Unknown Bacterium Identification.pdf

University of the Philippines Los Baos |1st Semester AY 2014-2015

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Cultural and Physiological Characterization of Unknown Bacterium, B9

Ruth C. Abanador | 2012-10766 | MCB 101 | F-2L | Dr. Vernans V. Bautista

1st Semester AY 2014-2015

A Gram-positive, facultative anaerobic bacterium, comprising of endospore-forming motile rods was provided by the laboratory instructor for identification. B9 grew at mesophilic temperature and did not require growth factors. It was able to produce cellular inclusions such as starch and metachromatic granules as reserves. Based on its physiological characteristics, the unknown bacterium, B9, belongs to genus Bacillus, probably Bacillus gibsonii specifically.

An unknown bacterial culture, B9, probably belonging to genus Bacillus, was provided by the laboratory instructor in order for the student to identify the assigned microorganism. Series of tests were conducted consecutively to describe the unknown bacterium morphologically and physiologically.

The genus Bacillus (family Bacillaceae) is comprised of around ninety recognized species, including Bacillus subtilis as one of the well-studied species of Bacillus. Most of the species belonging to this genus were isolated from soil, although Bacillus can be isolated from a wide range of sources (Whittman, et al., 2009).

The main objective of this study was to identify the genus where the unknown bacterium, B9, belongs based on cultural and physiological characterizations. Molecular analyses must be used to determine the exact identity of the unknown bacterium in species level. However, this study is focused on the cultural and physiological methods of characterizing the unknown bacterium.

A pure culture of an unknown bacterium, B9, was provided by the laboratory instructor in order to be identified. Macroscopic characteristics of the unknown bacterium were observed by inoculating it in different media Nutrient Agar (NA), Nutrient Broth (NB), Kings Medium B Agar (KMBA), Tetrazolium Chloride Agar (TZCA), and Yeast Dextrose Carbonate Agar (YDCA). The tubes and plates were incubated at 28-30oC for 24 hours. The colonies had an undulate margin and were convex, dull, opaque and white in color.

Microscopic characteristics of the unknown bacterium were also observed. Nutrient agar was used as the medium in most of the tests conducted to identify the unknown bacterium. The plates and tubes were incubated at room temperature for 24-48 hours.

The cell wall characteristic of the unknown bacterium was determined using Gram staining and Gregersens Method. These tests were conducted to know whether the unknown bacterium was Gram positive or Gram negative. The shape and arrangement of the culture under oil immersion objective were also noted.

The presence of different cell surface structures in the organism was also determined. The test for the presence of capsule was conducted using three different


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staining procedures- negative staining, Anthonys method, and Manevals Staining Method. The unknown bacterium was grown in two nutrient agars with different sugar content, 2.0% glucose and 2.0% sucrose respectively.

The motility of the organism was determined by incubating the unknown bacterium in nutrient agar slant at 5oC below the optimum temperature. This will facilitate the formation of flagella. To observe the actual motility of the unknown bacterium, hanging drop technique was conducted. Also, the motility bands it formed after 24-48 hours incubation was observed by inoculating the unknown bacterial suspension to motility medium. The distanced travelled by the unknown bacterium from the point of inoculation was measured if the unknown bacterium is motile.

Bacterial storage granule formation of the unknown bacterium was also identified by inoculating it on N-limited Glucose Yeast Extract Agar slant (GYEA) at 30-35oC for 24-48 hours. The presence of intracellular lipids, glycogen and starch granules, and metachromatic granules were determined using different staining procedures. For staining intracellular lipids, Sudan black was used as the main stain. Glycogen and starch granules were stained primarily by Lugols iodine. On the other hand, methylene blue was used to observe the presence of metachromatic granules in the unknown bacterium.

The ability of the unknown bacterium to form endospore was also determined. It was incubated at 30oC for 36-48 hours to facilitate endospore formation. Cold method of Shaeffer-Fulton Spore Staining was conducted to determine the presence of endospore.

The oxygen requirement of the unknown bacterium was assessed by stabbing it to a molten Thioglycollate Agar. The tube was incubated at 30oC for 24-48 hours. Catalase and oxidase tests were also conducted using 3% hydrogen peroxide and oxidase reagent respectively.

The growth factor requirement of the unknown bacterium was measured by growing it in two different media: Growth Factor Test Media A, where no growth factor was present, and Growth Factor Test Media B, where yeast extract was added. The plates were then observed after 2-7 days of incubation at 30oC.

