MCB 3020L Lab Report.docx - Identification of an Unknown...

Identification of an Unknown
Pathogen and Recommended
Treatment
Hussien Mohamed
MCB 3020L, SEC.008
UNKNOWN #10

Mohamed 1
Summary
The purpose of this experiment was to use the various test and laboratory techniques
learned throughout the semester to identify an unknown pathogen and to propose a
recommended treatment. The series of tests needed to identify the unknown was established by
the cell wall composition of the unknown bacterium. The unknown bacterium was determined to
be Gram-negative through the Gram stain and KOH test. To complete identification of the
unknown bacterium, MacConkey (Mac) and Salmonella-Shigella (SS) plates, as well as Triple
Sugar Iron (TSI) and Citrate slants were inoculated with the unknown bacteria.
Isolates produced bright pink colonies on the Mac agar and pink-red colonies on the SS
agar. The TSI slant had a result of A/A/+/-, which indicates an acidic slant and butt, visible gas
production, and no H
2
S gas production. The citrate slant appeared green. A Kirby-Bauer test was
also conducted to determine the susceptibility and resistance of the unknown bacteria to three
antibiotics: Neomycin, Polymyxin B, and Tetracycline. The unknown was susceptible to all three
antibiotics.
The unknown bacterium was conclusively determined to be Escherichia coli upon
analysis of the results . Although E. coli was determined to be susceptible to all three antibiotics,
Polymyxin B was chosen as the recommended treatment because the bacterium exhibited the
greatest susceptibility to this antibiotic.
Materials and Methods
All methods were adapted from the Microbiology Lab Manual (CMMB, 2017).
The Gram Stain and the KOH Test

Mohamed 2
The first test conducted to determine the identity of the unknown bacterium was the
Gram stain. To begin, a bacterial smear was prepared by placing a drop of water and a small
amount of the unknown sample on a glass slide using an inoculating loop. The slide was then
heat-fixed by passing the slide through a Bunsen burner flame a few times. The smear was
covered with crystal violet for one minute and then rinsed with water. The smear was then
covered with Gram’s iodine for one minute and rinsed with water again. The smear was
decolorized by adding 95% ethanol drop-wise. After rinsing with water a third time, the smear
was covered with the counterstain, safranin, for one minute. The smear was rinsed with water a
final time and was blot dried. The slide was examined under oil immersion, and the color and
shape of the bacteria was recorded.
The KOH test was used to verify the results of the Gram stain. A small amount of the
unknown sample was placed on a glass slide and a drop of 3% KOH was added to it. The slide
was left to sit for one minute. A toothpick was used to mix the fluid so that the viscosity could be
determined. Gram-negative bacteria appear as viscous and stringy and Gram-positive bacteria
appear as cloudy.