Isolating, Analyzing, and Identifying a Soil Bacteria Sample collected from Flagstaff, Arizona

Alexander Matic

Bio 305W Section 4

Angelique Krencius

16 October 2015

INTRODUCTION

Soil samples are commonly filled with a plethora of bacteria that are immensely useful in

societal pursuits such as industry, pharmaceuticals, and agriculture. One of the uses of bacteria in

industrial is the utilization of lipase functions in bacteria to initiate esterification, which

ultimately produces plastic or artificial flavoring (Willerding et al. 2011). Among the other uses

of bacteria is the pharmaceutical testing and development of antibiotics used to treat human-

related diseases. Bacterial components, such as dual stranded DNA molecules known as

plasmids, are studied for the purpose of antibiotic development. Plasmids encode various

secondary functions within soil bacterial samples, such as the ability to resist antibiotics.

Thereby, trials involving the application of experimental antibiotics to bacteria with antibiotic-

resistance-encoded plasmids aim to denature the bacterial plasmids via a chemical gradient.

Once the antibiotic resistant capabilities of the bacteria are diminished, the other components of

the antibiotic work to kill the bacteria. (Herrick et al. 2014). Additionally, agriculture can benefit

from the use of bacteria, such as those that break down harmful substances and resist stress.

Scientists in Japan were able to successfully extract dinitrotoluene (DNT) degrading bacterial

genera such as Bacillus, Burkholderia, Variovorax, Raistonia, Methylobacterium, Arthrobacter,

Mycobacterium, Pseudomonas, and Rhodanobacter, which can survive unfavorable conditions

(Koichi, Yoshinori 2014). These bacteria eliminate DNT within regions affected by warfare and

maintain a stress resistance that applies to temperature and pH. The DNT-degrading bacteria, due

to their versatility, are introduced to plant micro-biomes in order to encourage conjugation and

transformation horizontal transfers that spread resistance-encoded plasmids into these

environments (Koichi, Yoshinori 2014). Thereby, plants with modified micro-biomes are able to

grow in stressful environments and thrive in regions with contaminated soils (Thijs et al. 2014).

In the pursuit of bacterial studies, the specific process of collecting and growing bacteria

via environmental isolate or enriched cultures is imperative in providing bacterial samples for

industrial, pharmaceutical, and agricultural purposes. One process of bacteria retrieval that is

used for a handful of strains is the collection, isolation, and growth of a desired strain by

applying a collected sample to a plate of trypticase soy agar (Fitchett 2015, unreferenced

personal communication). Bacteria may be cultivated by the process of enriching cultures, where

conditions are set up to promote the growth of a specified strain of bacteria while preventing

other, unwanted strains from forming. The enrichment culture technique focuses on creating or

maintaining conditions such as pH, temperature, bacterial competition, moisture, and bacterial

food sources. The purpose of these conditions is to force the growth of bacteria that fit into a

very specific niche defined by the set conditions (Chawla et al. 2013). The purpose of the

environmental isolate culture technique is to identify and utilize naturally occurring bacteria

towards practical aforementioned purposes. Furthermore, once a potentially useful bacterium is

found and isolated in a specific region, the enrichment culture technique is used to ensure the

growth of said bacteria (Chawla et al. 2013). Therefore, with the application of these culture

techniques, bacteria can be manipulated to serve the purposes of the modern world.

The purpose of this study was to isolate, characterize, and identify an unknown species of

bacteria collected from soil in Flagstaff, Arizona. Morphological and physiological

characteristics of an unknown species of soil bacteria were compared with characteristics of

known bacterial species using Bergey’s Manual of Systematic Bacteriology (Holt 1984). The

bacteria cultivated in this experiment are predicated to be bacteria adapted to high altitude,

because Flagstaff has alkaline soil, moderate to high slopes, and high precipitation. These

conditions are caused by proximity to the atmosphere, an absence of nitrogen, and topographical

variance, which influence the growth of bacteria such as Deschampsia flexuosa, Dicentra

peregrina, Pinus pumila, and Stellaria nipponica within mountainous regions, like those found

in Japan (Koichi and Yoshinori 2014).