The temperature at which the unknown organism can grow was also determined. Three nutrient agar plates containing streaked unknown bacterium were individually incubated at low, room, and high temperature. Their growth in the respective plates after 2-5 days was noted.

The ability of the unknown bacterium to ferment or oxidize different sugars was determined using six Hugh and Leifson media. Glucose, lactose, and sucrose were separately added to two tubes. One of the two tubes containing a particular sugar was covered with water agar, providing low oxygen supply. The tubes were incubated for 24-48 hours at 30oC and the change in color in each tube was observed.

The production of different compounds by the unknown bacterium was also determined. Mixed acid production was tested through Methyl Red Test. 2-ketogluconate production, on the other hand, was determined by adding Benedicts


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reagent to the unknown bacterium in Haynes Medium. Indol production was tested using Tryptone Broth (TB). Production of hydrogen sulphide was determined by inoculating the unknown bacterium to Lead Acetate Agar (LAA).

Lipolytic activity of the unknown bacterium was known by growing the bacterium in Sierras Medium (SM) with Tween 80. Deamination of phenylalanine was tested by adding 10% ferric chloride solution to inoculated Phenylalanine Agar (PA). Lysine decarboxylation by the unknown bacterium was also tested by growing it in Lysine Decarboxylase Broth (LDB).

Utilization of citrate, malonate, and nitrogen were also determined. Simmons Citrate Agar (SCA), Malonate Broth (MB), and Nitrogen Test Media A (NMA) and B (NMB) were used respectively. The unknown bacteriums ability to hydrolize starch, gelatin, and urea was also tested. Starch agar (SA), Nutrient Gelatin (NG), and Christensens Medium (CM) were used respectively. All of them were incubated at room temperature for 24-48 hours.

All of the media were incubated at room temperature for 24-48 hours. The results of the tests conducted were collected to determine the identity of the unknown bacterium using Bergeys Manual of Systematic Bacteriology.

Cultural characteristics are concerned to the macroscopic appearance of a microorganism in different types of media (Brown, 2001). They cannot immediately determine the identity of the organism since most bacterial species can be morphologically similar to other bacterial species of different taxa. Nevertheless, by using different media, the capability of the organism to utilize or not utilize certain molecules, or produce or not produce certain products can give a hint on their identity. The cultural characteristics of the unknown bacterium, B9, grown in different media were summarized in Table 1. Figure 1 shows the growth of B9 in different media.

Table 1. Cultural Characteristics of unknown bacterium, B9, cultivated on different media and incubated at 28-30oC for 24 hours.

Characteristics NA plate KMBA YDCA TZCA NA slant NB

Size (mm) 2 - 2 - - -

Abundance moderate abundant moderate slight moderate -

Opacity opaque opaque opaque opaque opaque -

Form concentric - concentric - - -

Surface dull dull dull smooth dull -

Margin undulate - undulate - - -

Consistency hard hard hard hard hard -

Color white white white red violet white -

Elevation convex convex convex drop-like - -

Form of growth on agar slope

- - - - echinulate -

Growth characteristics on NB

- - - - - pellicle w/

Sedim-entation


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Different reference organisms were used as basis for the results in different tests. The reference organisms used are as follows: Escherichia coli, Pseudomonas fluorescens, Pseudomonas polymyxa, Pseudomonas aeroginosa, Pseudomonas vulgaris, Bacillus subtilis, Bacillus amiloliquefasciens, Enterobacter aerogenes, Shigella sonnei, Alcaligenes feacalis, Serratia marcesens, Lactobacillus casei,Bacillus cereus and Pediococcus cerevisiae. Reference organisms do not only give the possible identity of the organism. They are also used to determine if the reagents and media used were of good condition to provide true results.

Gram staining, an important differential staining, was conducted to know the characteristic of the cell wall present in the unknown bacterium. Gram staining was developed Hans Christian Gram in 1884, classifying bacteria into Gram-positive and Gram-negative (Tortora, Funke, & Case, 2013). After being examined under oil immersion objective in the light microscope, the unknown bacteria was identified to be a Gram-positive bacterium.

Cell surface structures of the unknown bacterium B9 were assessed. Capsule was present in B9. Negative staining clearly showed the presence of capsule (Figure 2). The presence of flagella was not observed under the light microscope due to an error during staining. Nevertheless, B9 exhibited true motility when subjected to hanging drop observation.

