Final Testing!

Today in lab we prepared the final tests for determining the identity of "F". We also were able to work with antibiotics for the first time! Read on for the exciting procedure!

Citrate Test: 


The purpose of this test was to determine whether our bacteria uses citrate as its sole carbon and energy source. This can be determined by the green medium turning to a blue color. If this happens our bacteria is able to break down citrate into pyruvate and carbon dioxide. We inoculated "F" using the inoculating loop, while utilizing the aseptic technique. We then placed it into the incubator until further instruction.

Indole (Tryptophan Degradation) Test: 


The purpose of this test is to determine if our bacteria can split the amino acid Tryptophan. If our bacteria can split this amino acid, yielding pyruvate and indole, a red layer will form at the top of the broth. This indicates the presence of indole. As usual, in order to prepare the sample we inoculated the broth using the aseptic technique and proceeded to put the culture into the incubator until next class.

Nitrate Reduction Test: 


The purpose of this test is to determine if our bacteria has the enzyme nitrate reductase. This enzyme reduces nitrate ions to nitrite ions and nitrogen gas. We prepared by inoculating the sample with aseptic technique and placing it in the incubator.

Urea Hydrolysis Test: 


The purpose of this test is to test for the presence of the enzyme urease. If this enzyme is present urea will be converted into carbon dioxide and ammonia. The presence of ammonia raises the pH changing the color of the broth from dark yellow to a bright pink color. We prepared this using aseptic inoculation techniques and placing it into the incubator until next lab.

After finishing these tests we created a new slant in order to have a sample to test for the presence of oxidase in our bacteria next class. Then we proceeded to make a streak plate to test "F"'s response to certain common antibiotics.

When streaking the plate we used a certain procedure, making sure that every part of the plate was covered with bacteria. We had to streak in multiple different directions, using this model:

This picture was taken from Techniques in Microbiology: A Student Handbook. After streaking the plate, we carefully placed each of the types of antibiotic discs onto the freshly streaked agar plate. The antibiotics used include:
- Penicillin
- Vancomycin
- Novabiocin
- Tetracycline
- Erythromycin
- Chloramphenical
- Neomycin

These antibiotics attack different structures and processes of the bacteria. Penicillin and Vancomycin attack the cell wall. They only attack bacteria because a bacteria's cell wall contains Peptidoglycan. However, Novabiocin attacks nucleic acid synthesis. The last four attack protein synthesis in 30S and 50S ribosomes. Since all of these antibiotics attack different structures and processes, they are selective to certain bacteria. This test will help us to decipher which bacteria "F" could be!

Join us next blog for the exciting results of these tests!