Identification of Unknown #19
Organism 1: Escherichia coli
Organism 2: Staphylococcus aureus
Brandi Beckman
Introduction
Biochemical tests are greatly important in the health care field. The tests allow for the
identification of microbes and can be used to determine what specific microbe is causing an illness in
humans and other animals. Performing tests on pathogenic microbes can also lead to the understanding
of the microbes which can result in better treatment methods of the diseases caused by them. Rachel
Watson, M.S. of the University of Wyoming states that using biochemical tests is also important in the
area of food microbiology (1). She says that the tests can help accurately identify microbes that cause
food spoilage.
After receiving the two unknown organisms, one stock culture was inoculated for each unknown
in TSA slants. Next, a MacConkey plate was divided in half. One half of the plate was inoculated with
unknown 1 and the other half with unknown 2. Both unknowns were incubated at 37 degrees Celsius.
A gram stain was also performed on both unknowns and the results were recorded.
Results
For Organism 1, the colonies on the TSA slant were thick, round, and flat. The results for the
MacConkey test showed growth which indicated that the organism was gram negative. This meant that
Organism 1 must have a thin layer of peptidoglycan in the bacterial cell wall. The colonies were also a
pink color which meant that the organism must ferment lactose. The bacteria also appeared to have a
round shape. From this characteristic, the organism was determined to be bacillus. The results of the
gram test also came back negative because the bacteria were dyed a pink color.
Next, a SIM (Sulfide, Iodine, Motility) test was performed. The organism was determined to be
negative for sulfide production since there was no black precipitate to be found in the SIM tube. Also,
the organism tested positive for iodine production. This proves that the organism can produce the
enzyme tryptophanase to break down tryptophan. Since growth was visible moving away from the stab
line in the SIM tube, the organism tested positive for motility, meaning that the organism must be
motile.
After the SIM test, a lysine decarboxylase test was conducted. After incubation, a deep purple
color was visible. This was interpreted to be a positive result for the test and meant that the organism
was capable of removing the carboxyl end from lysine which would shift the pH to alkaline.
Since the gram negative, lactose fermenting, bacillus organism had a positive result for the
iodine, motility and lysine decarboxylase tests, the identity of unknown Organism 1 must be Escherichia
coli.
For Organism 2, the colonies on the TSA slant appeared thick, round, elevated and bright. There
was no growth of this unknown on the MacConkey plate which indicates that the organism was gram
positive. The gram stain also produced a gram positive result due to the colonies appearing purple
underneath a microscope. This gram positive result means that the bacteria must have a thick layer of
peptidoglycan. The bacteria had a circular or coccus shape when observed under a microscope.
The next test performed on Organism 2 was a catalase test. When hydrogen peroxide was
added, oxygen bubbles were produced immediately. The bubble production indicates a positive
reaction to the catalase test. This means that the organism produces the enzyme catalase, which also
means that it is probably an aerobic respirator.
After the catalase test, a TSIA (triple sugar iron agar) test was performed. The TSIA test tests for
the fermentation of lactose, sucrose and/or glucose. The butt of the TSIA tube after incubation was a
bright yellow so, the unknown tested positive for glucose fermentation.
A urease test was performed after the TSIA test. A urease test tests for urea hydrolysis,
meaning the enzyme urease is produced by a microbe to break down urea into carbon dioxide and
ammonia. Organism 2 tested positive for the urease test because of a bright pink color along the top of
the tube’s slant, however, due to incorrectly reading the flowchart written for Organism 2, this test was
unnecessarily performed. The urease test didn't aid in the identification of Organism 2.
The last test performed on Organism 2 was a mannitol carbohydrate fermentation test. After
incubation, the color of the tube’s contents was yellow. This was interpreted to be a positive result
meaning that the unknown must ferment mannitol.
Since unknown Organism 2 was a gram positive and had positive results on the TSIA test for
glucose, the catalase test, and the mannitol fermentation test, the organism must be Staphylococcus
aureus.
Discussion
According to author of Microbiology Fundamentals: A Clinical Approach Marjorie Kelly Cowan,
only a fraction of the dozens of strains of E. coli in existence cause illness in humans (2). Most of the
pathogenic strands of E. coli cause various degrees of intestinal distress while some can cause urinary
tract infections. Some strands of E. coli have a special virulence factor of producing shiga toxins, which
interrupt the protein synthesis of cells. A person is diagnosed with having E. coli after a stool sample is
performed. E. coli can spread by the fecal-oral route of transportation or by touching contaminated
surfaces.
According to the United States Department of Health and Human Services’ FoodSafety.gov, S.
aureus is found on the skin, hair, noses, and throats of up to 25 percent of humans (3). S. aureus is
responsible for most staph infections. S. aureus is a cause of food poisoning when contaminated food is
not properly cooked or refrigerated. The incubation period for the microbe is one to six hours.
Symptoms of food poisoning by S. aureus include nausea, vomiting, diarrhea, abdominal cramps and a
mild fever. The U.S. National Library of Medicine’s Medline Plus states that S. aureus can also cause skin
infections, pneumonia, toxic shock syndrome, and blood poisoning (4).
References
1. Watson, R. (2013). The Virtual Edge. Retrieved December 1, 2014.
2. Cowen, M. (2013). Infectious Diseases Affecting the Gastrointestinal Tract. In Microbiology
Fundamentals: A Clinical Approach (pp. 561 - 562). New York: McGraw-Hill.
3. U.S. Department of Human Services. (2014). Staphylococcus. Retrieved December 1, 2014.
4. U.S. National Library of Medicine. (2014, October 20). Staphylococcal Infections. Retrieved December
1, 2014.