Cellular inclusions and endospore present in the bacterium was also assessed. Cellular inclusions or storage granules serve energy reserves for the bacterium (Madigan, Martinko, Stahl, & Clark, 2012). B9 was able to form starch and metachromatic granules (Figure 3). The bacterium was also able to produce endospore and was observed using Shaeffer-Fulton Spore Staining.

B9 was identified to be a facultative anaerobe after growing in Thioglycollate Agar (Figure 4). B9 showed positive reaction for catalase and oxidase tests. Also, B9 did not require growth factors since it was able to grow in GFA (Figure 4).

Temperature greatly affects the growth and survival of microorganisms (Madigan, Martinko, Stahl, & Clark, 2012). Any decrease or increase of temperature in the environment may lower the organisms metabolism or worse, cause its death. Growth in different temperatures can give an idea on the identity of the microorganism being identified. In the experiment, B9 did not exhibit growth in low temperature. Low temperature inhibited the growth of the unknown bacterium. However, growth was observed among the plates that were incubated at room and high temperature, making the unknown bacterium to belong to mesophile group.

The metabolism of carbohydrates and organics acids by the unknown organism B9 was analysed through different tests. First were the oxidation and/or fermentation of sugars. The unknown organism was able to utilize glucose, sucrose, and lactose and was identified to be anaerogenic fermentative microorganism since the Hugh and Leifson Media were turned into yellow in both tubes (Figure 5).


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Figure 5. Fermentation of glucose, sucrose, and lactose by B9 in Hugh and Leifson Media.

Figure 1. Growth of unknown bacterial, B9,

culture in different media.

Figure 2. Negatively stained cells of

unknown bacterium B9 under light

microscope (1200x).

Figure 3. Starch and metachromatic granules under light microscope (1200x).

Figure 4. Facultative anaerobic growth of unknown bacterium B9 in Thioglycollate Agar (1) and Growth of B9 in Growth Factor Test Media A (2).


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B9 was able to produce mixed acid when methyl red indicator was added to NBG and pink color was observed. However, it was not able to utilize citric acid and malonate when incubated in SCA and MB respectively. Consecutively, starch was not hydrolyzed by B9 and potassium gluconate was not oxidized.

Different tests were also conducted to know the ability of the unknown bacterium B9 in decomposing large molecules as its nutrient source. One is its ability to produce gelatinase that would degrade gelatin. The inoculated Nutrient Gelatin remained liquid after a cold ice bath, which indicated the degradation of gelatine into polypeptides (Leboffe & Pierce, 2010). Another test was its ability to degrade lipids which can be tested using Sierras Medium with Tween 80. The precipitation of calcium oleate indicated by the formation of opaque zone around and under the culture is a positive result. However, lipolytic activity was not exhibited by B9.

Further series of tests were performed to know the ability of B9 in using nitrogen and sulphur for its metabolism. One important test is to know the ability of the unknown bacterium in utilizing inorganic and organic nitrogen. B9 was able to grow in both NMA and NMB, indicating its ability to utilize both organic and inorganic nitrogen.

On the other hand, B9 was not able to hydrolyze urea when grown in CM Urea Broth. Also, red violet interphase was not observed upon addition of Kovacs reagent to inoculated Tryptone Broth, indicating the inability of B9 to produce indol. Neverheless, B9 was able to produce hydrogen sulphide when grown in LAA. Phenylalanine deamination and lysine decarboxylation was not exhibited by the unknown bacterium B9.

The obtained results were collected and were summarized in Table 2 including the results exhibited by the reference organisms in their respective tests. B9 was identified to belong in genus Bacillus using Bergeys Manual of Systematic Bacteriology. B9 shared similar characteristics with the members of genus Bacillus, with the possible identity of Bacillus gibsonii. The inability to perform molecular analyses in this study made it impossible for the unknown bacterium, B9, to be identified up to species level. Nevertheless, Table 3 shows the comparison of B9 to its closely related species belonging to genus Bacillus based on the tests conducted.

