LABORATORY REPORT

COURSE: MICROBIOLOGY 2B

LECTURER: MRS. F. ENGLBRECHT

LAB TECHNOLOGIST: MR. H. SHITALENI

STUDENT: JACKSON THOMAS

#222067713

Table of Contents
1
Materials and Methods..............................................................................................................................3
Results.........................................................................................................................................................5
Discussion...................................................................................................................................................6
Conclusion...................................................................................................................................................8
References..................................................................................................................................................9

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Introduction
The aim of this practical experiment was to identify the organisms present in two specimens of
spoiled food. The specimens, labeled as A and B, were subjected to a series of microbiological
tests to determine the types of bacteria present. These tests were selected based on their
ability to identify specific characteristics and virulence factors associated with different bacterial
species (Vila-Farrés, 2017). This report presents the results of these tests and discusses the
identification of the organisms found in the specimens.

Materials and Methods

INOCULATION
To perform this laboratory experiment, 4 different specimens are required. Using the 4-
way streaking method, the samples were inoculated onto 2 blood agar plates, one plate
was incubated aerobically at 37 degree celsius and the other anaerobically at 37 degree
celsius.
MacConkey agar (no crystalline violet) and TSA agar were inoculated with both
organisms and incubated aerobically at 37 degree celsius.
GRAM STAINING
Each organism should be applied to a glass slide and allowed to air dry. Subsequently,
each slide should be stained using standard Gram staining procedures. Firstly the
crystal violet solution is poured onto the slide for 1 minute, followed by rinsing with tap
water. Secondly, the slide is soaked with mordant for 1 minute and rinsed with tap
water. The smear is than discolored with acetone and then washed with tap water.
Finally, Carbol Fuchsin is used for counter staining, followed by rinsing with tap water,
and allowing air drying.
The catalase test is to be performed using the provided culture plates, where a colony is
transferred from the culture medium onto a slide. The colony is then exposed to 1 drop
of 3% hydrogen peroxide. The immediate observation of bubbles is indicative of positive
a result. Conversely, the absence of bubbles signifies a negative result. The process
should be repeated for both samples.
The motility test involves the impalement of each sample with the organism into a semi-
solid motility agar tube, with repetition of this process for both organisms, followed by
overnight incubation (at 37 degree celsius) of the tubes.
The peptone and MRVP assays are carried out by inoculating each sample into the
respective tubes and then incubated overnight at 37 degree celsius.
To conduct the carbohydrate assay, glucose, maltose, lactose, sucrose, and fructose
are needed in the Durham tube medium. Each individual carbohydrate tube is

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inoculated using aseptic technique with a microbiological loop. This process is repeated
for each organism, and the tubes are incubated overnight at 37 degree celsius.
Lastly, for the urease production test, colonies from the provided culture are inoculated
into 1 tube of solid urea medium, with the procedure repeated for both organisms,
followed by overnight incubation at 37 degree celsius.

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Results
The following table summarizes the results of the tests performed on specimens A, B, C
and D

Media/Test Organism A Organism B Organism C Organism D

Positive Positive Positive
growth growth Positive growth
growth
No Beta Beta
BA (O2) haemolysis haemolysis No haemolysis haemolysis
Positive Scanty No growth
growth growth
Scanty growth
Beta No
BA (AnO2) haemolysis haemolysis
4+ growth 3+ growth
3+ growth 4+ growth non-
non-lactose
Lactose non-lactose lactose
fermenter
MacConkey fermenter fermenter fermenter
Chocolate Agar 4+ growth 4+ growth 4+ growth 4+ growth
Negative Negative Negative
Gram stain bacilli bacilli Negative bacilli bacilli
Catalase Negative Positive Positive Positive
Oxidase Negative Negative Negative Positive
Glucose Fermentation Positive Positive Positive Positive
Lactose Fermentation Positive Positive Positive Positive
Sucrose Fermentation Positive Positive Positive Positive
Mannitol utilization Positive Positive Positive Positive
Positive Positive
Gas Gas
Motility producing Positive Positive producing
Urease Negative Negative Negative Negative
Salmonella P.
Organism Identification E. coli P. mirabilis species aeruginosa

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Discussion
The laboratory tests conducted provided a comprehensive approach to differentiating
and identifying organisms A, B, C and D based on their distinct characteristics and
virulence factors. The combination of morphological, biochemical, and growth-related
tests allowed for a systematic deduction of their identities.

TEST PRINCIPLES
Gram Stain
Principle: reaction is based on the difference of the physical or chemical properties in
the bacterial cell wall. Gram positive bacteria have a thick cell wall with high
peptidoglycan content, and it is able to retain crystal violet with iodine (Moyes, 2009).
Meanwhile gram negative bacteria have a thinner cell wall and lower peptidoglycan
content and are surrounded by an outer membrane composed of various lipids, and it is
unable to retain crystal violet- iodine complexes after decolourization.
Oxidase test
The test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of
the bacterial electron transport chain. The principle of the oxidase test depends upon
the mechanism of oxidation reaction, in which the organisms possessing cytochrome
oxidase enzyme can oxidize the tetramethyl-p-phenylenediamine dihydrochloride
(TMPD) reagent to indophenols, a purple or dark blue color end product. When the
enzyme is not present, the reagent remains reduced and is colorless (Cobos-Triguero,
2017).
Carbohydrate Use Test
Principle: This test determines an organism's ability to ferment specific carbohydrates.
Microorganisms can metabolize carbohydrates, producing various metabolic end-
products, such as acids and gases. The test involves inoculating a medium containing a
specific carbohydrate and observing for the production of acid (a drop in pH) or gas
(usually measured by gas production or by the presence of gas bubbles) as a result of
fermentation.
Voges-Proskauer (VP) Test
Principle: The VP test is used to detect the presence of acetoin, a metabolic product of
glucose fermentation. Some organisms produce acetoin when they ferment glucose.
This test involves adding reagents that oxidize acetoin, producing a red color in a
positive reaction. A negative reaction remains yellow.
Methyl Red Test

