THE UNIVERSITY OF DAR ES SALAAM.

COLLEGE OF NATURAL AND APPLIED SCIENCES.

DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY.

PRACTICAL REPORT.

COURSE CODE: MC 130

COURSE NAME: METHODS AND SAFETY IN MICROBIOLOGY.
STUDENT’S REGISTRATION NUMBER: 2020 – 04 – 06606
STUDENT’S NAME: MCHARO, JONATHAN ABINERI
DEGREE PROGRAM: BSc. Microbiology
DATES OF PRACTICALS: 14/01/2021, 20/12/2021, 21/12/2021, 27/01/2021 and 28/01/2021.
DATE OF SUBMISSION: 10/02/2021
PRACTICAL NUMBER: Practical 4
PRACTICAL SESSION: IDENTIFICATION OF MICROORGANISMS USING CULTURE
CHARACTERISTICS AND BIOCHEMICAL METHODS.
NAME OF INSTRUCTORS: DR. MPINDA C. B
ABSTRACT
The aim of this practical with all it’s related activities of different ways of performing
biochemical tests is to test and identify different types of bacteria based on their physiology
and the substances they produce or utilize. The biochemical tests include catalase test and
oxidase test for identifying bacteria that produce catalase and oxidase respectively. Other tests
include Indole test, methyl red test, Voges-proskauer test, catalase test and coagulase test .
The tests are all performed in direct procedures and the results after each test is usually
described using color, substance formed or growth and the types of growth.
INTRODUCTION
BIOCHEMICAL tests are the tests performed for the aim of identifying bacteria species based on
their differences in their biochemical activities. Bacteria physiology differs from one species to
the other, these differences in carbohydrates, proteins and fat metabolism and production of
certain enzymes and ability to utilize a particular compound provide a basis for the types of
biochemical tests performed.(See: Benson. 2001. Microbiological application lab manual. McGraw-Hill
companies. 8th edition.)

