Microbiology Lab Report

EM

IDENTIFYING AN UNKNOWN MICROORGANISM

The identification of microorganisms is a process which occurs throughout a standard

scientific experiment which will eventually help to determine the actual microbe within a diverse

type of microorganisms. The goal of this experiment was to identify an unknown bacterium

within six possible microorganisms that was assigned by the instructor in which nine tests were

performed in order to identify the unknown microorganism in question within a given testing

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chart seen in the picture below. The nine tests were Gram stain, Morphology, Starch, Oxidase,

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Urease, Citrate, Mannitol Salt Agar (MSA), Nitrate Reduction, and Methyl Red (MR) and

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Voges-Proskauer (VP) which were the sole pathway of determining the unknown bacterium.
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 Microbiology Lab Report
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First of all, the sample given by the instructor was called sample #4 and the whole

experiment was done with a lab partner in which each party obtain their respective data based on

their own experiment performance. A bit of sample #4 was obtained with a heated sterile loop

and placed into watered area of the slide thru the method called preparation of smears. Then, the

slide was placed for two seconds over the flame burner alternatively within two to three time in

order to fix the smear on the glass slide which would proceed with the Gram staining.

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In fact, Gram staining is the primordial step into the phenotypic identification of

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microorganism in order to differentiate the indefinite bacterium as gram-positive or gram-
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negative. The fixed sample #4 or smear was washed with crystal violet for approximatively 30

seconds then washed away with distilled water. Then, it was washed with Gram’s iodine for
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approximatively 30 seconds and next eroded with distilled water. The next step was quickly
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washing the smear with the decolorizer 95% ethanol and then washed with distilled water. The

final step of the Gram Staining was the usage Safranin over the smear for 30 seconds and washed
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with distilled water. The slide was completely blot dried up with absorbent papers; Then, it was

placed under the microscope in order to visually identify the color and morphology of sample #4
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which would automatically cover two of the nine tests.

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The unknown bacteria were definitely a gram-negative because it demonstrates a red/pink

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color under the microscope. At the same time, the morphology of the bacterium revealed to be

rod shape. Based on these results, two of the six microorganisms which were gram-positive were

eliminated from the unknown microorganism determination such as: Bacillus subtilis and

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 Microbiology Lab Report
EM

Staphylococcus aureus according to the chart. The next test was Starch testing in order to

determine the production of amylase. Sample #4 was collected with a heated sterile loop and

placed into Starch Agar plate as the actual plate was separated in two sides by the respective

partner working on it. Then, iodine was poured all over the plate and was incubated for a week.

Later on, it was observed that the black color surrounding the bacterial growth presented the

absence of starch hydrolysis which concluded to be negative according to the chart.

The oxidase test was used to identify bacteria that contain cytochrome oxidase. Sample

#4 was independently smeared accordingly on a glass slide wherein oxidase reagents was poured

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in as drops. As a result, smeared sample #4 stayed intact which demonstrated a negative oxidase.

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According to the given Chart, Pseudomonas aeruginosa was eliminated as the possible

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unknown bacteria because it only showed a positive oxidase result which left three more bacteria
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to be tested in the following tests. The next step was to perform Urease testing where a small
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amount of sample #4 was collected with a heated sterile loop and placed into the Urease tube,
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after been inverted several times with the intention of mixing the sample with the solvent. Thus,

the color was visible changed to pink color which indicated that it was positive and the bacteria
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obviously produced urease enzyme. According to the given Chart, Enterobacter aerogenes and
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Escherichia coli were eliminated from the list as the possible unknown because they showed

negative results which left one more microorganism to be further tested.

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Then, the next step was to perform Citrate test as tool to determine if the microorganism

could utilize sodium citrate as a carbon source. A small amount of sample #4 was collected with
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a heated sterile loop which was now shaped as a needle and placed into the citrate agar in a test

tube. However, the test tube was closed and stood on the rack for approximatively a week until

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 Microbiology Lab Report
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observation was made. Hence, sample #4 stayed green and intact in the tube which concluded

that it was negative.

