
ns
903
Brev
ibac
illus
agr
i
0.99
A
J586
388
154
(Tab
le c
ontin
ued:
Enr
ichm
ent i
sola
tes)
E
nric
hmen
t Iso
late
Dilu
tion
Med
ia
Dep
th
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
S
imila
rity
Acc
essi
on n
o.
AT0
4-15
2-B
18
-1
NA
30
cm
of
fwhi
te/y
ello
w c
onve
x w
ith d
ip in
cen
ter ~
trans
90
0 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
B20
-2
1/
10 P
CA
su
rfac
e lt
pk g
loss
y co
nvex
89
3 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
152-
B23
-2
1/
10 P
CA
30
cm
ye
llow
clu
mp
with
whi
te o
n to
p co
nvex
77
2 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
B24
-1
1/
10 P
CA
10
cm
w
hite
fuzz
y br
own
cent
er c
onve
x op
aque
83
2 St
rept
omyc
es sp
. LK
1221
.3
0.96
A
Y46
5252
A
T04-
152-
B25
-2
1/
10 P
CA
10
cm
w
hite
~fu
zzy
opaq
ue c
onve
x 89
0 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
B26
-2
1/
10 P
CA
10
cm
ye
llow
ish
flat ~
opaq
ue
817
Stre
ptom
yces
sp. I
M-7
082
0.
97
AF1
3154
9 A
T04-
152-
B27
-1
1/
10 P
CA
20
cm
w
hite
fuzz
y op
aque
con
vex
904
Amyc
olat
opsi
s sp.
1B
dz
0.97
A
F479
268
AT0
4-15
2-B
28
-1
MA
80
cm
ye
llow
opa
que
conv
ex
671
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-B
29
-1
MA
70
cm
w
hite
opa
que
conv
ex fu
zzy
brow
nish
mid
dle
883
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
2-B
30
-2
MA
70
cm
w
hite
opa
que
conv
ex w
ith d
ip in
mid
dle
yello
w c
ente
r 74
5 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
152-
B31
-2
M
A
60 c
m
lt pk
opa
que
conv
ex g
loss
y 42
4 Am
ycol
atop
sis s
p. 1
Bdz
0.
98
AF4
7926
8 A
T04-
152-
B32
-2
M
A
40 c
m
smal
l cre
amis
h gl
ossy
con
vex
opaq
ue g
row
s in
agar
81
6 Am
ycol
atop
sis s
p. 1
Bdz
0.
97
AF4
7926
8 A
T04-
152-
B33
-2
M
A
20 c
m
crea
m g
loss
y tin
y ~c
onve
x ~t
rans
75
0 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
B34
-2
M
A
20 c
m
whi
te c
onve
x cl
umpy
opa
que
yello
w m
iddl
e 77
1 U
ncul
t firm
icut
e cl
one
SM1E
07
0.92
A
F445
685
AT0
4-15
2-B
35
-2
MA
30
cm
of
fwhi
te c
onve
x op
aque
gro
ws i
n ag
ar
788
Stre
ptom
yces
sp. A
S 4.
1182
0.
97
AY
1141
79
AT0
4-15
2-B
36
-1
MA
10
cm
cr
eam
gro
win
g in
aga
r 76
4 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
B37
-1
M
A
10 c
m
whi
te d
ots o
paqu
e co
nvex
78
8 St
rept
omyc
es sp
. AS
4.11
82
0.98
A
Y11
4179
A
T04-
152-
B38
-1
M
A
40 c
m
crea
m ~
glos
sy c
onve
x op
aque
80
3 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
152-
B39
-1
M
A
surf
ace
lt pk
opa
que
conv
ex g
loss
y 58
3 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
152-
B40
-1
M
A
surf
ace
gree
nish
bro
wn
grow
ing
flat o
n ag
ar
984
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
2-B
41
-1
MA
30
cm
cr
eam
with
whi
te to
p gr
owin
g in
to a
gar o
paqu
e 69
5 St
rept
omyc
etac
eae
0.