These considerations are due to the tendency of bacteria being rarely recognizable by their
microscope morphology alone. Bacteria have a tendency of growing in groups of different
species on culture media but they can be isolated and grown as a pure culture. Some of the
biochemical tests performed to identify certain types of bacteria are as the following:
Catalase test; This type of test is important in testing of bacteria Staphylococcus from
Streptococcus by their ability to produce catalase enzyme which is an enzyme produced in
some organisms for catalyzing the release of Hydrogen Peroxide. What happens in testing and
culturing is that in a culture, the enzyme Superoxide dismutase catalyzes the formation of
Hydrogen Peroxide while the catalase enzyme breaks down the formed Peroxide to water and
molecular oxygen.
Oxidase test; This test is used to test for the ability of some bacteria to produce an enzyme
called oxidase, which catalyzes the transport of electrons. The test organism are usually species
of Naisseria, Vibrio, Pseudomonas, Alkaligenes and Aeromonas which give positive reactions.
Indole test; This test is used in demonstrating the ability of some bacteria in decomposing an
amino acid called tryptophan to a substance called Indole where the product's presence is the
one tested for using a reaction with Kovac's reagent (P-methylaminobenzaldehyde).
Methyl red; This test is used to distinguish various coliform bacteria from each other. In this
test, when a drop of Methyl red reagent is added after 48 hours of incubation of the organisms,
the culture of positive results will be found to be acidic due to the ability of the bacteria of test
to produce glucose strongly this leading to a pH fall in the medium.
Citrate Utilization test; This test is used to test for the ability of some bacteria to use or
utilize Citrate as the main source of carbon and an energy source for enabling metabolism and
growth together with ammonium salt as the main source for Nitrogen. In this test, Koser's liquid
medium is commonly used. Enterobacter specie and Klebsiella species grow in citrate while
Escherichia Coli and Pseudomonas fail to grow in citrate medium.
Coagulase test; Coagulase test is an enzyme produced by Staphylococcus aureus that
performs the conversion of fibrinogen to fibrin. This reacts with prothrombin in blood to form
“Staphylothrombin” which causes blood to clot by converting fibrinogen to fibrin. This test is
used to test for the ability of the bacteria to produce coagulase enzyme and forming fibrin in
the process.
METHODOLOGY
ACTIVITY 1: CATALASE TEST
Materials Used:
Materials used in this activity were; Hydrogen peroxide solution, 6glass slides, culture plates
with organisms inoculated from hair, bench and the skin and a Bunsen burner.
Procedures:
1. A drop of Hydrogen peroxide was put on a glass slide in a drop on one side of the slide
and one on the other.
2. Using the edge of a sterile glass slide, a speck of growth from the culture of hair sample
was picked and suspended into one of the drops of Hydrogen Peroxide.
3. The procedures were repeated for all the cultured organism, that is for bench and skin
using the remaining glass slides.
4. Observations were made after the procedures.
ACTIVITY 2: OXIDASE TEST
Materials Used:
The materials used were; OXIDASE reagent, a moist filter paper, 4 glass slides and cultured
organism from hair, skin and bench.
Procedures:
1. A few milliliters of Oxidase reagent were added on a folded filter paper in 3 different
drops linearly and laid on a clean glass slide.
2. Using an edge of a clean glass slide, a speck of growth of the cultured organism from
hair were picked from the media plate and smeared on the soaked filter paper.
3. The procedures were done for the remaining organisms into the remaining soaked
portions and observations were done almost immediately.
ACTIVITY 3: INDOLE TEST
Materials Used:
The materials used for this activity were 3 test tubes containing 4ml of Tryptophan broth which
had a composition of [Casein enzymatic hydrolysate(Trypton) 10g/L, Sodium Chloride 5g/L, DL –
Tryptophan 1g/L, pH 7.5], a wire loop, cultures of Staphylococcus aureus, E.coli and bacteria
from bench sample and Kovac’s reagent.
Procedures:
1. A sterilized test tube containing 4ml of tryptophan broth was taken and inoculated
aseptically by taking the growth from 18 to 24 hours culture.
 2. After, the inoculated test tube was incubated at 37°C for 24 – 28 hours.
3. After the successful incubation time, a 0.5ml of Kovac’s reagent was added to the broth
culture.
4. The procedures were repeated for all cultured organisms.
5. The test tubes were then settled and observations were made for the absence or
presence of ring.
ACTIVITY 4: METHYL RED (MR) TEST
Materials Used:
Materials used were 3 test tubes, methyl red indicator, a conical flask containing culture media,
agar plate containing cultured organism and a Bunsen burner.
Procedures:
1. The medium was divided into three test tubes each by 5mls and then the test tubes
were sterilized in order to be inoculated.
2. Prior to inoculation, the medium was allowed to equilibrate at room temperature.
3. Test tubes containing media were then incubated aerobically at 37°C for 24hours.
4. After successful incubation time, 1 ml of the broth was taken to a clean test tube.
5. The remaining growth was incubated again for another 24hours.
6. After a successful re-incubation time, 3drops of Methyl red indicator were added into
