The Pursuers: More Tests on Mystery Bacteria J

More Tests on Mystery Bacteria J

Class: Thursday, October 17

In class today, we looked at the results of a few tests on Bacteria J as well as complete a few more tests.

On Tuesday, we completed the Citrate Utilization Test to determine if our mystery bacteria possessed the membrane-associated transporter citrate permease. Our test came back negative.

Mystery bacteria negative Citrate test

Dr. Pathakamuri enjoying Max & Sam's positive Citrate test

Next, we checked our results for the Urea Hydrolysis test. The reason for this test is to see if the bacteria secretes an enzyme called urease.

Mystery bacteria negative Urea test

For our next test, called the Indole test, we wanted to determine if our bacteria would split an amino acid called tryptophan into indole and pyruvic acid. To do this, we added ten drops of Kovac's reagent into a broth culture of our mystery bacteria.

Adding Kovak's reagent to our mystery bacteria

If our mystery bacteria produced the byproduct of indole, then it would be positive for undergoing the reaction for splitting the amino acid tryptophan.
If positive, a red layer at the top of the broth tube would quickly appear. If negative, there would be no presence of a red layer, meaning that tryptophan was not hydrolyzed.

Mystery bacteria negative Indole test

As the picture indicates, our mystery bacteria tested negative for Indole.

Another test we completed is a respiration test, specifically called a Nitrate Reduction test. For this test, its purpose is to determine whether our bacteria is able to reduce nitrate ions to either nitrite ions or ammonia (nitrogen gas). To test this, last class on Tuesday we inoculated a nitrate broth tube with our mystery bacteria. Today, we added five drops of sulfanilic acid and five drops of dimethyl-a-naphthylamine, and gently mixed the broth.

Adding sulfanilic acid to inoculated nitrate broth

Adding dimethyl-a-naphthylamineto inoculated nitrate broth

If there is a presence of nitrate, the broth turns a red color. However, if there is no color change this does not mean that the nitrate test is negative. It is possible that the nitrate ions were not reduced to nitrite ions. In this case, a small amount of zinc would be added to reduce the nitrate ions to nitrite ions. We did not need to do this last step, because our broth turned red after adding the two reagents.

Negative nitrate test

Matt checking out our initial nitrate test

The next test is another respiration test, called Oxidase test. This test is to determine whether our bacteria has the cytochrome oxidase, which plays a role in electron transport during respiration.

To do this test, we swabbed our mystery bacteria and dripped oxidase reagent on the swab. Within ten seconds, if the test is positive, the bacteria should turn blue/purple, indicating the reaction is positive for oxidase. If nothing happens, then the bacteria is negative for oxidase.

Adding oxidase reagent to mystery bacteria

Negative oxidase test

For our next test called Methyl Red Mixed Fermentation test, we tested the pH of our bacteria after adding methyl red pH indicator. This would indicate products of mixed-acid fermentation of glucose. To do this, we added 5 drops of methyl red to our mystery bacteria-inoculated broth tube.

Negative mixed fermentation test

Another test we completed is called the Voges-Proskauer test or Butanediol Fermentation, which would determine the ability of our mystery bacteria to ferment glucose through butanediol fermentation.

To test this, on Tuesday, we inoculated a methly red Voges-Proskauer broth tube with our mystery bacteria. Today, we added 15 drops of Barritt's reagent A and 5 drops of Barritt's reagent B to the tube.