97
X87
320
AT0
4-15
2-B
42
-2
MA
10
cm
cr
eam
~tra
ns ~
conv
ex g
row
n in
aga
r 76
7 A
ctin
obac
teriu
m R
G-5
1
0.97
A
Y56
1610
A
T04-
152-
B43
-1
M
A
20 c
m
crea
m ~
trans
~co
nvex
gro
wn
in a
gar
826
Stre
ptom
yces
ney
agaw
aens
is
0.97
A
J399
493
AT0
4-15
2-B
44
-2
NA
su
rfac
e or
ange
/pk
glos
sy o
paqu
e fla
t thi
n la
yer c
over
s pla
te
575
Pseu
dono
card
ia sa
turn
ea
0.98
A
J252
829
AT0
4-15
3-B
1 -1
N
A
70 c
m
lt pk
/ora
nge
~con
vex
glos
sy o
paqu
e 50
4 Bl
asto
cocc
us sp
. BC
521
0.
99
AJ3
1657
3 A
T04-
153-
B2
-2
NA
70
cm
lt
pk/o
rang
e co
nvex
glo
ssy
opaq
ue
843
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
3-B
3 -1
N
A
80 c
m
crea
m ~
conv
ex o
paqu
e gl
ossy
65
7 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
153-
B4
-2
NA
80
cm
cr
eam
/lt p
k co
nvex
glo
ssy
~tra
ns
701
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-15
3-B
5 -2
N
A
surf
ace
oran
ge ~
conv
ex g
loss
y ~o
paqu
e 69
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B6
-2
NA
su
rfac
e cr
eam
~co
nvex
glo
ssy
trans
65
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B7
-1
NA
su
rfac
e or
ange
glo
ssy
trans
~co
nvex
61
2 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
153-
B8
-1
NA
60
cm
pk
tran
s glo
ssy
~con
vex
842
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
3-B
9 -1
N
A
60 c
m
yello
w ~
glos
sy ~
trans
84
3 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
B11
-1
N
A
30 c
m
crea
m o
paqu
e gl
ossy
con
vex
930
Stre
ptom
yces
cal
iforn
icus
stra
in D
SM 4
0058
0.
98
AY
9998
45
AT0
4-15
3-B
12
-1
NA
50
cm
cr
eam
ish
opaq
ue ra
ised
/~fla
t ~gl
ossy
85
4 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B13
-1
M
A
20 c
m
crea
m o
paqu
e ~c
onve
x gl
ossy
56
8 B
acte
rium
Elli
n602
3
0.96
A
Y23
4675
A
T04-
153-
B15
-1
M
A
60 c
m
lt pk
con
vex
~clu
mpy
tran
s dul
l 50
6 Bl
asto
cocc
us sp
. BC
521
0.
99
AJ3
1657
3 A
T04-
153-
B16
-1
M
A
70 c
m
crea
m ~
conv
ex g
loss
y ~o
paqu
e 65
6 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B17
-2
M
A
70 c
m
lt pk
opa
que
conv
ex g
loss
y 85
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B18
-1
M
A
80 c
m
offw
hite
con
vex
glos
sy o
paqu
e 75
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B19
-2
M
A
70 c
m
tiny
~cle
ar g
loss
y co
nvex
96
8 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
153-
B20
-2
M
A
30 c
m
lt pk
opa
que
glos
sy ~
conv
ex
601
Blas
toco
ccus
sp. B
C44
8
0.96
A
J316
571
AT0
4-15
3-B
21
-1
MA
40
cm
m
auve
con
vex
opaq
ue ~
glos
sy
601
Geo
derm
atop
hilu
s obs
curu
s obs
curu
s 0.
