bacteria id

Identification of Unknown BacteriaBarsabal, Ibanez, de Jesus, Ladra, Regunton, YorobeGroup III, Bio120, Dr. Sia Su, Lab1BJuly 29, 2013

I. ABSTRACT

Various tests were performed by the researchers to identify the genera and species of the unknown bacteria. Cultural and morphological classifications were performed first in order to narrow down the tests that will be performed in further investigation of the unknown bacteria. The bacteria were characterized as gram positive, spore-producing, encapsulated bacteria and are classified under the Bacillus genera. Further methods such as physiological and metabolic classification were carried out to identify the species of the unknown bacteria. The unknown showed positive results on Anthony’s test, Voges-Proskauer test, Motility band test, catalase test, citric acid utilization, and casein hydrolysis while it showed negative results on urea hydrolysis, indole production from tryptophan, gelatin liquefaction, phenylalanine deamination, and methyl red test. The triple sugar iron test gave the following results: A/KG+. The researchers cross-referenced laboratory data and existing literature on bacteria identification (Bergey’s Manual of Determinative Bacteriology) and found out that the unknown bacteria are Bacillus megaterium.

II. KEY WORDS: bacteria, characterization, bacillus, Bacillus megateriumIII. Introduction

Bacteria is one of the Three Domains of Life. It consists of small prokaryotic unicellular organisms enclosed by a cell wall of peptidoglycan. Identification of bacteria is the determination of whether an isolate should be placed within a group known to fit some classification scheme. The taxonomy order is Kingdom to Phylum to Class to Order to Family to Genus to Species to Type, and finally, to Strain. Identification is very significant particularly in gaining information on the pathogenicity of the given bacteria, diagnosis of any disease caused by it, formulation of treatment for infection, and other practical purposes. In this experiment, Bacterial Identification was done through Cultural, Morphological, and Physiological Characterizations.

One of the earliest bacteria described was “Vibrio Subtillis”. This description was made by Christian Gottfried Ehrenberg in 1835. In 1872, Ferdinand Cohn, a contemporary of Robert Koch, renamed the organism “Bacillus subtillis”. This microorganism is a charter member of a large and diverse genus that is part of the family Bacillaceae. The distinguishing feature of this family is the production of endospores, which are round, oval or cylindrical, highly refractile structures formed within bacterial cells (Hemphill, Slepecky, 2006). The term “endospores” was due to the fact that they are formed intracellularly, although they are eventually released from their mother cell, or sporangium, as free spores. Because of their chemical impermeability, colorizing these spores in the early days required stains and special conditions. However, it was proven that the Gram Stain is sufficient to determine the presence of spores, as they remain unstainable while the vegetative cells

or the vegetative part of the sporangia will stain. Because of this easy technique, several endosporeformers were determined. Vegetative cells and other resting forms such as cysts and exospores are usually killed by heating the isolate at 80°C for 10-30 minutes. As a result, at this temperature, endospores can be isolated. These endospores were first described by Cohn in B. subtillis, and later on by Koch in B anthracis, the latter being a major pathogen to vertebrates. The heat resistance of spores of B. subtillis and the developmental cycle of sporeformers (vegetative cell to spore, and vice versa) were determined by Cohn and Koch, respectively. Because of its unusual spore resistance to chemical and physical agents, the developmental cycle, the ubiquity of its members, and the pathogenicity of B. anthracis, the genus Bacillus attracted early interest which has continued on through the years.

Fig 1. Original photomicrographs of Bacillus anthracis, done by Robert Koch in 1876.

The family Bacillaceae, which encompassed all endosporeformers, was first formulated in 1895 by Fisher. What separate members of the genus Bacillus from other Bacillaceae are their aerobic nature (may be strict or facultative), rod shape, and catalase-production

Experiment 8 | Identification of Unknown Bacteria

ability. The other genera include Sporolactobacillus (microaerophilic and catalase-negative), Clostridium (anaerobic and does not reduce sulfate), Desulfotomaculum (anaerobic and does reduce sulfate), Sporosarcina (coccus), and Thermoactinomycetes (displays typical actinomycete characteristics).