MATERIALS AND METHODS

The original environmental isolate bacteria were isolated from soil within Flagstaff using

a cotton swab, which was applied to a trypticase soy agar plate via a growth streak. After the

initial culture of environmental isolate bacteria was grown, additional trypticase soy agar plates

were saturated with the environmental isolate bacteria via an isolation streak. The isolation

streaks eventually produced a singular type of bacteria; the purity of the culture was confirmed

via wet mount slides, a simple stain, and a Gram stain. The environmental bacteria were

simultaneously observed on trypticase soy agar plates for growth patterns and morphology, and

trypticase soy agar slants were used to observe growth patterns. The environmental isolate

bacteria were streaked onto two screw-top trypticase soy agar slants and left to grow before the

first slant was stored at room temperature for two weeks while the second slant was stored at

fridge temperature for two weeks. The screw-top trypticase soy agar slants were taken out of

storage after the two week period and restreaked onto new trypticase soy agar slants, resulting in

a growth period that determined the favorable storage condition. Bergey’s manual volume 2 was

used to identify the environmental isolate bacteria because the bacteria were positive for the

Gram stain.

Aseptic technique was used with all stains. The simple stain was performed on bacteria

obtained from a trypticase soy agar plate. The purpose of using the simple stain is to observe the

morphology of the environmental isolate bacteria. The bacterial slide was dry mounted, heat

fixed, and stained with methylene blue dye. The slide was observed using bright-field

microscopy with 1000x magnification and oil immersion. The expected positive result of the

simple stain is the observation of blue stained cells.

The capsule stain was performed on bacteria obtained from a trypticase soy agar plate

towards the purpose of confirming whether or not the environmental isolate bacteria has a

capsule surrounding the cell membrane. The positive control organism used for the capsule stain

is Klebsiella pneumonia, which was obtained from a trypticase soy agar plate. The bacterial slide

was dry mounted, where the bacteria was emulsified with congo red dye and spread across the

slide. After the bacteria were emulsified, the sample was air dried and stained with Maneval's

dye. The slide was observed under bright field view with 1000x magnification and oil

immersion. The expected positive result of the capsule stain is the observation of clear halos

around the bacteria, which is stained red along with the background of the sample. The expected

negative result of the capsule stain is the absence of clear halos around the bacteria, which is

stained red along with the background of the sample.

The Gram stain was performed on bacteria obtained from a trypticase soy agar plate. The

purpose of using the Gram stain is to identify the presence or absence of an outer membranous

layer and to identify the thickness of the peptidoglycan layer of the environmental isolate

bacteria. The positive control organism used for the Gram stain is Bacillus megaterium, and the

negative control organism used for the Gram stain is Escherichia coli, both of which were

obtained from a bacterial broth. The bacterial slide was dry mounted and heat fixed, after which

crystal violet dye was used as a stain, Gram's iodine was used as a mordant, 70% ethanol was

used as a decolorizing agent, and safranin dye was used as a counterstain. The slide was

observed under bright field view with 1000x magnification and oil immersion. The expected

positive result of the Gram stain is the observation of purple stained cells, while the expected

negative result of the Gram stain is the observation of pink stained cells.

The acid-fast stain was performed on bacteria obtained from a trypticase soy agar plate.

The purpose of obtaining these bacteria is to observe the morphology of the environmental

isolate bacteria. The positive control organism used for the acid-fast stain is Mycobacterium

smegmatis and the negative control organism used for the acid-fast stain is Bacillus megaterium,

where the Mycobacterium smegmatis was obtained from trypticase soy agar while the Bacillus

megaterium was obtained from a bacterial broth. The bacterial slide was dry mounted and heat

fixed before carbol fuchsin dye was driven into the cells via heat. After the heating, acid alcohol

was used to decolorize the bacteria and methylene blue dye was used to counterstain the sample.

The slide was observed under bright field view with 1000x magnification and oil immersion. The

expected positive result of the acid-fast stain is the observation of pink stained cells, while the

expected negative result of the acid-fast stain is the observation of blue stained cells.

The endospore stain was performed on bacteria obtained from a trypticase soy agar plate.