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Principle: The Methyl Red test assesses an organism's ability to perform mixed acid
fermentation of glucose. Organisms that produce a significant amount of acid from
glucose fermentation will lower the pH of the medium. Methyl red is added, and if the pH
remains below a certain threshold, the medium turns red (positive result). A higher pH
results in a yellow color (negative result).
Citrate Test
The citrate test is a biochemical test used to determine the ability of bacteria to utilize
citrate as a source of energy. The test is based on the principle that some bacteria have
the ability to use citrate as the sole carbon source and inorganic ammonium salts as the
sole source of nitrogen. The citrate utilization test is possible only if the organisms are
capable of fermenting citrate via the enzyme citrase.
Indole Test
Principle: The indole test detects the ability of an organism to produce indole from the
amino acid tryptophan. This test is performed by adding Kovac's reagent after
incubation. A pink/red color in the reagent layer indicates a positive result, indicating
indole production.
Nitrate Reduction Test
Principle: The nitrate reduction test assesses an organism's ability to reduce nitrate
(NO3-) to nitrite (NO2-) or further to nitrogen gas (N2) or ammonia (NH3). The test
involves the addition of nitrate reagents, and the presence of a red color indicates a
positive reaction, which suggests the reduction of nitrate to nitrite. Further testing may
be needed to differentiate between complete nitrate reduction and incomplete reduction.
Urease Production Test
Principle: This test determines whether an organism produces the enzyme urease,
which hydrolyzes urea to produce ammonia and carbon dioxide. A positive reaction is
indicated by a color change (often from yellow to pink) due to the alkaline conditions
created by ammonia production. This test is commonly used to identify organisms like
Helicobacter pylori.
Hydrogen sulphide
The hydrogen sulfide (H2S) test is a biochemical test used to detect the ability of
bacteria to produce hydrogen sulfide gas. The test is based on the principle that some
bacteria have the ability to reduce sulfur-containing compounds to sulfides during
metabolism. The test is commonly employed for the identification of bacteria in
laboratories. The test is performed by inoculating the bacteria onto a medium containing
sulfur compounds such as thiosulfate or cysteine. If the bacteria produce hydrogen
sulfide, it reacts with iron compounds in the medium to produce ferrous sulfide, a black
precipitate.

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Oxidation/Fermentation
The Oxidation/Fermentation (OF) test is a biochemical test used to differentiate
between oxidative and fermentative metabolism of glucose by bacteria. The test is
based on the principle that bacteria can metabolize glucose and produce acid either via
oxidation (aerobically) or via fermentation (anaerobically) or both. The OF test is
performed using a special medium called the oxidative-fermentation (OF) medium,
which contains glucose and a pH indicator, bromothymol blue.

Specimen A: Specimen A is identified as a Gram-negative bacillus that exhibits positive

growth on Blood Agar (anaerobic) with beta hemolysis. It is also a lactose fermenter on
MacConkey agar, catalase-positive, and capable of fermenting glucose, mannitol,
lactose, and sucrose. It is motile and produces gas, while it tests negative for urease.
Specimen A may belong to the Enterobacteriaceae family, possibly Escherichia coli.
Specimen B: Specimen B is identified as a Gram-positive bacillus that exhibits scanty
growth on Blood Agar (anaerobic) with no hemolysis. It is a non-lactose fermenter on
MacConkey agar, catalase-positive, and capable of fermenting glucose, mannitol,
lactose, and sucrose. Specimen B is most likely to be Proteus mirabilis.
Specimen C: Specimen C is identified as a Gram-positive bacillus that exhibits scanty
growth on Blood Agar (anaerobic) with no growth. It is a non-lactose fermenter on
MacConkey agar, catalase-positive, and capable of fermenting glucose, mannitol,
lactose, and sucrose. Specimen C belongs to the genus Salmonella.
Specimen D: Specimen D is identified as a Gram-positive bacillus that exhibits positive
growth on Blood Agar (aerobic) with beta hemolysis. It is a non-lactose fermenter on
MacConkey agar, catalase-positive, and capable of fermenting glucose, mannitol,
lactose, and sucrose. Specimen D is motile, produces gas, and tests positive for
oxidase, making it likely a member of the Pseudomonas species., P. aeruginosa to be
exact.

Conclusion
In conclusion, based on the results obtained from various tests, Specimen A is identified
as Escherichia coli, Specimen B identifies as Proteus mirabilis, Specimen C belongs to
the genus Salmonella, and Specimen D is identified as Pseudomonas aeruginosa.
These identifications are consistent with the observed characteristics of each specimen
in the tests conducted.

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9
References
Cobos-Triguero, N. Z. (2017). Time-to-positivity, type of culture media and oxidase test performed on
positive blood culture vials to predict Pseudomonas aeruginosa in patients with Gram-negative
bacilli bacteraemia. Revista espanola de quimioterapia : publicacion oficial de la Sociedad
Espanola de Quimioterapia.30(1), 9–13.

Moyes, R. B. (2009). Differential staining of bacteria: gram stain. Current protocols in microbiology,
Appendix 3.

Vila-Farrés, X. P.-M.-E.-A.-I. (2017). Combating virulence of Gram-negative bacilli by OmpA inhibition.

Scientific reports, 7(1).

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