The Bacillus species can be found in various habitats such as soil, water, and foods. The aerial distribution of dormant spores could be responsible for their presence in these habitats. The diversity of the species was apparent even with classical phenotypic characterization, nutrition, growth characteristics, different substrate utilization, and physiological assessments.

Table 1. Origins of isolates of Bacillus species

Table 1 shows the origins of isolates of Bacillus species based on Claus and Berkeley (1986).

Understanding of the genus Bacillus has advanced through augmentation of the phenotypic characterizations with the measurements of the DNA base composition, particularly the G+C content, and DNA-DNA hybridization. In the first edition (1986) of Bergey’s Manual of Systematic Bacteriology or BMSB, 40 recognized species are

listed. Several validly published new species that were shown to be genetically and phenotypically distinct from other Bacillus species that have not been described in the manual include B. pulvifaciens, B. chrondrotinus, B. smithii, B. thermoleovorans, B. benzoevorans, and B. gordonae. More than 200 species are in the category “Species Incertae Sedis”, all of which have been inadequately described or the original isolates have been lost.

Table 2. DNA base composition and sources of the type strains of Bacillus species

Table 2 shows the DNA base composition and sources of the type strains of Bacillus species, based on BMSB (1986). aTm corresponds to GC content by thermal melting; bBD corresponds to GC content by buoyant density; cND corresponds to not determined; dATTC corresponds to American Type Culture Collection; DSM corresponds to Deutsche Sammlung von Mikroorganismen; NCIB corresponds to National Collection of Industrial Bacteria; NCTC corresponds to National Collection of Type Culture; and NRRL corresponds to Northern Regional Research Laboratory.

In the second edition of BMSB, phylogenetic classification schemes landed on the two most prominent endospore-forming bacteria (Clostridium and Bacillus) into two different classes of Clostridia and Bacilli. Clostridia includes Order Clostridiales and Family Clostridiaceae with 11 genera, including Clostridum. Bacilli, on the other


hand, includes the Order Bacillales and the Family Bacillicaea, in which 37 new genera are on the level with Bacillus. This provides explanation to the G+C content heterogeneity observed in the 1986 genus Bacillus.

Table 3. Important reassignments in the genus Bacillus

Table 3 shows the important reassignments in the genus Bacillus.

One species in the genus Bacillus is the Bacillus Megaterium, which is commercially available, commonly found in soil and is considered a saprophyte. It was first described by Anton De Bary in 1884. Its name came from the two Greek words “mega” and “tetratis” which means “large” and “monster or beast”, respectively. Hence, “megaterium” means “big beast”. It has the largest cell diameter, 1.2-1.5 μm, among any aerobic spore-former. Its colonies grow in chains due to the presence of sticky polysaccharides in the cell wall. Prior to the introduction of B. subtillis as a Gram-positive model organism, B. megaterium was used in the studies of Biochemistry and even in bacteriophages. In 1925, Maurice Lemoigne discovered polyester polyhydroxybutyrate in the species’ cell as an essential energy storage molecule. In the same year, Andre Lwoff discovered UV induction of bacteriophage in a lysogenic B. megaterium strain.

It is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution dynamics, and plasticity in the bacilli (Eppinger, et.al, 2011). Its large cell size makes it well-suited for research on cell morphology, such as synthesis of cell wall and cytoplasmic membrane, sporulation, structure and cell organization of spore, DNA partitioning, and localization of protein. Furthermore, it is a nonpathogenic host for the biotechnological production of several substances such as Vitamin B12, penicillin acylase, and amylases. It has been extensively subjected to genetic studies and is amenable to genetic manipulation.

In this experiment, different methods of characterization were performed to identify the unknown bacterium, which was found to be Bacillus megaterium.