96
L406
20
AT0
4-15
3-B
22
-1
MA
40
cm
tin
y pk
con
vex
glos
sy o
paqu
e 82
1 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B23
-2
M
A
20 c
m
lt pk
opa
que
glos
sy ~
conv
ex/~
flat
757
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
3-B
24
-2
MA
10
cm
cr
eam
glo
ssy
opaq
ue fl
at g
row
s in
agar
61
3 St
rept
omyc
es sp
. AS
4.11
82
0.97
A
Y11
4179
155
(Tab
le c
ontin
ued:
Enr
ichm
ent i
sola
tes)
E
nric
hmen
t Iso
late
Dilu
tion
Med
ia
Dep
th
Col
ony
Mor
phol
ogy
Seq
uenc
e L
engt
h
BL
AST
Res
ult
S
imila
rity
Acc
essi
on n
o.
AT0
4-15
3-B
25
-1
MA
10
cm
lt
oran
ge/p
k op
aque
con
vex
glos
sy
822
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
3-B
26
-1
MA
su
rfac
e lt
pk c
onve
x gl
ossy
opa
que
tiny
597
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-15
3-B
27
-1
MA
su
rfac
e or
ange
con
vex
glos
sy o
paqu
e 85
2 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B29
-1
M
A
30 c
m
lt pe
ach
conv
ex g
loss
y op
aque
70
5 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
153-
B30
-2
1/
10 P
CA
40
cm
lt
oran
ge c
onve
x op
aque
~du
ll 70
3 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B31
-1
1/
10 P
CA
70
cm
tin
y of
fwhi
te tr
ans ~
conv
ex g
loss
y 59
3 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
153-
B32
-1
1/
10 P
CA
70
cm
lt
oran
ge fl
at th
in la
yer c
over
ing
plat
e 84
7 Bl
asto
cocc
us a
ggre
gatu
s 0.
97
AJ4
3019
3 A
T04-
153-
B33
-1
1/
10 P
CA
60
cm
lt
pk g
loss
y tra
ns ~
conv
ex/~
flat
661
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
3-B
34
-2
1/10
PC
A
30 c
m
pale
pk
trans
glo
ssy
conv
ex
683
Blas
toco
ccus
sp. B
C44
8
0.97
A
J316
571
AT0
4-15
3-B
35
-2
1/10
PC
A
80 c
m
clea
r lay
er c
over
ing
plat
e - ~
lt pk
69
4 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
B36
-1
1/
10 P
CA
30
cm
cr
eam
tran
s lay
er c
over
ing
plat
e 68
8 Bl
asto
cocc
us sp
. BC
448
0.
97
AJ3
1657
1 A
T04-
153-
B37
-1
1/
10 P
CA
20
cm
lt
pk c
onve
x tra
ns g
loss
y 64
9 Bl
asto
cocc
us sp
. BC
448
0.
96
AJ3
1657
1 A
T04-
153-
B38
-1
1/
10 P
CA
40
cm
w
hite
con
vex
glos
sy ~
trans
94
5 Ps
eudo
noca
rdia
satu
rnea
0.
98
AJ2
5282
9 A
T04-
153-
B39
-1
1/
10 P
CA
40
cm
lt
oran
ge tr
ans l
ayer
cov
erin
g pl
ate
619
Blas
toco
ccus
sp. B
C44
8
0.96
A
J316
571
AT0
4-15
3-B
40
-1
1/10
PC
A
80 c
m
lt or
ange
tran
s lay
er c
over
ing
plat
e w
ith w
hite
spot
s 84
0 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B42
-1
PC
A
80 c
m
peac
h ~c
onve
x gl
ossy
~op
aque
70
7 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
B43
-1
PC
A
surf
ace
brig
ht o
rang
e op
aque
con
vex
~glo
ssy
716
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
3-B
44
-1
PCA
60
cm
lt
peac
h bu
nch
of ti
ny d
ots m
akin
g th
in o
paqu
e la
yer
704
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
3-B
45
-1
PCA
70
cm
lt
oran
ge/c
lear
glo
ssy
laye
r 67
1 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
B47
-2
PC
A
70 c
m
peac
h gl
ossy
opa
que
conv
ex
716
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
3-B
48
-2
PCA
su
rfac
e lt
yello
w g
loss
y op
aque
con
vex
681
Baci
llus c
ereu
s stra
in M
SU13
10
0.99
A
Y64
7292
A
T04-
153-
B49
-2
PC
A
surf
ace
flat o
paqu
e gl
ossy
off
whi
te
707
Baci
llus c
ereu
s stra
in M
SU13
10
0.99
A
Y64
7292
A
T04-
153-
B50
-2
PC
A
surf
ace
brow
n op
aque
gro
win
g in
aga
r 70
0 Ba
cillu
s cer
eus s
train
MSU
1310
0.