The next step was Mannitol Salt Agar (MSA) with the sole purpose of identifying the

endurance of microorganisms into high concentration levels of salt. A small amount of sample #4

was collected with a heated sterile loop and placed into the MSA agar plate. Yet, the plate was

incubated for a week before observing the outcome of the test. Therefore, the Mannitol Salt Agar

(MSA) plate haven’t demonstrated any changes which came into the conclusion that the test was

negative. Then, Nitrate Reduction test was used to determine the possible production of nitrate

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reductase enzyme by microorganisms. A small amount of sample #4 was collected with a heated

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sterile loop and placed into to the nitrate broth test tube and was inverted several times with the

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intention of mixing the sample with the solvent. Hence, five drops of Nitrate A were added and
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thereafter five drops of Nitrate B were added into the nitrate broth test tube. Therefore, the nitrate
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test tube turned red color which was tested positive.

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The final ninth test was performed with Methyl Red (MR) and Voges-Proskauer (VP)

thru a MRVP broth. First, five drops of Methyl Red (MR) was utilized into the (MRVP) test tube
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to identify bacteria capacity of producing stable acids thru acid fermentation of glucose as a pH
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indicator. As a result, the medium indicated to be at the PH 4.2 in which the (MRVP) test tube
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was visibly red indicating a positive result. Then, Voges-Proskauer (VP) test was performed
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within two sets of V-P reagent I and II which were alternatively added into another MRVP Broth.

The process was done with the counting of 12 drops of V-P reagent I and 3 drops of V-P reagent
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II into the MRVP Broth which was exposed to the air for 30 minutes in order for the acetoin to be

oxidized. Consequently, a yellow-brown color appeared to the test tube broth which indicated a

negative result.

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 Microbiology Lab Report
EM

Proteus Vulgaris

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In Conclusion, the final result of the experiment indicated on the unknown

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microorganism was accurately referred to Proteus Vulgaris according to the chart guideline.

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Actually, Proteus vulgaris (P. vulgaris) is a genus of Gram-negative bacteria with rod shaped

commonly found in the environment and in normal gut flora of the human body. It produces
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secreted protease enzymes from fimbriae which helps into the digestion of immunoglobulins.
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Proteus Vulgaris bacteria is motile by Peritrichous Flagella which is member of the

Enterobacteriaceae which is part of Gram-negative bacteria family also found in fecal matter. It
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can also cause pyelonephritis or kidney stones and Bladder stones in which it stimulates the
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urease enzyme overproductions of ammonia into these organs which origins possible urinary
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tract infections. On the other hand, Proteus vulgaris also causes diarrhea especially in infants.
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Although, Proteus Vulgaris is extremely resistant against penicillin, the best treatment options

against this bacterium remains penicillin or aminoglycosides. Henceforth, the current effective
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drugs used to cure Proteus Vulgaris infections are Ureidopenicillin, Cephalosporins,

Aminoglycosides, Imipenem, Ciprofloxacin, and Trimethoprim/sulfamethoxazole.

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 Microbiology Lab Report
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References:

1. Preparation of smear and Gram staining steps. Johnson, T. R., & Case, C. L. (2016).
Laboratory Experiments In Microbiology Eleventh Edition. United States: Pearson.
Retrieved December 4th, 2016 between pgs 51-63.

2. Summary of Biochemical Tests. Retrieved December 4th, 2016, from

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http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm#catalase
3. Medical terms and definition. Retrieved December 4th, 2016, from http://medical-

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dictionary.thefreedictionary.com/Proteus+vulgaris

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4. Peer review on Proteus Vulgaris; Journal Issue: International Journal of Genetics and
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Genomics. Retrieved December 4th, 2016, from

http://escholarship.org/uc/item/0r7691jq#page-1
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5. Proteus Vulgaris Morphological, structural and physiological. Retrieved December 4th,

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2016, from http://ww3.bergen.edu/faculty/jsmalley/pvulgaris.html

6. Proteus Vulgaris Scanning Electron Micrograph (SEM) picture. Retrieved December 4th,

2016, from http://fineartamerica.com/featured/5-proteus-vulgaris-bacteria-sem-thomas-

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deerinck-ncmir.html
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