IV. Methodology

I. Cultural CharacterizationThe unknown bacterial cultures were plated

into a suitable culture media and were incubated accordingly. Once pure, it was inoculated onto plate, broth and slant and was then incubated at suitable temperature for 24 hours. The broth and slant culture was characterized using the diagram below:

Fig 2. Growth patterns in slant

Plate colonies were characterized as follows:- Size of colonies- Shape of colonies (punctiform, circular, rhizoid,

irregular, filamentous)- Surface (dull, shiny, smooth, contoured)- Edge (irregular, entire, undulate, lobate, curled)- Elevation (flat, round, convex, pulvinate,

umbonate)- Consistency (doughly, hard, sticky)- Capacity to transmit light (iridescent,

translucent, opaque)


Fig 3. Form, elevation, and margin patterns in plates

II. Morphological Characterization

A. Preparing a smear

A very small drop of distilled water was placed over a circled area on a glass slide. After aseptically removing material from a culture, it is then mixed with the drop and then completely air-dried. While holding the slide with a clothes pin, it was quickly passed through a flame thrice.

B. Study of the Bacterial Cell Wall

Gram staining was performed. The previously prepared bacterial smear was flooded with crystal violet and gram’s iodine followed by rinsing after each step. It was decolorized with 95% ethanol for 15 seconds and was immediately rinsed. It was then flooded with safranin for 45 seconds, was rinsed and blotted dry. The specimen was observed under OIO.

C. Study of Bacterial Capsule

Anthony’s method was performed. A bacterial smear was prepared air-dried but not fixed by heat. It was flooded with crystal violet for 2 minutes and washed with 20% copper sulfate solution. It was air-dried and then examined under OIO. The bacterial capsule will appear blue-violet or colorless while the cells stain dark blue.

D. Study of flagella

The motility band method was performed. Using an inoculating needle, the motility medium tubes were stab-inoculated with the unknown bacterial culture. It was incubated at 5° C below optimum growth temperature for 24-48 hours. Bands of growth movement from the point/line of inoculation were observed. No movement would mean that the unknown bacteria are non-motile.

III. Physiological Characterization

A. Oxygen Requirement

Catalase test was performed (control: (-) lactic acid bacteria; (+) Staphylococcus). A colony of bacteria was transferred on a clean glass slide. 1-2 drops of hydrogen peroxide was added and bubble formation was observed, which is positive for catalase test.

B. Carbohydrate Metabolism

1. MRVP Test (control: E. coli = IMVIC profile is ++--)

The bacterial culture was inoculated onto MRVP broth and was incubated at suitable temperature for 24-48 hours. 1-2 mL of culture broth was transferred into an empty clean tube and 1-2 drops of methyl red reagent was added. A red color is positive for MR test, indicating acid production from glucose. On the other tube, 5 drops of 5% α-naphtol was added and mixed. Then 7-10 drops of 40% KOH was added, mixed, and let stand for 20 minutes. Color change was observed. A pink to red color is positive for VP indicating acetoin production.

2. Citric Acid Utilization (control: E. coli = IMVIC profile is ++--)

An overnight culture of bacteria was inoculated onto Simmon’s Citrate Agar slate and was incubated at suitable temperature for 24-48 hours. Color change was observed. A blue color is positive for citrate utilization

C. Decomposition of Large Molecules

1. Gelatin Liquefaction (control: (+) Bacillus)

An overnight culture was inoculated onto Nutrient Gelatin (NG) tubes and was incubated at suitable temperature for 24-48 hours. It was then placed in an ice bath for a few minutes. Liquefied tubes are positive for gelatin liquefication.

2. Casein Hydrolysis (control: (+) Bacillus)

An overnight culture was inoculated onto casein agar plate and was incubated at suitable temperature for 24-48 hours. Clearing of the previously milky-white medium was observed. This indicated hydrolysis of casein.

D. Nitrogen and Sulfur Metabolism

1. Urea Hydrolysis (control: (+) Proteus)


An overnight culture was inoculated onto Urea broth and was incubated at suitable temperature for 4-6 hours. A change to deep red from light orange color media means positive for urea hydrolysis.

