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Identification of the Unknown Bacterium by Cultural, Morphological and Physiological Characterization Tests

Ma. Christine B. Cabatlao Group 1, UV-2L

March 13, 2012

A scientific paper submitted in partial fulfillment of the requirements in

Microbiology 101 laboratory under Ms. Shiela Marie G. Selisana, 2nd sem., 2011-2012.

ABSTRACT In identification, organisms are placed into previously established classes that are named by deductive procedures. The main objective of this study is to identify the given unknown bacterium. Different tests were performed for cultural characteristics, morphological characteristics and physiological characteristics. The unknown bacterium is a gram positive, supplement requiring, mannitol fermenting, mesophilic, facultative anaerobic cocci which appears beige on Nutrient Agar. Using Bergeys Manual of Determinative Bateriology, results show that the unknown bacterium is Staphylococcus aureus.

INTRODUCTION Prokaryotes are the most diverse group among organisms. This diversity is made sense by grouping microorganisms together and organizing them in non-overlapping hierarchal arrangement. Taxonomy is the branch of biology that names and classifies organisms in groups of increasing depth. This consists of three main separate but interrelated parts: classification, nomenclature and identification (Bauman, 2004). Classification is the ordering of populations and groups of populations at all levels by inductive procedures. Another, nomenclature is the application of distinctive names to each of the groups recognized in the classification; and the third is identification, where individuals are placed into previously established classes that are named by deductive procedures (Soligam-Hadsall et al, 2007). Taxonomy is thought to be significant for several reasons. First, organization of huge amounts of knowledge about organisms is allowed because all members of a particular group share many characteristics. Second, making predictions and framing hypothesis for further research is performed based on knowledge of similar organisms; 2

and third, microorganisms are placed in meaningful groups with precise names so that microbiologist can work with them and communicate effectively. And lastly, this is essential for the identification of unknown microorganisms (Prescott, 2005). Systematics, which is the nearest field related with taxonomy, is the scientific study of kinds and diversity of the organisms and of any all relationships among them. The background and knowledge in the taxonomy of organisms is based on systematics, which encompasses multiple disciplines such as morpho-anatomy, ecology,

epidemiology, biochemistry, molecular biology, physiology and evolutionary biology (Soligam-Hadsall et al., 2007). Microorganisms can be classified based on the two major classification systems: the artificial classification and the natural classification. In the artificial classification, microorganisms are classified based on arbitrary chosen criteria; while in the natural classification system, microorganisms are arranged into groups whose members share many characteristics and reflect the biological nature of microorganisms. The natural classification system can be subdivided into two: the phenetic system, which classifies microorganisms together based on the mutual similarity of their phenotypic characteristics; and the phylogenetic system, which is based on the evolutionary relationships rather than general resemblance. In microbial systematics, the Bergeys Manual of Systematic Bacteriology is the standard that provides phylogenetic information on bacteria and archaea. The approved lists of known prokaryotes are published on the International Journal of Systematic Bacteriology (Tortora, Funke and Case, 2004). This study focuses on the identification aspect of microbial taxonomy. Cowan (1965) described the practice of identification to be the utilitarian aspect of taxonomy. According to Steel (1965), identification, which can also be termed as diagnosis, is the practical application of taxonomic knowledge which makes use of standardization 3

methods and characterization tests. The Bergeys Manual of Determinative Bacteriology provides a standard scheme for identifying bacteria and archaea (Tortora, Funke and Case, 2004). The identification of microorganisms must be done in the shortest possible time, however, speed should be always considered as secondary to accuracy. There are five main objectives in microbial identification which was stated by Nungester in 1963: (1) to determine the susceptibility of the microorganisms to antimicrobial drugs (2) to gain information which may have prognostic value (3) to identify pathogens in terms of their potential danger (4) to aid epidemiologists in tracing sources of infection (5) to accumulate data of interest to those studying infectious diseases. Many techniques are performed in the identification of unknown bacteria including morphological identification, differential staining, serological methods,

biochemical analysis, phage typing and usage of differential media. This study is limited in identifying microorganisms through morphological identification, differential media and simple biochemical/ physiological tests (Ingraham, 2004). This study aims to identify the given unknown bacterium. The specific objectives were the following: 1. To be able to perform different tests for the identification of unknown bacterium. 2. To be able to explain the concepts behind the results on each performed test. 3. To be able to map the identity of the unknown bacterium based on Bergeys Manual of Determinative Bacteriology. The study was performed at Room 307, Wing B of the Institute of Biological Sciences, University of the Philippines- Los Baos, College, Laguna, Philippines from December 13, 2011 to March 1, 2012. 4