98
AY
6472
92
AT0
4-15
3-B
51
-2
PCA
su
rfac
e ta
n op
aque
dul
l ~fu
zzy
conv
ex
659
Baci
llus c
ereu
s stra
in M
SU13
10
0.98
A
Y64
7292
A
T04-
153-
B52
-2
PC
A
surf
ace
oran
ge tr
ans g
loss
y co
nvex
69
1 Ba
cillu
s cer
eus s
train
J-1
0.
99
AY
3052
75
AT0
4-15
3-B
53
-1
NA
10
cm
tra
ns y
ello
w g
row
ing
in a
gar
692
Stre
ptom
yces
sp. A
S 4.
1182
0.
98
AY
1141
79
AT0
4-15
3-B
54
-2
MA
40
cm
m
auve
opa
que
conv
ex ~
clum
py g
loss
y 65
8 G
eode
rmat
ophi
lus o
bscu
rus o
bscu
rus
0.97
L4
0620
A
T04-
153-
B56
-2
1/
10 P
CA
70
cm
tra
ns fl
at la
yer c
over
ing
plat
e 69
1 Bl
asto
cocc
us a
ggre
gatu
s 0.
98
AJ4
3019
3 A
T04-
153-
B57
-1
1/
10 P
CA
su
rfac
e fa
intly
pk
trans
laye
r cov
erin
g pl
ate
732
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
3-B
58
-2
1/10
PC
A
surf
ace
yello
w g
loss
y op
aque
con
vex
704
Mic
roco
ccus
lute
us
0.95
A
J409
096
AT0
4-15
3-B
59
-1
1/10
0 PC
A 1
0 cm
tin
y w
hite
tran
s con
vex
glos
sy
687
Blas
toco
ccus
sp. B
C44
8
0.98
A
J316
571
AT0
4-15
3-B
61
-1
1/10
0 PC
A s
urfa
ce t
hin
trans
laye
r cov
erin
g pl
ate
(may
just
be
soil)
61
9 Bl
asto
cocc
us sp
. BC
448
0.
98
AJ3
1657
1 A
T04-
153-
B62
-2
1/
100
PCA
60
cm
tiny
trans
lt p
k gl
ossy
con
vex
686
Rhi
zosp
here
soil
bact
eriu
m is
olat
e R
SI-2
1 0.
95
AJ2
5258
8 A
T04-
153-
B63
-2
1/
100
PCA
sur
face
thi
n tra
ns la
yer y
ello
wis
h 56
3 Br
evib
acill
us a
gri s
train
NC
HU
1002
0.
98
AY
3193
01
AT0
4-15
3-B
64
-1
1/10
0 PC
A 7
0 cm
th
in tr
ans p
each
laye
r cov
erin
g pl
ate
877
Pseu
dono
card
ia sa
turn
ea
0.97
A
J252
829
AT0
4-15
3-B
65
-1
1/10
0 PC
A 8
0 cm
th
in tr
ans p
each
laye
r cov
erin
g pl
ate
693
Blas
toco
ccus
sp. B
C44
8
0.96
A
J316
571
AT0
4-15
3-B
66
-1
1/10
0 PC
A 4
0 cm
lt
pk ~
conv
ex g
loss
y op
aque
88
5 Bl
asto
cocc
us sp
. BC
448
0.