the test tubes.
7. The test tubes were then left to settle and immediately, observations for the presence
or absence of red color were done.
ACTIVITY 5: VOGES-PROSKAUER (VP) TEST.
Materials Used:
The materials used were 3 test tubes, conical flask containing culture medium, a wire loop, a
Bunsen burner and Potassium hydroxide solution and cultures organisms in agar plate medias.
Procedures:
1. Culture media were divided into 3 test tubes each at 5mls and sterilized, ready for
inoculations
2. Prior to inoculations, the medium was allowed to equilibrate at room temperature.
3. The medium was then inoculated by using a sterilized wire loop by organism from the
pure cultures.
4. The test tubes containing medium were incubated aerobically at 37°C for 24 hours.
5. After 24 hours of incubation, 2ml of broth was taken into a clean test tube.
6. After re-incubation, 2 drops of 40% Potassium hydroxide were added and then mixed
well to aerate.
7. The resulting mixtures were then shaken vigorously for a 30 minutes straight.
ACTIVITY 6: CITRATE UTILIZATION TEST.
Materials Used:
Materials used for this activity were 3 test tubes containing a slant containing citrate, a wire
loop, a Bunsen burner and isolated colonies of E.coli and Saccharomyces aureus.
Procedures:
1. The slant for each tube was streaked back and forth with a wire loop from the center of
a well isolated colony of E. Coli and the same was done in the second test tube
containing slant for S. aureus organism.
2. One test tube containing slant was left un-inoculated and was labeled control.
3. The slants were then incubated aerobically at 35 – 37°C for 7 days straight.
4. After 7 days of incubation, the slant was examined to observe any changes in the slant
and for any growth on the slant.
ACTIVITY 7: COAGULASE TEST.
Materials Used:
Materials used were a glass slides, a wire loop, a Bunsen burner, human plasma, normal saline
and cultured organisms of Staphylococcus aureus and Staphylococcus epidermis both in pure
cultures
Procedures:
1. At the two ends of a slide, a drop of normal saline was added.
2. Aseptically by using a sterilized wire loop, a portion of isolated colony was emulsified in
each drop and made a thick suspension
3. After that, a drop of human plasma was added on the suspensions at both ends and
mixed gently.
4. After mixing gently, the suspensions were left for 10 seconds to observe for clumping of
the organisms.
5. On the other suspension, plasma was not added in order to differentiate between the
two suspensions.
RESULTS
ACTIVITY 1: CATALASE TEST
The results obtained were all positive for the organism from hair, skin and bench by
producing bubbles on each slide that indicates break down of Hydrogen Peroxide into
oxygen and water.
ACTIVITY 2: OXIDASE TEST
The results obtained showed bench organism turned purple right after being placed on
the Oxidase reagent moist parts of the filter paper.
The organism from skin and hair turned purple slowly on the moist paper after being
placed.
ACTIVITY 3: INDOLE TEST
The results observed showed that in the test tube inoculated with E. Coli formed a red
ring on the meniscus of the mixture thus indicating positive results.
The remaining test tubes inoculated with Staphylococcus aureus and organism from the
bench sample formed yellow rings on their mixtures indicating negative result.
ACTIVITY 4: METHYL RED TEST
The results observations showed that in the test tube inoculated with organism from
the bench sample turned red, indicating positive results (Methyl red positive)
The test tube inoculated with E. coli and Staphylococcus aureus after addition of Methyl
red retained the yellow color of the reagent thus indicating negative test results.
ACTIVITY 5: VOGES-PROSKAUER TEST
The results obtained after the experiment indicated no change of color in all the test
tubes from the bench, E. coli and Staphylococcus aureus.
ACTIVITY 6: CITRATE UTILIZATION TEST
The slant inoculated with Staphylococcus aureus and E. coli both showed positive results
by turning the forest green color of the slant blue and growth of the organisms in the
inoculated area is observed.
The control slant showed no changes after inoculation but the forest green color of the
slant was retained.
ACTIVITY 7: COAGULASE TEST
The results showed Staphylococcus aureus being positive because it’s plasma clotted
while for the plasma placed on Staphylococcus epidermis showed no changes
DISCUSSION
ACTIVITY 1: CATALASE TEST
Catalase test is a biochemical test for aerobic organisms that detect the production of
catalase enzyme in the organism. Catalase enzyme is a common enzyme found in all living
beings found that can survive in the presence of oxygen and catalyzes the decomposition of
Hydrogen Peroxide releasing water and oxygen.
The main purpose of catalase test is to detect the ability of organisms to produce the
catalase enzyme as well as to differentiate catalase positive organisms like Staphylococci from
catalase negative organisms like Streptococci.
The principle involved in a catalase test is that the metabolic activity of aerobic and
facultative anaerobic microorganisms produce toxic product’s like Hydrogen Peroxide and
Superoxide. These products are toxic to the organism and might even result in cell lysis if not
broken down thus bacteria capable of synthesizing the enzyme catalase use it to hydrolyze
Hydrogen Peroxide into water and Oxygen which results in the liberation of gas bubbles.
(catalase)H2O2 ➝ H2O + O2

