Lab Day 7

May 22

Part 1 of today's lab: checking our experimental results from yesterday

1: Indole (Tryptophan Degradation) Test

Purpose: to determine the ability of some bacteria to split the amino acid trypotphan into indole and pyruvic acid.

Result: positive

We added 15 drops of Kovac's reagent. A red layer formed on the top of our sample and this indicates a positive test for the presence of indole. This means that Trypotphan was hydrolyzed.

2: Urea Hydrolysis Test

Purpose: to determine the ability of a bacterium to hydrolyze a urea.

Result: Negative

A few bacteria use the enzyme urease to rapidly degrade urea into CO2 and Ammonia. The color didn't change from from the original color and this signified a negative test. If ammonia was produced, the color would've been pink in alkaline ph due to the breaking down of urea with the urease enzyme.

3: Citrate Utilization Test

Purpose: to determine if bacterium can utilize citrate as its sole source of carbon and energy.

Result: negative

The ph indicator bromothymol blue turns blue in alkaline ph. A change in color of the medium from green to blue denotes a positive test. Our color did not change and therefore, our bacteria didn't have the enzyme to break down citric acid.

4: Nitrate Reduction Test

Purpose: to determine if a bacterium is able to reduce nitrate ions to either nitrite ions or to nitrogen gas.

Result: positive

Nitrate accepts electrons instead of oxygen. We were looking for a nitrate reduction enzyme.

This test can be two part:

Nitrate ----> nitrate

Nitrite ---> ammonia

However, because our test was positive for nitrites upon adding Sulfanilic Acid and Dimethyl-alpha-naphthylamine, we did not have to test further in case our bacterial nitrate broth had undergone both reactions and turned into ammonia. It turned a cherry red which signifies that it produced nitrites and is therefore positive.

5: MR-VP

Purpose: to determine the ability of some bacteria to ferment glucose via mixed-acid fermentation.

MR result: positive – VP result: negative

If our MR-VP broth used glucose and fermented it for acid, upon adding methyl red (a ph indicator), you should see a color change to red. Ours reacted this way when it was added. VP test tests butanediol for non-acidic end product but if MR test is positive, VP is negative.

6: Litmus Milk Reactions

Purpose: to differentiate among bacteria as to their ability to utilize lactose, protein, and litmus in litmus milk.

Results: The curds that were present yesterday, were even more of a pink color today. The pink color signifies that our bacteria can ferment lactose. The cracks in the curds signify the presence of gas.

When testing for acidic and non-acidic curds, we turned the tube upside down. If they were to come down, it would be non-acid curd but being that our curds remained immotile when turned, we can tell that our bacteria form an acid curd in litmus milk.

Part 2 of todays lab: Selective and Differential Plates

The procedures for each of these tests were the same. Practicing aseptic technique throughout, we inoculated each of the following plates.

1: MacConkey Agar

Purpose: to detect and differentiate among gram-negative enteric bacilli, based on their ability to grow on the medium and to ferment lactose.

2: Eosin Methylene Blue Agar

Purpose: To isolate and differentiate gram-negative enteric bacilli.

3: Phenylethyl Alcohol Agar

Purpose: To isolate gram-positive bacteria from a specimen containing a mixture of gram-positive and -negative bacteria

4: Blood Agar Plate

Purpose: 1. To isolate and support the growth of fastidious bacteria. 2. To differentiate among bacteria based on their ability to lyse red blood cells.

5: Mannitol Salt Agar

Purpose: to isolate bacteria based on their salt tolerance and differentiate among these isolates for mannitol fermentation.

After inoculating each of these plates, we placed them in an incubator set at 37 degrees Celsius. 

Joanna Dawyot, Cassie Livingston, Mary Rose Capara 

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