Description of genus Bacillus represented by B9

Cells are facultative anaerobic, Gram-positive, motile rods that are endospore-forming. The colonies in NA are white opaque, with an undulate margin (2mm in diameter), convex, and dull after 24 hours of incubation at 28-30oC. Has starch and metachomatic granules as nutrient reserves and does not require growth factors for growth. Grows in room temperature and high temperature. Positive in tests for the assimilation of glucose, lactose, sucrose, gelatine, organic nitrogen, and inorganic nitrogen, but not citrate and malonate. Produces hydrogen sulphide, catalase, oxidase but not indol, lipase, and 2-ketogluconate. No hydrolysis of starch and urea, as well as phenylalanine deamination and lysine decarboxylation.


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Table 2. Some characteristics of unknown bacterium, B9, and reference organisms used based on the tests conducted.

Strain: 1, B9; 2, Escherichia coli; 3, Pseudomonas fluorescens; 4, Pseudomonas polymyxa; 5, Pseudomonas aerogenosa; 6, Pseudomonas vulgaris; 7, Bacillus subtilis; 8, Bacillus amiloliquefasciens; 9, Enterobacter aerogenes; 10, Shigella sonnei; 11, Alcaligenes feacalis; 12, Serratia marcesens; 13, Lactobacillus casei; 14, Bacillus cereus; 15, Pediococcus cerevisia; f, facultative anaerobe; oa, obligate aerobe; m, microaerotolerant; blank data means the particular species was not tested with the corresponding test.

Characteristics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Cell Wall + + - Capsule + + + Motile + Storage Granules

Intracellular Lipid - + Glycogen - + Starch + Metachromatic + +

Endospore + + Oxygen Requirement f f oa m Catalase + + - - Oxidase + - + Growth Factor - - + Growth at: Low temperature - - - Room temperature + + + High Temperature + + + Oxidation - - + - - Fermentation

Anaerogenic + - - + - aerogenic - + - - -

Acid from: Glucose + + - Lactose + + - Sucrose + +* -

Methyl red test + + - + Utilization of:

Citrate - - + + + Malonate - - + Organic Nitrogen + + + + + + Inorganic Nitrogen + - + + + +

Hydrolysis of: Starch - - + + + Gelatin + - + +

(continued)


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Table 2. (continued)

Characteristics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Hydrolysis of: Lipase - + + - Urea - - +

Production of: 2-Ketogluconate - - + Indol - + - - Hydrogen sulfide + - +

Phenylalanine Deamination

- + +

Lysine Decarboxylation

- + +

* With agar plug.

Table 3. Some characteristics of unknown bacterium B9 and closely related species of genus Bacillus.

Strain: 1, B9; 2, Bacillus gibsonii; 3, Bacillus licheniformis; 4, Bacillus vedderi; 5, Bacillus simplex; 6, Bacillus niacin. +, positive; -, negative; nd, no data; d, variable; s, swollen; n, not swollen; f, facultative anaerobe; a, aerobic.

Characteristics 1 2 3 4 5 6

Motile + nd + + + d Endospore n n n s n v Oxygen Requirement f nd f f f a Catalase + + + + + + Oxidase + nd nd + - d Growth at:

Low temperature - + + - - + Room temperature + + + + + + High Temperature + + + + - -

Acid from: Glucose + + + + + + Lactose + + - - - d Sucrose + nd + + + +

Utilization of: Citrate - - + nd d d

Hydrolysis of: Starch - - + - + d Gelatin + + + + + +

Phenylalanine Deamination - - - nd nd - Lysine Decarboxylation - nd - nd - -


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References:

Brown, A. E. (2001). Benson's Microbiological Applications Laboratory Manual in

General Microbiology (8th ed.). The McGrawHill.

Leboffe, M. J., & Pierce, B. E. (2010). Microbiology Laboratory Theory and Application

(3rd ed.). USA: Morton Publishing Company.

Madigan, M. T., Martinko, J. M., Stahl, D. A., & Clark, D. P. (2012). Brock Biology of

Microorganisms (13th ed.). USA: Pearson Education, Inc.

Tortora, G. J., Funke, B. R., & Case, C. L. (2013). Microbiology: An Introduction (11th

ed.). USA: Pearson Education, Inc.

Whittman, W. B., De Vos, P., Garrity, G. M., Jones, D., Krieg, N. R., Ludwig, W., et al.

(2009). Bergey's Manual of Systematic Bacteriology (2nd ed., Vol. III). (A. C. Parte,

Ed.) New York, USA: Springer Science+Business Media.

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Unknown Bacterium Identification.pdf