MATERIALS AND METHODS In the identification of unknown microorganism, a culture of the unknown bacterium was provided by the instructor. Series of morphological and physiological tests, and observations on the cultural characteristics were performed. The morphology of the unknown bacteria was studied through analysis of its cell wall, capsule, flagella and endospore formation. On the physiological characterization of the unknown bacterium, oxygen requirement, growth factors, catalase reaction and temperature requirements and mannitol fermentation were studied. The cultural characteristics of the bacteria are also significant in the determination of the unknowns identity. The cultural characteristics of the bacteria were determined by growing the unknown bacterium on Nutrient Agar. The plate was incubated at 28- 30C for 24-48 hours. Microscopic observations were performed for determining the morphological characteristics. All tests were observed under oil immersion objective. The tests performed for the study of cell wall were Gram staining, cepacol staining and Gregersens method. All of which provided the gram reaction of the unknown bacterium. The unknown bacterium was incubated into two different media: Nutrient agar with 0.1% glucose and Nutrient agar with 20% sucrose to determine the presence of capsule. The media will provide conditions favorable for the formation of capsule. After which, negative staining, Anthonys method and Manevals Method were done to confirm the presence of capsule.

Hanging drop technique and flagella staining were performed to determinine the bacterial motility and presence of flagella. The observation of motility band in the Motility Medium was not observed under the microscope; however the length of the band was measured in order to determine the distance traveled from point of inoculation after two days. The endospore formation of the unknown bacterium was also determined by incubating the unknown bacterium in high temperature to provide favorable conditions for the formation of endospores. Schaeffer- Fulton spore staining method was performed in order to determine the presence of endospores. The unknown bacterium was stabbed into Thiogycollate Agar and incubated to determine the oxygen requirement. A smear of the unknown bacterium was added with hydrogen peroxide to determine catalase reaction. The temperature requirement was obtained by incubating plates on different temperatures: 10C, 30C, 45C and 60C. This method provided the temperature range essential for the growth of the unknown microorganism. The unknown bacterium was placed on Growth Factor Test Media A and B (GFA and GFB) plates. GFA did not contain any additional growth factors while GFB did contain additional 3 grams of yeast extract to serve as the growth factor. The plates were observed for growth. Presence of mannitol fermentation was determined by inoculating the unknown bacterium into 2 tubes of Hugh and Leifson medium with mannitol. One tube was sealed by water agar to provide a low oxygen environment, while the other was not.

After the results were gathered, they were mapped on the Bergeys Manual of Determinative Bacteriology to determine the bacterial group where the microorganism belongs and to determine the identity of the unknown bacterium.

RESULTS AND DISCUSSIONS The table below, Table 1 shows the results on the cultural characteristics of the unknown bacterium which was grown on Nutrient Agar. Figure 1 shows image of the unknown bacterium grown on Nutrient Agar. The cultural characteristics can provide an idea on the identity of the unknown microorganism since certain microorganisms are distinguishable especially by pigmentation. For example, Micrococcus luteus is distinguishable because of the yellow punctiform colony when grown on Nutrient Agar. Table 1. Cultural characteristics of the unknown bacterium grown on Nutrient Agar. CHARACTERISTICS Size Form Surface Margin Pigmentation Elevation RESULTS 1 mm punctiform shiny and smooth Entire Cream/ beige convex

