Unknown #16 Lab Report

Presented by:
Ley-Senan Qualls

April 30, 2019

MICROBIOLOGY NON-SCI MAJORS

The study of microbiology requires not only an academic understanding of the microscopic world but also a practical
understanding of lab techniques and procedures used to identify, control, and manipulate microorganisms. The proper identification of
a microorganism is important in the medical, industrial, and pharmaceutical fields. In this lab report, lab techniques and procedures
learned during this course were performed to assess practical knowledge in microbiology. If I understand the methods and lab
techniques that I learned during the semester, then, I will be able to explain the tests that I performed, properly identify my unknown,
and give a background on the characteristics of my unknown organism.

Materials and Methods

1. Negative stain

Materials: microscope slide, inoculating loop, flint Stricker, Bunsen burner, water bottle, bacteria culture, nigrosin, microscope.

Method: A small spot of nigrosine was put on the clean microscope slide. The culture was then aseptically transferred to the spot
and spread out thinly and evenly. The smear was later allowed to air dry completely. Lastly, the slide was examined microscopically at
1000X magnification to visualize morphology

2. Heat-fixed smear

Materials: microscope slide, inoculating loop, flint Stricker, Bunsen burner, water bottle, bacteria culture.

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 Method: A loopful of water was transferred to the clean microscope slide. The bacterial culture was aseptically transferred to the
water on the slide, and a smear the size of a nickel was made. The smear air dried completely and was heat-fixed by passing it 3 to 4
times through the top portion of the Bunsen burner flame.

3. Gram stain

Materials: microscope slide, inoculating loop, flint Stricker, Bunsen burner, water bottle, bacteria culture, staining tray and rack,
clothespin, test tube rack, crystal-violet, iodine, safranin, Gram’s decolorizer, microscope, immersion oil.

Method: A heat-fixed smear was made. The smear was then covered by crystal-violet 60 seconds. The slide was rinsed with water
to remove unbound dye. The smear was covered with iodine for 60 seconds and the slide was rinsed with water to remove iodine.
Holding the slide at an angle, decolorizer was added one drop at the time, until unbound dye is gone, and the slide was immediately
rinsed with water to stop decolorization. The smear was covered with safranin for 1.5 minutes and was rinsed with water to remove
unbound dye. The smear was finally blotted dry with bibulous paper and examined microscopically at 1000X magnification.

4. 3% KOH test

Materials: 3% KOH microscope slide, inoculating loop, flint Stricker, Bunsen burner, bacteria culture.

Method: A tiny drop of 3% KOH was deposited on the clean microscope slide. Then, a visible clump of the culture was aseptically
transferred to the drop. The culture was mixed with the KOH in a small, slow, circular motion with the loop parallel with the slide (for
up to 60 seconds). The loop was periodically lifted to look for stringing.

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 5. Vancomycin test

Materials: Mueller-Hinton (MH)Agar plates, sterile swabs, flint Stricker, Bunsen burner, bacteria culture, forceps, vancomycin.

Method: Using a sterile swab, a small sample of bacteria was collected. The surface of the MH agar plate was lightly swabbed in a
way that ensured complete coverage. A 5-microgram vancomycin disk was placed on the culture. Sterilized forceps were used to
ensure contact between the disk and the agar surface. The plate was then inverted and incubated at 37 degree Celsius for 18 to 24
hours. The results were read following incubation.

6. IMViC series: Indole test

Materials: SIM (sulfur reduction, indole, and motility) medium, flint Stricker, Bunsen burner, bacteria culture, Kovac’s reagent,
inoculating needle, test tube rack.

Method: The medium was straight stab inoculated and incubated at 37 degree Celsius for 24 to 48 hours. Following incubation, 5
to 10 drops of Kovac’s reagent was added to the SIM tube.

7. IMVic series: Methyl Red-Voges Proskauer (MRVP) tests

Materials: MRVP broth, inoculating loop, flint Stricker, Bunsen burner, bacteria culture, Methyl Red pH indicator, Alpha-naphthol
(VP A) reagent, Potassium hydroxide (VP B) reagent, empty sterile tubes with caps, sterile graduated pipettes.

Method: The MRVP broth was aseptically inoculated with organism and incubated at 37 degree Celsius for 24 to 48 hours.
Following incubation, after checking for growth, a pipette was used to transfer 2 ml of the original tube into an empty test tube. 5 to 10
drops of Methyl Red PH indicator were added to the original tube and the result was read immediately. Then, 10 drops of VP A and
10 drops of VP B were added to the 2ml of MRVP; the cap was put on, and the tube was gently shaken for 5 to 10 minutes.

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 8. IMViC series: Citrate test

Materials: Simmon’s citrate agar, inoculating needle, flint Stricker, Bunsen burner, bacteria culture.