The purpose of obtaining these bacteria is to observe the morphology of the environmental

isolate bacteria. The control organism used for the endospore stain is Bacillus megaterium. The

bacterial slide was dry mounted and heat fixed before malachite green was driven into the cells

via heat. After the heating, the sample was decolorized with the use of RO water and

counterstained with the use of safranin dye. The slide was observed under bright field view with

1000x magnification and oil immersion. The expected positive result of the endospore stain is

the observation of red or pink cells with green spores, while the expected negative result of the

endospore stain is the absence of visible spores. All methods are from Shand and Fitchett (2015).

The purpose of the catalase test is to evaluate if the bacteria have the capability to defuse

reactive oxygen species via the catalase enzyme, which the bacteria may or may not have. The

catalase test requires that hydrogen peroxide to be used as the primary reagent, where H2O2 is

applied to a glass slide with a sample of environmental bacteria. In the catalase test

Staphylococcus epidermidis is used as the positive control, and Streptococcus lactis is used as

the negative control. The expected positive result of the catalase test is the presence of bubbles,

while the expected negative result of the catalase test is the absence of bubbles. The results for

the catalase test were recorded on October 16th, 2015.

The purpose of the oxidase test is to evaluate if there are oxidase enzymes in the

cytochrome of the environmental isolate bacteria. The catalase test involves applying the

environmental bacteria to p-phenylenediamine squares to potentially initiate a chemically-based

color change. The positive control organism for the oxidase test is Pseudomonas aeruginosa.

The expected positive result for the oxidase test is a color change to dark purple, and the

expected negative result for the oxidase test is the absence of a color change. The oxidase test

results were recorded on October 16th, 2015.

The purpose of the carbohydrate test is to determine if the environmental isolate

bacteria has the capability to ferment various types of sugars. The carbohydrate test involves the

application of glucose, sucrose, mannose, and lactose; the test requires that the environmental

bacteria be emulsified with each carbohydrate broth. The expected positive result for the

carbohydrate test is a color change to yellow while the expected negative result for the

carbohydrate test is an absence of color change with the sample remaining red, and along with

the positive test, the solution can be scored for the production of gas along with acid. The results

for the carbohydrate test were recorded on October 16th, 2015.

The purpose of the oxygen requirement test is to evaluate what type of respiration the

environmental bacteria uses, whether the method used is anaerobic or aerobic. The oxygen

requirement test involves the use of thioglycollate broth with emulsified environmental isolate

bacteria, which is meant to grow at the level at which the bacteria best survives at based on

oxygen availability. The expected positive result for the oxygen requirement test is the growth of

bacteria at the bottom of the test tube, the top of the test tube, or across the entire test tube. The

expected negative control for the oxygen requirement test is an absence of bacterial growth. The

test results were recorded on October 16th, 2015.

The purpose of the nitrate reduction test is to determine if the environmental isolate has

the capability to reduce nitrate to nitrite, ammonia, or nitrogen. The nitrate reduction test uses

nitrate broth, where the environmental isolate bacteria is emulsified within the nitrate broth, then

exposed to Nitrite A/B reagent and zinc powder. The expected positive result of the nitrate

reduction test is the presence of gas in the Durham tube, a color change to red after the

application of Nitrite A/B reagent, and an absence of color change after the application of zinc

powder. The expected negative result of the nitrate reduction test is the absence of gas in the

Durham tube, an absence of color change after the application of Nitrite A/B reagent, and a color

change to red after the application of zinc powder. The results of the nitrate reduction test were

recorded on October 21st, 2015.

The purpose of the motility test is to evaluate if the environmental isolate has flagella

and, thereby, a means to travel. The motility test involves the environmental isolate being

stabbed into a motility medium of triphenyltetrazolium chloride. The expected positive result is

the presence of a red cloud around the stab pathway, while the expected negative result is the

absence of a red cloud around the stab pathway, where the red coloration is contained within the

stab pathway. The test results for the motility test were recorded on October 16th, 2015.

The purpose of the Simmons’ citrate test is to determine whether the environmental

isolate has the capability to transport citrate and use citrate as a carbon source. The Simmons’

citrate test utilizes Simmons’ citrate agar with brom thymol blue as the medium which is stabbed

and streaked with environmental isolate. The expected positive result for the Simmons’ citrate

test is a color change from green to blue, the expected negative result for the Simmons’ citrate

test is the absence of a color change. The test results for the motility test were recorded on

October 16th, 2015.

