sci paper mcb 101
DESCRIPTION
a sample microbiology 101 fileTRANSCRIPT
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.
1
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;
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 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
3
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.
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 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.
5
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.
6
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
7
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
8
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
9
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
10
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
11
(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
12
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
13
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
14
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.
15
Figure 2. Bergey’s identification flowchart for gram positive cocci bacteria.
SUMMARY AND CONCLUSIONS
16
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.
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.
17
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 Baňos, 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 Baňos, 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.
18
Salanitro, J.P., I.G. Fairchilds, and YD Zgornicki. (1974). Isolation, Culture
characterization and Identification of Anaerobic Bacteria from the Chichen
cecum. Appl. Microbiol.. 24(7):678-687
STEELK, . J. (1965). The practice of bacterial identification. Symp. Soc. gen. Microbiol.
12,405.
NUNGESTER,. F. (1963). Contributions of microbiology and immunology to medicine
and some unfinished business. Tcxas Rep. Biol. Med. 21, 315
19