Method: The medium was inoculated using the stab and streak technique. With the test tube cap loose, the culture was incubated at
37 degree Celsius for 24 to 48 hours. The results were read following incubation.

9. Lysine decarboxylase test

Materials: Lysine medium, mineral oil, inoculating loop, flint Stricker, Bunsen burner, bacteria culture.

Method: The Lysine medium was aseptically inoculated using an inoculating loop. Several drops of mineral oil were added to the
tube to provide an anerobic environment for glucose fermentation to acid. The broth was incubated at 37 degree Celsius for 24 to 48
hours.

10. Phenyl Red Lactose

Materials: PR lactose tube, inoculating loop, flint Stricker, Bunsen burner, bacteria culture.

Method: The culture was aseptically inoculated into the broth. The broth was then incubated at 37 degree Celsius for 24 to 48
hours.

11. Phenyl Red Mannitol

Materials: PR mannitol tube, inoculating loop, flint Stricker, Bunsen burner, bacteria culture.

Method: The culture was aseptically inoculated into the broth, which was incubated at 37 degree Celsius for 24 to 48 hours.

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 Results

Tests Reagents and media Observations Results Interpretations

Neg stain Nigrosin Rod shape - Bacteria is bacillus

Gram stain Crystal violet, Gram’s iodine, Pink rods Gram neg rods Organism is gram negative

decolorizer, safranin

KOH 3% KOH Stringing (DNA)/cell lysis - Organism is gram negative

Vancomycin Vancomycin Growth throughout medium R Organism is resistant to vancomycin; hence it

is Gram negative

Indole Kovac’s Top of the medium turned Indole positive Indole is present in medium as the end-

cherry red product of tryptophan hydrolyzation by

tryptophanase

Methyl Red MR-VP broth, methyl red dye Medium turned red Positive pH is 4 or lower

VP MR-VP broth, VP reagent A, No color change in media Negative Bacteria does not produce acetoin as an end-

VP reagent B product of glucose fermentation

Citrate Simmons Citrate agar Medium color stayed green + no Citrate permease Bacteria does not produce citrate permease

growth negative and cannot break down citrate

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Lysine D. Lysine medium broth Medium turned purple Positive Decarboxylase enzyme present and end-

product is alkaline (cadaverine)

PR lactose Phenol red lactose broth Medium turned yellow and gas A;G Organism was able to ferment lactose. End-

in Durham tube product is acidic, and gas was produced

PR mannitol Phenol red mannitol broth Medium turned yellow and gas A;G Organism was able to ferment mannitol. End-

in Durham tube product is acidic, and gas was produced.

Legend:

Neg stain: Negative stain

KOH: Potassium hydroxide

VP: Voges-Proskauer

Lysine D: Lysine decarboxylase

PR Lactose: Phenyl Red lactose

PR Mannitol: Phenyl Red mannitol

MRVP: Methyl Red Voges-Proskauer

Gram neg: Gram negative

R: Resistant

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A: Acid

G: Gas

Negative stain is very effective on determining organisms’ morphology. Here, the negative stain revealed that the organism is rod
shaped. This means that tube 16 was a colony of bacilli.

When the Gram stain procedure was performed and observed under the light microscope the colony appeared to be pink as the
organism could not retain the crystal-violet stain, meaning the organism is Gram negative.

To be surer about the gram result, Vancomycin sensitivity and KOH string test were performed. The organism showed
resistance to Vancomycin by growing throughout the medium and showed DNA stringing when mixed with KOH. Gram negative
organisms are resistant to Vancomycin and lysis in the presence of KOH.

The Gram-negative result prompted me to perform IMViC tests. IMViC stands for Indole, Methyl red, Voges-Proskauer, and
Citrate. In the case of Indole, the SIM medium turned cherry red at the top, after Kovac’s reagent was added, indicating a positive
result that means that the organism was able to break the protein tryptophan down into indole. For methyl red, the MRVP medium
showed a red color after the Methyl red PH indicator was added. This means that the organism can carry out a mixed acid
fermentation, is positive for glucose fermentation, and enough acid has been produced to lower the pH to 4 or below. The Voges-
Proskauer test was negative and this is explained by the fact that no color change was observed after VPA and VPB reagents were
added to the culture. The result indicates that there was no trace of acetoin as an end-product of glucose fermentation. The citrate test
ended up in a negative result for the fact that there was neither growth, nor color change in the media. This means that the organism is
not able to utilize sodium citrate as its only carbon source. The overall result of the IMViC tests was + + - -.

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 Due to the IMViC result, I was prompted to perform the Lysine decarboxylase test, as per the chart on page 353 of the test
book. The medium’s purple color suggested a positive result, which means that the organism was able to use the amino acid lysine as
a source carbon and energy for growth. The pH rose because the end-product was alkaline. At this point, the chart suggested that the
organism is either Escherichia coli or Edwardsiella tarda.