The purpose of the urea hydrolysis test is to evaluate whether the environmental isolate

bacteria contain urease and whether the bacteria have the capability to break down urea. The urea

hydrolysis test utilizes a urea broth that consists of phenol red, yeast extract, and urea, where the

environmental isolate bacteria is emulsified into the broth and left to grow. The expected positive

result of the urea hydrolysis test is a color change from yellow to pink, while the expected

negative result of the urea hydrolysis test is the absence of a color change. The test results for the

urea hydrolysis test were recorded on October 16th, 2015.

The purpose of the Kligler’s iron agar test is to determine whether the environmental

isolate bacteria is capable of producing hydrogen sulfide. The Kligler’s iron agar test requires

that the environmental isolate bacteria be stabbed into a medium containing ferrous sulfate,

glucose, lactose, and phenol red. The expected positive result for the Kligler’s iron agar test is

the presence of a dark precipitate, while the expected negative result for the Kligler’s iron agar

test is the absence of a dark precipitate. The test results were recorded on October 16th, 2015.

The purpose of the gelatinase test is to determine whether the environmental isolate can

utilize the polypeptides and amino acids contained within gelatin, which can be broken down

with the presence of the enzyme gelatinase. The gelatinase test requires that the environmental

isolate bacteria be stabbed into a gelatin media that contains protein, peptone, beef extract, and

gelatin. The expected positive result of the gelatinase test is that the gelatin remains in liquid

form after being chilled, and the expected negative result of the gelatinase test is that the gelatin

forms into a solid after being chilled. The test results for the gelatinase test were recorded on

October 21st, 2015.

The purpose of the starch hydrolysis test is to evaluate if the environmental isolate

bacteria is capable of breaking down starch via a-amylases enzymes. The test involves applying

the environmental isolate bacteria, as well as other bacteria, to a plate of starch medium, waiting

for growth, and applying Gram’s iodine. The positive control for the starch hydrolysis test is

Bacillus Megaterium and the negative control for the starch hydrolysis test is Escherichia coli.

The expected positive result of the starch hydrolysis test is the presence of clear zones around the

bacteria, and the expected negative result of the starch hydrolysis is the absence of clear zones

around the bacteria. The test results for the starch hydrolysis test were recorded on October 21st,

2015.

The purpose of the casein hydrolysis test is to evaluate the capability of the

environmental isolate bacteria to break down casein. The casein hydrolysis test involves

applying the environmental isolate bacteria, along with other bacteria, to a plate of casein that

contains protein. The positive control organism for the casein hydrolysis test is Bacillus

megaterium, and the negative control organism for the casein hydrolysis test is Escherichia coli.

The expected positive result of the casein hydrolysis is the presence of clear zones around the

bacteria, and the expected negative result of the casein hydrolysis is the absence of clear zones

around the bacteria. The test results for the casein hydrolysis test were recorded on October 21st,

2015.

The purpose of the lipid hydrolysis is to determine the capability of the environmental

isolate to break down lipids via the use of lipase enzymes. The lipid hydrolysis test involves the

application of environmental isolate bacteria, along with other bacteria, to a plate of spirit blue

agar. The positive control organism for the lipid hydrolysis test is Serratia spp., and the negative

control organism for the lipid hydrolysis test is Staphylococcus epidermidis. The expected

positive result of the lipid hydrolysis test is the presence of a clear zone around the bacteria,

while the expected negative result of the lipid hydrolysis test is the absence of a clear zone

around the bacteria. The test results for the lipid hydrolysis test were recorded on October 16th,

2015.

The purpose of the facultative anaerobe test is to evaluate if the environmental isolate

bacteria is capable of surviving under anaerobic conditions. The facultative anaerobe test

involves placing a streaked trypticase soy agar plate in an anaerobic chamber. The expected

positive results of the facultative anaerobe test is the presence of bacterial life after the

incubation period, the expected negative results of the facultative anaerobe test is the absence of

bacterial life after the incubation period. The test results for the facultative anaerobe test were

recorded on October 21st, 2015.

The purpose of the biofilm test is to determine whether or not the environmental isolate

bacteria contain a biofilm. The biofilm test requires that glucose TSB be emulsified with

environmental isolate bacteria and then mixed with crystal violet solution. The positive control

organism used for the biofilm test is Staphylococcus aureus, and the negative control organism

used for the biofilm test is Staphylococcus epidermidis. The expected positive result for the

biofilm test is the presence of a biofilm layer on the top of the solution, the expected negative

result for the biofilm test is the absence of a biofilm layer. The test results for the biofilm test

were recorded on October 21st, 2015.