99
AJ3
1657
1 A
T04-
153-
B68
-1
1/
100
PCA
30
cm
tiny
whi
te o
paqu
e gl
ossy
con
vex
678
Blas
toco
ccus
sp. B
C44
8
0.99
A
J316
571
AT0
4-15
3-B
69
-1
1/10
0 PC
A 6
0 cm
tin
y w
hite
opa
que
glos
sy c
onve
x 95
0 A
ctin
omyc
etal
es b
acte
rium
HPA
66
0.97
D
Q14
4230
A
bbre
viat
ions
in c
olon
y m
orph
olog
y co
lum
n ar
e as
list
ed: l
t - li
ght,
pk –
pin
k, tr
ans –
tran
sluc
ent,
~ - s
light
ly
156
APPENDIX C TABLE OF PHOSPHATE, NITRITE, AND BROMIDE CONCENTRATIONS (MG/L)
“NA” means that nitrate was not detected (detection limit is 0.012 mg/L)
Surface Site Phosphate Nitrite Bromide Soil Pit Sample Phosphate Nitrite BromideAT03-33 0.2540 NA NA AT04-150 surface NA NA NA AT03-34 NA NA NA AT04-150 10cm NA NA NA AT03-35 0.2920 NA NA AT04-150 20cm NA NA NA AT03-36 0.2840 NA NA AT04-150 30cm NA NA NA AT03-37 0.0800 NA NA AT04-150 40cm NA NA NA AT03-38 NA NA NA AT04-150 50cm NA NA NA AT03-39 0.5700 NA NA AT04-150 60cm NA NA NA AT03-40 NA NA NA AT04-150 70cm 0.1260 NA NA AT03-41 NA NA NA AT04-150 80cm 0.4000 NA NA AT03-42 0.2920 NA NA AT04-150 90cm 0.2080 NA NA AT03-43 0.1080 NA NA AT04-152 surface 0.2360 NA NA AT03-44 0.3600 NA NA AT04-152 10cm NA NA NA AT03-45 0.0800 NA NA AT04-152 20cm NA NA NA AT03-46 0.2700 NA NA AT04-152 30cm NA NA NA AT03-48 NA NA NA AT04-152 40cm NA NA NA AT03-49 NA NA NA AT04-152 50cm NA NA NA AT03-50 NA NA NA AT04-152 60cm NA NA NA AT04-151 NA NA NA AT04-152 70cm NA NA NA AT04-154 NA NA NA AT04-152 80cm 0.4960 NA NA AT04-155 NA NA NA AT04-153 surface NA NA NA AT04-156 0.3120 NA NA AT04-153 10cm NA NA NA AT04-157 0.1740 NA NA AT04-153 20cm NA NA NA AT04-158 NA NA NA AT04-153 30cm NA NA NA AT04-161 NA NA NA AT04-153 40cm NA NA NA AT04-162 NA NA NA AT04-153 50cm NA NA NA AT04-163 NA NA NA AT04-153 60cm NA NA NA AT04-164 NA NA NA AT04-153 70cm NA NA NA AT04-165 NA NA NA AT04-153 80cm 0.6040 NA NA AT04-166 NA NA NA AT04-159 surface NA NA NA AT04-167 0.3980 NA NA AT04-159 10cm NA NA NA AT04-168 NA NA NA AT04-159 20cm NA NA NA AT04-169 NA NA NA AT04-159 30cm NA NA NA AT04-170 NA NA NA AT04-159 40cm NA NA NA Cornfield NA NA NA
157
APP
EN
DIX
D
ME
ASU
RE
ME
NT
S O
F SA
LIN
TY
, TD
S, A
ND
CO
ND
UC
TIV
ITY
IN T
AB
LE
OF
SOL
UB
LE
SA
LT
S W
ITH
IN S
OIL
S
Surf
ace
Sam
ple
Salin
ity (p
pt)
Con
duct
ivity
(mS)
T
DS
(ppt
) Pi
t Sam
ple
Salin
ity (p
pt)
Con
duct
ivity
(mS)
T
DS
(ppt
)
A
T03
-33
1.10
00
2.20
00
1.54
00
AT
04-1
50 su
rfac
e 1.