After performing the Lysine decarboxylase test, Phenyl red lactose and Phenyl red mannitol tests were carried out as the next
steps. The organism tested positive for acid end-products and production of gas in both tests. The medium turned yellow in both case
and that indicates that the organism was able to ferment lactose and mannitol.

The results from all the tests cited above point out that organism 16 is Escherichia coli.

Discussion

The outcome of the series of tests that were conducted led to discover that the unknown organism 16 was E. coli. The above
statements therefore support the hypothesis that “If I understand the methods and lab techniques that I learned during the semester,
then, I will be able to explain the tests that I performed, properly identify my unknown, and give a background on the characteristics
of my unknown organism.” Escherichia is a genus of gram negative non spore-forming, facultative anaerobic rod-shaped bacteria
from the family Enterobacteriaceae. Escherichia coli, commonly known as E. coli, is a Gram-negative, facultative anaerobic, bacilli
bacterium of the genus Escherichia that is commonly found in the lower intestine of humans and animals.

Most E. coli strains are harmless, but some strains can cause serious food poisoning in their hosts. The harmless strains of E.
coli are part of the normal microbiota of the intestines, and because of the mutualism relationship with their host, they can benefit their
hosts by producing vitamin K2, and preventing colonization of the guts with pathogenic bacteria. E. coli is expelled into the
environment within fecal matter. According to the Center of Disease Control (CDC), “Although most strains of E. coli are harmless,

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 others can make one sick. Some strands of E. coli can cause diarrhea, while others cause urinary tract infections, respiratory illness
and pneumonia, and other illnesses” (CDC). An individual can contract E. coli from eating raw or undercooked meat or from
encountering animal feces, particularly cattle.

E. coli possesses the ability to transfer DNA via bacterial conjugation or transduction, which allows genetic material to spread
horizontally through an existing population.

The time between ingesting the bacteria and feeling sick (incubation period) is usually 3 to 4 days after exposure but may be as
short as 1 day or as long as 10 days. The symptoms often begin slowly with mild belly pain and worsens over several days.

The main way of treating E. coli is through rehydration and rest. Using antibiotics has been shown to shorten the course of
illness and duration of excretion of enterotoxigenic E. coli, though they are generally not recommended. It is also recommended to
abstain from treating with anti-diarrheal. The body generally clears the bacterial infection on its own within 7-10 days.

No specific vaccine has been licensed for E. coli but there are other ways to prevent the pathogenic type. Handwashing
prevents transmission. Improved sanitation and drinking water are other preventative methods, as transmission occurs through fecal
contamination of food and water supplies. Additionally, thoroughly cooking meat and avoiding consumption of raw, unpasteurized
beverages, such as juices and milk are other proven methods for preventing E. coli.

Works Cited

Carrington Ph.D, E. (2013). Microbiology Lab Manual: For Students Entering Into The Health Sciences Professions (3e ed.).
McGraw-Hill Education.
Pg64-65

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 Carrington Ph.D, E. (2013). Microbiology Lab Manual: For Students Entering Into The Health Sciences Professions (3e ed.).
McGraw-Hill Education.
Pg57-58

Carrington Ph.D, E. (2013). Microbiology Lab Manual: For Students Entering Into The Health Sciences Professions (3e ed.).
McGraw-Hill Education.
Pg73-74

Carrington Ph.D, E. (2013). Microbiology Lab Manual: For Students Entering Into The Health Sciences Professions (3e ed.).
McGraw-Hill Education.
Pg78-79

Carrington Ph.D, E. (2013). Microbiology Lab Manual: For Students Entering Into The Health Sciences Professions (3e ed.).
McGraw-Hill Education.
Pg228-233

Carrington Ph.D, E. (2013). Microbiology Lab Manual: For Students Entering Into The Health Sciences Professions (3e ed.).
McGraw-Hill Education.
Pg254-255

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 Carrington Ph.D, E. (2013). Microbiology Lab Manual: For Students Entering Into The Health Sciences Professions (3e ed.).
McGraw-Hill Education.
Pg147

Carrington Ph.D, E. (2013). Microbiology Lab Manual: For Students Entering Into The Health Sciences Professions (3e ed.).
McGraw-Hill Education.
Pg268-270

Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA (June 2005). "Diversity of
the human intestinal microbial flora". Science. 308 (5728): 1635–8. Bibcode:2005Sci...308.1635E. doi:10.1126/science.1110591.
PMC 1395357.

"Escherichia coli". CDC National Center for Emerging and Zoonotic Infectious Diseases. Retrieved 2 October 2012.

Lim JY, Yoon J, Hovde CJ (January 2010). "A brief overview of Escherichia coli O157:H7 and its plasmid O157". Journal of
Microbiology and Biotechnology. 20 (1): 5–14. PMC 3645889.

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