Identification of the Unknown Bacterium by Cultural, Morphological and

Physiological Characterization Tests

Ma. Christine B. Cabatlao

Group 1, UV-2L

March 13, 2012

1A scientific paper submitted in partial fulfillment of the requirements in

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

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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 Bergey’s 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;

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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 Bergey’s 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

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methods and characterization tests. The Bergey’s 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 Bergey’s

Manual of Determinative Bacteriology.

The study was performed at Room 307, Wing B of the Institute of Biological

Sciences, University of the Philippines- Los Baňos, College, Laguna, Philippines from

December 13, 2011 to March 1, 2012.

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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 unknown’s identity.

The cultural characteristics of the bacteria were determined by growing the

unknown bacterium on Nutrient Agar. The plate was incubated at 28- 30˚C 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

Gregersen’s 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, Anthony’s method and Maneval’s Method were done to confirm

the presence of capsule.

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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: 10˚C, 30˚C, 45˚C and 60˚C. 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.

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After the results were gathered, they were mapped on the Bergey’s 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 RESULTS

Size 1 mm

Form punctiform

Surface shiny and smooth

Margin Entire

Pigmentation Cream/ beige

Elevation convex

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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, Gram’s staining and Gregersens’s method. Only

Gram’s stain and Gregersen’s method confirmed that the unknown bacterium is a Gram-

positive 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:

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

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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).

Gregersen’s 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 Anthony’s

method and Maneval’s 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).

Anthony’s 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

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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).

Maneval’s 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).

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

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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: 10˚C, 30˚C, 45˚C and 60˚C. Growth was

observed on plates incubated at 30˚C and 45˚C, indicating that the unknown bacterium

is a mesophile. Mesophiles are organisms with growth optima around 20 to 45˚C, with

minimum temperature often 15- 20˚C and maximum temperature about 45˚C 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.

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

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(Raymundo, 2001). Therefore the unknown microorganism has a fermentative

metabolism of mannitol.

Table 2. The morphological characteristics of the unknown bacterium.

MORPHOLOGICAL

CHARACTER

TESTS OBSERVATIONS CONCLUSION

Gram reaction Cepacol staining No distinct cells

were observed; very

dark background

Erroneous data

Gram stain Purple cells Gram positive

Gregersen’s method No slime Gram positive

Capsule formation Negative staining Cells and capsules

were colorless

against dark

background

encapsulated

Maneval’s staining Cells were too small

for capsule

observation

erroeneous

Anthony’s method Cells were too small

for capsule

observation

erroneous

Presence of

Flagella

Flagella staining No flagella

observed

Non-flagellated

Hanging drop

technique

No flagella

observed; cells

Non- flagellated

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were suspended

Motility band test 1 mm difference

from the point of

inoculation

Cellular movement

is present.

Endospore

formation

Schaeffer-Fulton

method

No green spherical

structures were

stained; all were

vegetative cells

Doesn’t produce

endospore

Table 3. The physiological characteristics of the unknown bacterium.

PHYSIOLOGICAL

CHARCTERS

OBSERVATIONS CONCLUSION

Oxygen requirement more growth on top of the

tube; growth also present

throughout the tube

Facultative anaerobe

Growth factors Growth on GFB; no growth

on GFA

Requires growth factors

Temperature requirement Growth on 30-45˚C mesophile

Catalase test Bubble formation Presence of catalase

Mannitol fermentation Both tubes changed from

purple to yellow

Positive for mannitol

fermentation

After series of tests, the results were mapped on Bergey’s Manual of

Determinative Bacteriology to identify the unknown bacteria. The table below, Table 4

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shows the grouping of bacteria according to Bergey’s Manual of Determinative

Bacteriology.

Table 4. Bacteria groupings according to Bergey’s Manual of Determinative Bacteriology.

GROUP DESCRIPTION DIFFERENCES EXAMPLES

Group 4 Gram Negative,

Aerobic/Microaeroph

ilic rods and cocci

pigments/fluorescent,motility, growth requirements, denitrification, morphology, and oxidase, read Genera descriptions

Acinetobacter,

Pseudomonas,

Beijerinckia,

Acetobacter

Group 5 Facultatively

Anaerobic Gram

negative rods

growth factors,

morph., gram

reaction., oxidase

reaction., read

Genera

descriptions

Family

Enterobacteriace

ae and

Vibrionaceae

Group 17 Gram-Positive Cocci oxygen requirements, morph., growth requirements (45°C and supplements),read Genera

descriptions

Micrococcus,

Staphylococcus,

Streptococcus,

Enterococcus,

Lactococcus,

Aerococcus

Group 18 Endospore-Forming

Gram positive rods

oxygen

requirements,

Bacillus,

Clostridium

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and cocci motility,

morphology,

catalase reaction

Group 19 Regular,

Nonsporlating Gram

positive rods

morphology,

oxygen require,

catalase reaction

Lactobacillus,

Listeria

Group 20 Irregular,

Nonsporlating

Gram-positive rods

catalase, motility,

morph., read

Genera

descriptions

Actinomyces,

Corynebacterium

, Arthrobacter,

Propionibacteriu

m

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 (45°C 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 Bergey’s Manual of Determinative Bacteriology.

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Figure 2. Bergey’s 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 non-

endospore forming cocci. Further tests for physiological characteristics would reveal that

the unknown bacterium is a facultative anaerobe, catalase positive microorganism which

requires 30-45˚C and supplements for growth.

Results also reveal that the unknown microorganism belongs to Group 18.

Further tracing of results using Bergey’s identification flowchart for gram positive cocci

bacteria reveals that the unknown bacterium is Staphylococcus aureus.

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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:

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Prescott, L.M. 2002.Microbiology. Fifth Edition. USA: McGraw- Hill. p. 123-25, 234, 332.

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