RESULTS

The original plate grew colonies of various sizes, some of which were circular and other

of which were filamentous, with the circular colonies being yellow while the filamentous

colonies were white. Purity was confirmed by the absence of oddly shaped molecules and the

presence of clear rods within the wet mounted slide; the simple stain showed blue-stained rods

without the presence of other organisms; the Gram stain showed purple stained rods and did not

include the presence of black colored organisms such as yeast. The environmental isolate

bacteria were found to be white with a filamentous configuration, a filamentous margin, a flat

elevation, and a rhizoid growth pattern. After a two week period, the storage condition test of the

environmental isolate bacteria proved that the bacteria are best suited to (insert results).

The stains provided results based on the morphology and color of the environmental

isolate bacterial cells. The simple stain was rod-shaped, as indicated by the observation of rod-

shaped blue-stained cells (Table 1). The Gram stain was positive, as indicated by the observation

of purple cells (Table 1). The capsule stain was negative, as indicated by the observation of a

dark blue background, red cells, and an absence of a clear zone around each cell (Table 1). The

endospore stain was positive, as indicated by pink cells with green spores within and around the

cells (Table 1). The acid-fast stain was negative, as indicated by blue cells (Table 1).

Bubbles were observed for the catalase test, so the test was positive (Table 2). The

reaction area turned dark purple for the oxidase test, so the test was positive (Table 2). The

medium was yellow and gas was absent for the glucose test, so the test was positive for acid

(Table 2). The medium was yellow and gas was absent for the sucrose test, so the test was

positive for acid (Table 2). The medium was red and gas was absent for the lactose test, so the

test was negative for reaction but positive for growth (Table 2). The medium was yellow and gas

was absent for the mannose test, so the test was positive for acid (Table 2). The test tube retained

turbidity across the entire test tube and on a band around the top for the oxygen requirements

test, so the test was positive for facultative anaerobes (Table 2). (insert nitrate results here). A red

cloud formed around the stab pathway for the motility test, so the test was positive (Table 2). A

green medium was observed for the Simmons’ citrate test, so the test was negative (Table 2). The

medium was pink for the urea hydrolysis test, so the test was positive (Table 2). No dark

precipitate was formed within the stab pathway for the Kligler’s iron agar test, so the test was

negative (Table 2). (insert gelatinase results here). (insert starch results here). The test for casein

was inconclusive because the positive control and the casein plates in lab were all defective,

therefore no experiment could be performed. Clear zones were observed around the bacteria for

the lipid hydrolysis test, so the test was positive (Table 2). (insert fac anaerobe results). (insert

biofilm results).

Table 1. Gram, simple, capsule, acid-fast, and endospore stain results for environmental

isolate bacteria cells taken from Flagstaff soil.

Characteristics Results

Cell Morphology Rod

Gram Stain Positive

Capsule Stain Negative

Endospore Stain Positive

Acid-Fast Stain Negative

Table 2. Biochemical test results for environmental isolate bacteria taken from Flagstaff

soil.

Characteristics Result

Catalase Positive

Oxidase Positive

Carbohydrate Fermentation Positive (glucose, mannose, sucrose)

Growth Without Reaction (lactose)

Oxygen Requirements Facultative Anaerobe

Nitrate Reduction

Motility Positive

Simmons’ Citrate Negative

Urea Hydrolysis Positive

Kligler’s Iron Agar Negative

Gelatinase

Starch Hydrolysis

Casein Hydrolysis Inconclusive

Lipid Hydrolysis Positive

Facultative Anaerobe Positive

Biofilm

DISCUSSION

The purpose of the environmental isolate project is to identify a bacterium based on

biochemical tests performed on isolated bacterial samples obtained from soil in Flagstaff,

Arizona. The purpose was accomplished as environmental bacteria were successfully isolated

from Flagstaff soil and identified through a series of stains and biochemical tests. The ability to

utilize catalase allows the organism to defuse oxidative species that can cause harm to the cells.

