Identification of Unknown Report
The purpose of this lab was to determine the identity of an unknown bacteria slant culture. The unknown was identified using differential tests to determine its metabolic and morphology characteristics. Results from the differential tests indicated that unknown culture 5 is Staphylococcus aureus.
The purpose of this lab was to determine the identity of an unknown bacteria slant culture using a series of differential tests. The tests used to identify the unknown bacterial culture included: Gram stain, mannitol salt agar, coagulase tube test, and an antimicrobial susceptibility test. The tests selected were based on the results of a gram stain.
Gram staining, the most commonly used differential stain, allows for the fast and easy detection between gram negative and gram positive organisms based on the presence or absence of peptidoglycan. A gram positive organism resists the decolorization process due to a large amount of peptidoglycan and appears a purple color. A gram negative organism is successfully destained, counterstained with safranin and appears reddish-pink.
Mannitol salt agar is used to detect an organism’s ability to ferment mannitol, which lowers the pH of the phenol red containing medium. Phenol red is yellow below pH 6.8, red at 7.4-8.4, and pink at pH 8.4 and above.
The coagulase test uses rabbit plasma to determine if the organism produces the coagulase enzyme in either the free or bound coagulase forms. Staphylococcus aureus produces a positive test for the coagulase enzyme while other Staphylococcus organisms do not.
The last test performed was the test for the resistance to certain antibiotics. The results of the antimicrobial susceptibility test produced different sized zones of inhibition. The size of the zone of inhibition, or lack thereof, to a certain antibiotic determines it’s susceptibility to that antibiotic.
Materials and Methods:
Mannitol salt agar plate
Various antimicrobial infused discs Bunsen burner
Coagulase tube test
BHI agar culture plate
2 Nutrient agar plates
Gram stain test kit
1. First step was to inoculate the BHI plate from the slant culture and create a streak plate as described in Experiment 2, procedure part B, page 7 of my lab notebook. Incubated at 37°C for 24 hours. 2. Using sterile inoculation methods, a sample of the unknown organism was taken from the slant culture and used to create a gram stained slide as described in Experiment 5, pages 33-35. 3. After the streak plate was incubated for 24 hours at 37°C, it was used to perform the following tests (procedure for each test located in lab notebook on pages indicated): a. Mannitol salt agar fermentation – Experiment 7, page 43 b. Coagulase test – Experiment 10, part 2, page 62
c. Antimicrobial susceptibility test – Experiment 10, part 3, page 63
Gram Stain: The gram stain was able to show that the unknown organism was gram positive and a Staphylococcus, or round clusters of cells. Below is a picture of what the organism looked like under a light microscope at a magnification of 1000X.
Streak Plate: The streak of the unknown was useful in identifying the colony morphology of the organism. Below you can see the margin of the isolated unknown colonies is undulated, the size was moderate, and growth was white and opaque.
Mannitol Salt Agar Test: Most Staphylococci are able to grow on this salty medium nut do not ferment mannitol so the red color of the medium remains unchanged. Only a select few Staphylococci are able to ferment mannitol which effectively changes the pH level of the phenol red to a pH below 6.8 which changes the medium to a yellow color. The MSA test indicated (pictured below), through growth and a color change from red to yellow, that the unknown organism was a Staphylococcus (growth), and possibly a Staphylococcus aureus (pH/color change)....
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