2. Indole Production from Tryptophan (control: (+) E. coli = IMVIC profile is ++--)

An overnight culture was inoculated onto Tryptone broth and was incubated at suitable temperature for 24-48 hours. 2 drops of Kovac’s reagent was added. It was then allowed to stand without mixing. A red-colored ring on the interface indicates a positive reaction, that is, production of indole form tryptophan.

3. Phenylalanine deamination (control: (+) E. coli)

An overnight culture was inoculated onto Phenylalanine slants and was incubated at suitable temperature for 24-48 hours. 4 drops of 10% ferric chloride solution was added. The tube was then rotated to loosen growth. The immediate appearance of an intense green color indicates a positive reaction.

E. Lead Sulfide/ Hydrogen Sulfide Production (control: (+) Proteus)

An overnight culture was stab inoculated onto TSI slants and was incubated at suitable temperature for 34-48 hours. A development of a brown black color along the stab inoculated is indicative of hydrogen sulfide production from the breakdown of amino acids.

V. Results and Discussion

Cultural Classification

The culture was observed for its growth patterns in both slant and plate. Growth patterns in the slant exhibit effuse growth. Growth patterns in the plate are as follows: the shape of the colonies are irregular, the surface is dull, the edges of growth are irregular, the elevation of growth is flat, the consistency of the growth is sticky, and the capacity to transmit light is opaque.

Table 4. Summary of Cultural Characterization of Bacterial Colonies

ResultsGrowth pattern EffuseShape of colonies IrregularSurfaceEdge UndulateElevation FlatConsistencyCapacity to transmit Opaque

light

Fig 4. Bacterial colonies in Nutrient Agar

Morphological Classification

In the beginning of the experiment, Gram staining was conducted to narrow out the choices for the unknown into Gram-positive bacteria or Gram-negative bacteria. The result of the Gram staining is a purple color of the bacterial smear. This means that the bacterial unknown is a Gram-positive bacterium. Also, the bacterium takes on a rod shape. Endospores also appear in the bacterial sample. The endospores present in the unknown are not swollen. With this information, the Bergey’s Manual of Determinative Bacteriology was consulted to determine any bacteria with the given characteristics giben by the experiment results. This gives us a bacterial sample of the genus Bacillus.Bacillus are Gram-positive rod shaped bacteria with the capability to form endospores, endospores of which are not swollen.

Fig 5. Gram positive bacillus bacteria

To determine the presence of a flagellum, the Motility Band test was conducted. The medium used for the test is composed of a soft type of agar. Inoculation of a sample into the medium is done using stab inoculation. Bacteria which are non-


motile have growths only on the area of the stab. Motile bacteria grow outward from the stab. Inoculation of the unknown revealed a growth pattern that diffuses outward from the area of stabbing. This shows that the unknown is motile.

Fig 6. Growth of unknown along stab line of motility medium

Table 5. Summary of Morphological Characterization of Bacterial Colonies

ResultsGram Staining gram positiveAnthony’s Method positiveMotility Band positive

Physiological Classification

After determining the genus of the sample, the next phase is to determine the species of the unknown. Tests were made with reference from Bergey’s Manual of Determinative Bacteriology, specifically tests to determine Bacillus species. These include all physiological tests to determine the unknown sample.

The starch test is used to determine the use of starch as an energy source. Members of the genus Bacillus are positive of this test, indicating that Bacillus included species are starch metabolizers. Since there is no starch test due to contamination of the culture, tests that proceed after starch test are done instead.

The MRVP test is used to determine the metabolism of glucose through two pathways, the mixed acid pathway and the butylene glycol pathway. Different bacteria convert glucose to pyruvate through the two different pathways. The mixed acid pathway produces acidic end products like lactic acid and acetic acid. The butylene glycol pathway produces neutral end products, like acetoin

and 2,3-butanediol. The MRVP broth contains peptone, buffers (K2HPO4 is used), and dextrose or glucose.The Methyl red test (MR) is used to determine if the pathway used is the mixed acid pathway. The methyl red added to an aliquot of the culture solution turns into a red color at pH<4.4. The presence of the acid end-products decreases the pH, making the solution turn red, indicative that the bacterial sample uses the mixed acid pathway. The Voges-Proskauer test (VP) is used to determine if the pathway used is the butylene glycol pathway. The reaction of acetoin, the end product of glucose metabolism of bacteria using the butylene glycol path, and α-naphthol with potassium hydroxide produces a deep red color. The result of the MR test on the unknown is a yellow color. The result of the VP test on the unknown is a deep red color. This shows that the unknown uses the butylene glycol pathway.