The presence of oxidase allows the organism to perform the electron transport chain in a very

efficient manner. The ability to ferment monosaccharides allows the environmental isolate

bacteria to establish electron carriers that revitalize energy production (González-Cabaleiro et al.

2015). Because the environmental isolate is a facultative anaerobe, these bacteria can use both

fermentation and aerobic respiration when oxygen is present and absent. (Insert explanation of

nitrate). The presence of motility allows the organism to escape predators and to perform both

aerotaxis and chemotaxis. The absence of citrase means that the environmental isolate cell does

not have the potential to create bioluminescence, which is not universal among all citrase

positive cells. Citrase is typically used to form new colonies that establish symbiotic

relationships with animals; therefore the environmental isolate does not have symbiotic contact

with animals (Septer et al. 2015). In this case, the environmental isolate bacteria is unable to

accommodate host organisms with bioluminescence for camouflage, sexual attraction, and

predation, therefore this bacteria cannot receive safety or nutrition from a host organism (Pooley

2011). The environmental isolate is urea positive, meaning that the bacteria have a steady supply

of nitrogen for use in amino acids, which are constructed into proteins (Oliveira et al. 2009). The

inability to produce H2S for the Kligler’s iron agar test indicates that the organism is unable to

utilize cysteine found within sulfur compounds, which can be used as an antioxidant

(Maheswari, 2014). (insert explanation of gelatinase). (insert explanation of starch). The ability

to breakdown lipids indicates that the organism is able to separate fatty acids from glycerol,

which allows the organism to utilize another energy source. (insert explanation of biofilm).

REFERENCES

Herrick JB, Haynes R, Heringa S, Brooks JM, Sobota LT. 2014. Coselection for resistance to

multiple late-generation human therapeutic antibiotics encoded on tetracycline resistance

plasmids captured from uncultivated stream and soil bacteria. J Applied Microbiol.

117(2):380-389.

Thijs S, Weyens N, Sillen W, Gkorezis P, Carleer R, Vangronsveld J. 2014. Potential for plant

growth promotion by a consortium of stress-tolerant 2,4-dinitrotoluene-degrading

bacteria: isolation and characterization of military soil. Microb Biotechnol. 7(4):294-306.

Willerding AL, de Oliveira LA, Moreira FW, Germano MG, Chagas Jr. AF. 2011. Lipase

activity among bacteria isolated from Amazonian soils. Enzyme Res. 5(2):1-5.

Chawla N, Suneja S, Kukreja K. 2013. Isolation and characterization of chlorpyriphos degrading

bacteria. Indian J Agr Res. 47(5):381-391.

Shand R, Fitchett L. 2015. BIO 205L: fundamental techniques and experiments in microbiology.

Minneapolis, MN: bluedoor, LLC.

Koichi T, Yoshinori M. 2014. Effects of topographic and edaphic conditions on alpine plant

species distribution along a slope gradient on Mount Norikura, central Japan. Ecol Res.

29(5):823-833.

Holt JG, editor-in-chief. 1986. Bergey’s manual of systematic bacteriology, volume 2.

Baltimore, MD: Williams and Williams.

González-Cabaleiro R, Lema JM, Rodriguez J. 2015. Metabolic Energy-Based Modelling

Explains Product Yielding in Anaerobic Mixed Culture Fermentations. PLoS ONE.

10(5):1-17.

Septer AN, Bose JL, Lipzen A, Martin J, Whistler C, Stabb EV. 2015. Bright luminescence of

Vibrio fischeri aconitase mutants reveals a connection between citrate and the Gac/ Csr

regulatory system. Mol Microbiol. 92(2):283-296.

Pooley DT. 2011. Bacterial bioluminescence, bioelectromagnetics and function. Annu Rev

Microbiol. 87(2):324-328.

Oliveira EG, Santos ACF, Dias JCT, Rezende RP, Nogueira-Filho SLG, Gross E. 2009. The

influence of urea feeding on the bacterial and archaeal community in the forestomach of

collared peccary (Artiodactyla, Tayassuidae). J Appl Microbiol. 107(5):1711-1718.

Maheswari E, Ganesan RLS, Santhranii T. 2014. Hepatoprotective and antioxidant activity of N-

acetyl cysteine in carbamazepine-administered rats. Indian J Pharmacol. 46(2):211-215.