1000
2.
2100
1.
5400
A
T03
-34
1.16
00
2.33
00
1.63
00
AT
04-1
50 1
0cm
1.
0500
2.
1000
1.
4700
A
T03
-35
1.16
00
2.33
00
1.63
00
AT
04-1
50 2
0cm
1.
1000
2.
2100
1.
5400
A
T03
-36
1.14
00
2.29
00
1.60
00
AT
04-1
50 3
0cm
1.
1000
2.
2000
1.
5400
A
T03
-37
1.20
00
2.40
00
1.68
00
AT
04-1
50 4
0cm
1.
0900
2.
1800
1.
5200
A
T03
-38
1.09
00
2.19
00
1.53
00
AT
04-1
50 5
0cm
1.
3000
2.
6100
1.
8200
A
T03
-39
1.17
00
2.34
00
1.63
00
AT
04-1
50 6
0cm
2.
1600
4.
3200
3.
0200
A
T03
-40
1.24
00
2.49
00
1.74
00
AT
04-1
50 7
0cm
2.
6000
5.
2000
3.
6300
A
T03
-41
1.15
00
2.30
00
1.61
00
AT
04-1
50 8
0cm
5.
2900
10
.630
0 7.
4300
A
T03
-42
0.29
60
0.59
50
0.41
70
AT
04-1
50 9
0cm
4.
1600
8.
3500
5.
8400
A
T03
-43
0.31
00
0.62
00
0.43
30
AT
04-1
52 su
rfac
e 1.
1000
2.
2100
1.
5400
A
T03
-44
1.12
00
2.25
00
1.57
00
AT
04-1
52 1
0cm
0.
9980
1.
9990
1.
4000
A
T03
-45
1.07
00
2.14
00
1.49
00
AT
04-1
52 2
0cm
1.
1000
2.
2100
1.
5400
A
T03
-46
1.08
00
2.17
00
1.51
00
AT
04-1
52 3
0cm
1.
0700
2.
1400
1.
4900
A
T03
-48
1.16
00
2.33
00
1.63
00
AT
04-1
52 4
0cm
1.
3300
2.
6600
1.
8600
A
T03
-49
1.18
00
2.36
00
1.65
00
AT
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158
VITA
Danielle Bagaley was born in Baton Rouge, Louisiana, on May 6, 1979. She is the
daughter of Daniel and Robbie Bagaley and the sister of Jonathan Bagaley. She attended St.
Aloysius Catholic School in Baton Rouge from kindergarten until eighth grade and acquired her
high school degree in August of 1997 from Baton Rouge Magnet High School. Danielle began
her college career at Louisiana State University in 1997 and obtained a Bachelor of Science
degree in August of 2002. She participated in the National Student Exchange program and
attended the University of Georgia at Athens during the 1999/2000 school year. In her final
semester at LSU she enrolled in an undergraduate laboratory research course conducted by Dr.
Fred Rainey. She gained great interest in studies that the RaineyLab was undergoing at the time,
mainly a survey taking place in the Atacama Desert located in Chile. After graduating with her
bachelor degree she was hired as a research associate in the RaineyLab. After working for a year
and a half, Danielle decided to pursue a Master of Science degree from Louisiana State
University. She studied the extent of “Mars-like” soils in the Atacama Desert including
subsurface as well as surface samples. After graduating with a Master of Science degree,
Danielle Bagaley will presumably pursue a career with either a pharmaceutical corporation in
validation and quality control/assurance divisions, a biomedical research company, or with a
scientific research supply business as a sales representative.