Production of catalase therefore protects the organism against the lethal effect of Hydrogen
Peroxide accumulation at the end of the respiratory metabolism.

ACTIVITY 2: OXIDASE TEST

Oxidase test aims at determining the ability of the organism to produce the cytochrome
oxidase test.
The principle for oxidase test is that the test is designed for specifically detecting the
presence of the terminal enzyme system in aerobic respiration called Cytochrome C Oxidase or
Cytochrome a3. The ability of an organism to produce the cytochrome C Oxidase can be
determined by using the reagent tetramethyl-p-phenylenediammine dihydrochloride which
serves as an artificial substrate by donating electrons and thereby becoming oxidized to a deep
purple compound in the presence of the enzyme oxidase.
Development of pink, then maroon and finally dark purple coloration after rubbing the
organism in the Oxidase disc containing the reagent indicated positive reaction and no color
change usually indicates negative results.
ACTIVITY 3: INDOLE TEST
Indole test is a biochemical test conducted on bacterial species to detect their ability to
produce indole from Tryptophan in the presence of a group of enzymes called “Tryptophanase”
In the experiment, the test for E. coli proved to be positive by forming a red colored ring in
the reagent layer on top of the medium within seconds of adding the reagent because the
organisms have the ability to produce indole due to reductive deamination from Tryptophan is
the intermediate molecule indole pyruvic acid in which the other tested organisms
(Staphylococcus aureus and organisms from bench sample) thus giving negative results.

ACTIVITY 4: METHYL RED TEST

This test is used to differentiate two major types of facultative anaerobic enteric bacteria
based on the production of acid by identifying bacterial ability to produce stable acid end
products by means of a mixed acid fermentation of glucose.
The results of Methyl red test showed that the E. coli we’re methyl positive because their
medium turned red upon addition of methyl red. This could be because they have the ability to
produce and maintain stable acid end products from glucose fermentation. The organism
inoculated from the bench together with Staphylococcus aureus gave negative results by
retaining the color of the reagent.

ACTIVITY 5: VOGES-PROSKAUER TEST

This test is usually done together with the above Methyl red test although it is used to
determine the ability of some organisms to produce neutral end products from glucose
fermentation.
The results of this test indicated no change in color in all 3 mixtures even after shaking the
mixtures and this could be because the organisms have no ability to produce neutral end
products from glucose fermentation and other reasons may be the cultures not being pure thus
affecting the results, but also could be that there was a prolonged incubation of the organisms.

ACTIVITY 6: CITRATE UTILIZATION TEST

The results obtained showed positive results for E. coli although this isn’t theoretically
correct since normally, these organisms can not ferment citrate in the presence of citrase
enzyme. This result might be due to contamination by other microbes which can utilize citrate
as a sole source of carbon thus altering the results.
Few types of bacteria that can be differentiated by using citrate test include Klebsiella
pneumoniae, Proteus mirabilia, Enterobacteriacae and Salmonella typhii.
Limitations of this test can be that luxuriant growth on the slant without an accompanying color
change may indicate a positive test. Tests with ambiguous/equivocal results should be
repeated, the slant should not be stabbed since the test requires an aerobic environment. But
also inoculation should not be done from broth cultures due to the risk of carry over of media.

ACTIVITY 7: COAGULASE TEST

Coagulase test is used to differentiate Staphylococcus aureus from other Staphylococci
specie like S. epidermis as on this experiment where results varied on the basis of the ability to
produce the coagulase enzyme by S. aureus from S. epidermis.
CONCLUSION
Conclusively, the knowledge of biochemical tests is of vital importance to all fields that use
microbiology as the way of identifying bacteria can be difficult at times with the use of a
microscope but by the use of such tests can be a primary identification that could lead to
further examinations with a guided beginning.
Microbial biochemistry tests shorten the time required to identify microbes, reduce costs and
ensure or enhance the accuracy of identification of an unknown sample. Infact, it is the fastest
developing trend in microbial identification. Due to the different types of experiments
performed, the readouts for results are also different.
 REFERENCES

 Benson. 2001. Microbiological application lab manual. McGraw-Hill

companies. 8th edition.
 Martino M.J, Parker J. 1997. Brock biology of Microorganisms. Prentice
hall. 14th edition
 Leboffe M. J, Pierce E. B. 2010. Englewood, Colorado. Microbiology
Laboratory Theory & Application. Douglas N. Morton. 3rd Edition