Figure 1. The unknown bacterium grown on Nutrient Agar. The results for the morphological characteristics of the unknown bacterium are shown on Table 2. Three tests were done to determine the gram reaction of the unknown bacterium: cepacol staining, Grams staining and Gregersenss method. Only Grams stain and Gregersens method confirmed that the unknown bacterium is a Grampositive cocci since the data for cepacol staining was erroneous. The most probable source of error is the application of high amounts of dye which caused light not to pass through the slide. Cepacol staining is a technique performed to determine the differences in the thickness of the bacterial cell walls (Raymundo, 2001). This method contains three important stains: cepacol, congo red and methylene blue. Cepacol is a quaternary ammonium compound which serves as a cationic mordant that coats the cell wall with positive charges so that congo red, a negatively charged acidic dye which stains the cell wall red, can attach on it. Methylene blue is a positively charged dye which stains the cytoplasm blue. Gram staining is the most common differential staining technique for identifying the gram reaction of the bacteria. Four reagents/ stains were used in this technique: 8

crystal violet, which is the primary stain and basic dye; Gram Iodine, a mordant that increases the interaction between the cell and the dye; 95% ethanol, a decolorizing agent; and safranin which serves as a counterstain. According to Table 2, the unknown bacterium is a gram positive cocci which appeared purple to blue under the microscope. The context behind the observation is that gram positive bacteria contain thick peptidoglycan. Once the crystal violet and Gram iodine are added on the smear, a complex between the two reagents will form inside the peptidoglycan layer. Upon addition of ethanol, the pores of the peptidoglycan will close in which in effect the complex will be locked inside. Once inside, safranin cannot penetrate upon addition that is why cells appear blue to purple (Harley and Prescott, 2002). Gregersens method is the fastest method to determine the gram reaction of the bacteria. Three percent potassium hydroxide (KOH) was added on the smear and according to Table 2, no slime was observed. The reagent was added to disrupt the cell wall of the bacterium, which is supposedly evident as slime. However, since the bacterium is gram positive, the cell wall of the bacteria was not disrupted due to its thick peptidoglycan making the suspension watery (Appalaraju, Parvathi and Arthi, 2003). Only negative staining method provided a good result in determining the presence of capsule-- the unknown bacterium is encapsulated. The data for Anthonys method and Manevals method were erroneous since the distinction between the capsule and the cell was not clear. The microorganism appeared to be too small under the microscope. In negative staining technique, nigrosin or india ink was placed on the smear. Cells and capsules appeared colorless while the background appeared to be dark brown to black. The principle behind is that there was a tore repulsion between the cells and

the stain since both are negatively charged, therefore the stain cannot penetrate (Harley and Prescott, 2002). Anthonys method contains two important staining reagents: crystal violet, which serves as the primary stain; and copper sulfate, which acts as both decolorizing agent and counterstain. Theoretically, crystal violet provides the cell and the capsule a deep purple color. But since the capsule is non-ionic, then the capsule cannot adhere. When copper sulfate is added, this will remove the excess crystal violet and stain from the capsule. Since copper sulfate also acts as a counterstain, the capsule will absorb the reagent making capsules appear light blue to pink (Casida, 1971). Manevals method is composed of two dyes: acid fuschin and congo red. Acid fushin contains phenol, ferric chloride and acetic acid. Congo red serves as a

counterstain and also a pH indicator, which is blue when acidic and red when neutral and basic. Theoretically, cells will appear red since acid fuschin interacts with the

bacterial cell and capsules will appear colorless (Casida, 1971). Bacterial motility of the unknown bacterium was studied through hanging drop technique, motility band and flagella staining. Based on Table 2, the unknown bacterium does not contain flagella since cocci bacteria do not move through flagella but through Brownian movement. Brownian movement is the random movement of particles along the medium usually described as the zigzag motion of particles (Zumdahl, 1992). Endospore formation was determined using Schaeffer- Fulton method. Two different stains are used in this method: malachite green, which would stain the endospore; and safranin, which would stain the vegetative cells. All cells appeared pink under the microscope which would indicate a negative result for endospore formation (Raymundo, 2001). 10