Fig 7. MRVP Test (Left: MR; Right: VP)

The catalase test is used to determine the presenceof catalase, an enzyme used by bacteria to convert hydrogen peroxide into water and oxygen. The result of the catalase test upon application to the unknown is bubble formation, a positive result for catalase production.

The citric acid utilization test is used to determine if a bacterial sample utilizes citrate as a carbon source. Simmon’s citrate agar solution is a differential medium that contains sodium citrate as the source of carbon. Bromothymol blue is used as the pH indicator in the mediumBacteria which utilize citrate appear blue because of resulting alkaline end products indicated by the change in color of bromothymol blue from green (neutral) to blue (alkaline). The result of the citric acid utilization test on the unknown is a blue color formed on the point of inoculation. This indicates a positive test for citric acid utilization.


Fig 8. Blue-colored Simmon’s Citrate slant

The gelatin liquefaction test is used to determine if a bacterial sample hydrolyzes gelatin. This is due to the presence of gelatinase in the bacterial sample. The result of the gelatin liquefaction test for the unknown is a slightly soft gelatin on the point of inoculation. This is a negative result as it is not significant for gelatin hydrolysis. The casein hydrolysis test is used to determine if a bacterial sample utilizes casein as an energy source. A positive result for the casein test is a clearing out of the casein agar, which signifies the use of casein. The result of the test on the unknown is a slight clearing out of the casein agar. This signifies the use of casein as an energy source.

Fig 9. Slant for Gelatin Liquefaction

The urea hydrolysis test is used to determine if a bacterial sample produces urease, an enzyme used to hydrolyze urea into ammonia and carbon dioxide. Urea broth contains urea, a buffer (K2HPO4 is used), phenol red, peptone, NaCl, glucose, and distilled water. Hydrolysis of urea produces ammonia, which is basic. This turns the phenol red color from yellow to pink. The result of

the test on the unknown is a yellow color, indicative of a negative urea hydrolysis test.

Fig 10. Negative result for urea hydrolysis

The indole test determines if a bacterial sample degrades tryptophan and produces indole. Tryptophan undergoes deamination and hydrolysis by bacteria which have the enzyme tryptophanase. Kovac’s reagent reacts with indole forming a red ring on top of the solution. The result of the test on the unknown is a yellow solution, indicative of no indole production from tryptophan.

The phenylalanine deamination test determines of a bacterial sample produces the enzyme phenylalanine deaminase to be able to use phenylalanine as a carbon and energy source. The product from deamination of phenylalanine combines with iron compounds, present from the addition of ferric chloride, in an acidic environment to form a green colored solution. The result of the test is a yellow colored solution, indicative of a negative result.

Fig 11. Slant for phenylalanine deamination

The triple sugar iron agar test determines a bacterial sample’s ability to reduce sulphur and ferment carbohydrates. TSI contains lactose, sucrose, and glucose as the three sugars and ferric ammonium sulfate as the iron source. Glucose is


found at the slant part of TSI and lactose and sucrose are found at the butt part. If a bacterial sample can ferment any sugar, all of the TSI agar will turn yellow indicative of acid production. If an organism can only ferment glucose, the slant part will turn red indicative of an alkaline environment due to the reversion of the reaction which produces the acid in the slant in aerobic conditions. If a bacterial sample reduces sulfur, the product of reduction, hydrogen sulfide, reacts with the iron present in the TSI to form iron sulfide, which appears as a black precipitate. Gas formation can also be detected in the TSI slants. The result of the TSI test on the unknown is a yellow slant and a partially red butt, indicative of fermentation of sugars, but no growth in the butt area, and gas formation. No black precipitate is found in the TSI agar. The overall result for TSI agar is written as A/KG+.

Table 6. Physiological characterization of bacterial colonies

ResultsCatalase Test positiveMRVP Test negative/positiveCitric Acid Utilization positiveGelatin Liquefaction negativeCasein Hydrolysis positiveUrea Hydrolysis negativeIndole Production from Tryptophan

negative

Phenylalanine deamination

negative

TSI A/KG+

Based from Bergey’s Manual of Determinative Bacteriology, the unknown, already known to belong in the genus Bacillus, was tested according to the flowchart present in Bergey’s Manual. The first test, starch amylase test, is not conducted due to the absence of starch amylase that can be used. Tests which are usually conducted after starch amylase test were conducted. The result for the VP test is positive, which then leads to the measure of cell diameter. Since the cell diameter is greater than 1 μm, 4 species remain to determine the unknown. Bacillus cereus is a possible answer as it is VP positive, cell diameter ≥ 1μm, and motile. But due to some additional data, it was revealed that B. cereus is not the unknown sample. The VP test was assumed to have been a false positive result due to the instrument used for the VP test is not sterile. From the flowchart, the next part is the presence of a swollen endospore. The unknown sample has no swollen endospores, so two bacteria remain as the

unknown: Bacillus megaterium and Bacillus badius. Bacillus megaterium is positive for the citrate test while Bacillus badius is negative for the citrate test. The identity of the bacterium is Bacillus megaterium.

VI. Conclusions and Recommendation

The researchers conclude that the unknown bacteria are Bacillus megaterium. Cultural, morphological, cultural, physiological, biochemical, and metabolic tests and characterization can be performed to accurately identify an unknown bacteria up to the species level.

It is recommended that more methods of characterization and more tests be performed to be able to identify unknown bacteria accurately. The culture media that are to be used should be properly formulated and kept in areas with optimal conditions for the media so as to avoid contamination that may cause false results. Also, procedures and protocols when performing tests must be properly followed to avoid errors in the results.

VII. References

Ahmad, B. et. al. (2010). Psychotrophic bacteria isolated from -20oC freezer. African Journal of Bacteriology, 9(5), 718-724

Bacillus megaterium. Abis Encyclopedia. Visited on August 15, 2012 at www.tgw1916.net/Bacillus/megaterium.html

Eppinger, et.al (2011). Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147683/

Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319.

Hemphill, H., Slepecky, R. (2006). The Genus Bacillus—Nonmedical. Retrieved from http://www.ic.ucsc.edu/~saltikov/bio119l/readings/prokaryotes/Bacillus.pdf

Identification flow charts. Retrieved on August 15, 2012 from www.uiweb.uidaho.edu/micro_biology/250/IDIDFlowchar.pdf


Identifying Bacteria. Retrieved from http://www.premierbioso.com/tech_notes/bac-id.html

Levinson, H. & Hyatt, M. (1970). Effects of temperature on activation, germination, and outgrowth of bacillus megaterium spores [abstract]. Journal of Bacteriology, 101(1), 58-64

Microbiology 20 biochemical unknown – spring 2009. Retrieved on August 15, 2012 from www.lamission.edu/lifesciences/Steven/Biochemical%20Unknown%20guidelines.pdf

Puspasari, F., Nurachman, Z., Noer, A. S., Radjasa, O. K., van der Maarel, M. J., & Natalia, D. (2011). Characteristics of raw starch degrading a-amylase from Bacillus aquimaris MKSC 6.2 associated with soft coral Sinularia sp. Starch/Sta¨rke, 461-467.

Todar, K. (2012). The genus bacillus (page2). Todar’s Online Textbook of Bacteriology. Visited on August 15, 2012 at textbookofbacteriology.net/Bacillus_2.html

I hereby certify that I have given substantial contribution to this report,

______________________________________Barsabal, Marc Lharen

______________________________________Ibaňez, Jerica Margarita

______________________________________de Jesus, Rigel Lorenzo

______________________________________Ladra, Ma. Carmina

______________________________________Regunton, Precious Caree

______________________________________Yorobe, Marian Justine


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