The physiological characteristics of the unknown bacterium were studied by determining the oxygen requirement, growth factors, temperature requirement and mannitol fermentation. The oxygen requirement was determined by growing the unknown microorganism in Thioglycollate Agar tube and according to Table 3, more growth was observed on top of the tube and growth was also present throughout the tube. This indicates that the unknown bacterium is a facultative anaerobe. Facultative anaerobes are capable of producing energy with or without the presence of oxygen; however they prefer to live in the environment where oxygen is present since more ATP is produced (Tortora, Funke and Case, 2004). Growth factors are organic compounds of low molecular weight needed by microorganisms as essential cell components or precursors of these components that the organism cannot synthesize (Prescott, 2005). Growth factor requirement of the microorganism was determined by growing on Growth Factor test media A and B (GFA and GFB). GFA did not contain any growth factor while GFB was added with growth factor (yeast extract). The unknown microorganism did not grow on GFA but did grow on GFB indicating a growth factor requirement. The temperature requirement of the unknown bacteria was obtained by incubating plates on different temperatures: 10C, 30C, 45C and 60C. Growth was observed on plates incubated at 30C and 45C, indicating that the unknown bacterium is a mesophile. Mesophiles are organisms with growth optima around 20 to 45C, with minimum temperature often 15- 20C and maximum temperature about 45C or lower (Prescott, 2005). Catalase test was performed by adding hydrogen peroxide on the smear. Bubble formation was observed and indicates a positive result for presence of catalase.

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Catalase is an enzyme which protects the cell against toxic effects of hydrogen peroxide (Koolman and Roehm, 2005). The presence of mannitol fermentation was performed into two tubes of Hugh and Leifson medium with mannitol: one sealed with water agar the other was not. Both tubes changed in color, from purple to yellow indicating acid production on both tubes (Raymundo, 2001). Therefore the unknown microorganism has a fermentative metabolism of mannitol. Table 2. The morphological characteristics of the unknown bacterium. MORPHOLOGICAL CHARACTER Gram reaction Cepacol staining No distinct cells were observed; very dark background Gram stain Gregersens method Capsule formation Negative staining Purple cells No slime Cells and capsules were colorless against dark background Manevals staining Cells were too small for capsule observation Anthonys method Cells were too small for capsule erroneous erroeneous Gram positive Gram positive encapsulated Erroneous data TESTS OBSERVATIONS CONCLUSION

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observation Presence of Flagella Hanging drop technique Flagella staining No flagella observed No flagella observed; cells were suspended Motility band test 1 mm difference from the point of inoculation Endospore formation Schaeffer-Fulton method No green spherical structures were stained; all were vegetative cells Doesnt produce endospore Cellular movement is present. Non- flagellated Non-flagellated

Table 3. The physiological characteristics of the unknown bacterium. PHYSIOLOGICAL CHARCTERS Oxygen requirement more growth on top of the tube; growth also present throughout the tube Growth factors Growth on GFB; no growth on GFA Temperature requirement Catalase test Growth on 30-45C Bubble formation mesophile Presence of catalase Requires growth factors Facultative anaerobe OBSERVATIONS CONCLUSION

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Mannitol fermentation

Both tubes changed from purple to yellow

Positive for mannitol fermentation

After series of tests, the results were mapped on Bergeys Manual of Determinative Bacteriology to identify the unknown bacteria. The table below, Table 4 shows the grouping of bacteria according to Bergeys Manual of Determinative Bacteriology. Table 4. Bacteria groupings according to Bergeys Manual of Determinative Bacteriology. GROUP Group 4 DESCRIPTION DIFFERENCES EXAMPLES

pigments/fluorescent, motility, growth Aerobic/Microaerophilic requirements, denitrification, rods and cocci morphology, and oxidase, read Genera descriptions Facultatively Anaerobic Gram negative rods growth factors, morph., gram reaction., oxidase reaction., read Genera descriptions

Gram Negative,

Acinetobacter, Pseudomonas, Beijerinckia, Acetobacter Family Enterobacteriaceae and Vibrionaceae

Group 5

Group 17

Gram-Positive Cocci

oxygen requirements, morph., growth requirements (45C and supplements), read Genera descriptions

Micrococcus, Staphylococcus, Streptococcus, Enterococcus, Lactococcus,


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Aerococcus
Group 18

Endospore-Forming Gram positive rods and cocci

oxygen requirements, motility, morphology, catalase reaction

Bacillus, Clostridium

Group 19

Regular, Nonsporlating Gram positive rods

morphology, oxygen require, catalase reaction

Lactobacillus, Listeria

Group 20

Irregular, Nonsporlating Grampositive rods

catalase, motility, morph., read Genera descriptions

Actinomyces, Corynebacterium, Arthrobacter, Propionibacterium

Group 21

Weakly Gram-Positive Nonsporlating Acid Fast Slender Rods

acid fast, growth

Mycobacterium

Based on the data gathered results, the unknown bacterium belongs to Group 18, which is composed of Gram positive cocci bacteria which have key differences in

oxygen requirements, morphology and growth requirements (45C and supplements). Further tracing of the identity of the unknown bacterium was done using the flowchart below, Figure 2. The flowchart is also based on Bergeys
Manual of Determinative Bacteriology.

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Figure 2. Bergeys identification flowchart for gram positive cocci bacteria.

SUMMARY AND CONCLUSIONS

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The cultural characteristics of the unknown bacteria were observed to provide an idea on the identity of the microorganism. Series of tests were performed for morphological and physiological characteristics to further identify the unknown. Results show that the unknown bacterium appeared to be punctiform, convex, entire, smooth, beige colonies on Nutrient agar. After series of test for morphological characteristics, results show a gram positive, encapsulated, non-flagellated and nonendospore forming cocci. Further tests for physiological characteristics would reveal that the unknown bacterium is a facultative anaerobe, catalase positive microorganism which requires 30-45C and supplements for growth. Results also reveal that the unknown microorganism belongs to Group 18. Further tracing of results using Bergeys identification flowchart for gram positive cocci bacteria reveals that the unknown bacterium is Staphylococcus aureus.

LITERATURE CITED Books Bauman Jr., R.W.2005. Understanding Microbiology: An Introduction.USA: Pearsons, p. 314- 316. Harley, J.P and L.M. Prescott. 2002. Laboratory Exercises in Microbiology. Fifth Edition. USA; McGraw- Hill. p. 142- 143 Ingrahan, J.L. and C.A. Ingraham. 2004. Introduction to Microbiology. USA: Thomson. p. 304- 305.

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Koolman, J and K.H. Roehm. 2005. Color atlas of Biochemistry. Second Edition. NY: Thieme- Stuggart. p. 356. Prescott, L.M. 2002.Microbiology. Fifth Edition. USA: McGraw- Hill. p. 123-25, 234, 332. Raymundo, A.K. 2001. Microbial Identification Techniques (Microbiology 101). Microbiology Division, Institute of Biological Sciences, University of the Philippines Los Baos, Laguna.p. 22-24, 29-30. Soligam- Hadsall, and others. 2007. A Practical Guide to Introductory Biodiversity Volume 1: Systematic Survey of Biological Diversity. Institute of Biological Sciences, University of the Philippines Los Baos, Laguna. p.1. Tortora, G.J., B.R. Funke, and C.L. Case. 2004. Microbiology: An Introduction. Eighth Ed, Singapore: Perason Ed. South Asia Pte. Ltd, pp 402-403. Zumdahl, S.S. 1992. Chemical Principles. USA: D.C. Heath. p. 34.

Journals Arthi K., B. Appalaraju, and S. Parvathi (2003). Vancomycin sensitivity and KOH string test as an alternative to gram staining of bacteria. Indian J. Med Microbiol. (2):121-123 Casida Jr., L.E.(1971). Microorganisms in Unamended Soil as Observed by Various Forms of microscopy and Staining. Appl Microbiol Journ. 21(6): 1040-1045. COWAN, S. T. (1965). Principles and practice of bacterial taxonomy-a forward look. J. gen. Microbiol. 39, 143.

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