Week 2, Day 4

May 24, 2013

In lab today, after going through the usual routine, we checked our samples from the days before to determine the different substances our bacteria would react with. Our skim milk plate showed signs of a negative test for casein hydrolyzation. Our gelatin tube was still showing negative signs (still solid), so we will be incubating it longer to see if it liquefies. The triglyceride hydrolysis test gave negative results also, and the litmus milk and motility sample we are going to incubate for a longer period of time, so no results from those yet.

Next we moved on to our samples from our last lab session. Our carbohydrate (sugar) fermentation tests all showed negative signs except for glucose, which gave a positive test for glucose fermentation. No gas bubbles were present in the glucose Durham tube however, so our bacteria does not produce CO2 or H2 gases during fermentation. The sugars that gave negative tests (mannitol, lactose, and sucrose) we replaced in the incubator to check the next day to see if different results appear.

Mannitol

Lactose

Sucrose

                                                                          Glucose

Our citrate utilization test came back positive; our tube turned from green to blue on the surface and a large gas bubble formed behind the slant (CO2). This indicates that our bacteria can utilize citrate as its sole source of carbon and energy.

 

Citrate Utilization Slant

The TSIA (triple sugar iron agar) test is for differentiating among gram-negative enteric bacilli as to their ability to ferment glucose, lactose, and sucrose, and to produce H2S, so we can use it to double check our carbohydrate tests. The TSIA slant we had inoculated remained red on the slant, but turned yellow on the butt of the slant with a larger bubble under the slant (alkaline slant/acid butt). This indicates that only glucose was fermented, which matches our results from our carbohydrate tests in which only glucose tested positive.

TSIA Slant

For the indole (tryptophan degradation) test, we added 5 drops of Kovac's reagent to the incubated culture. A positive test would mean the formation of a red layer at the top of the tube, but our sample formed a yellow layer, meaning we had a negative test and our bacteria cannot split the amino acid tryptophan into indole and pyruvic acid.

Tryptophan (Negative)

Our urea test came back negative. A bright pink color would have indicated a positive sample, but ours only turned slightly orange. This test shows that our bacteria does not hydrolyze urea.

Urea (Negative)

For our nitrate test we had to wear gloves as we added five drops of reagent A (sulfanilic acid) and five drops of reagent B (dimethyl-α-naphthylamine - a carcinogen) to our sample and gently shook the tube to mix the reagents with the broth. No pink-ish color developed after 2 minutes (which would mean a positive test for nitrate reduction), so we added a small amount of powdered zinc to the tube. If our bacteria does not reduce nitrate ions, the zinc will show us by reducing them and activating the A and B reagents, producing a color. After 10 minutes there was no color change, indicating the bacteria reduced the nitrate ions to molecular nitrogen, a positive test for nitrate reduction.

dimethyl-α-naphthylamine

Nitrate (Positive) after Zinc

We divided our MR-VP sample into two separate tubes. The first tube we used for the methyl red test (mixed fermentation). We added 5 drops of methyl red to the tube and gently swirled the tube to mix the broth culture and the pH indicator.

 There was an orange-yellow color formed, the sign for a negative test. This means our bacteria does not ferment glucose via mixed-acid fermentation.

The second tube from the MR-VP sample we used for the Voges-Proskauer test (butanediol fermentation). We added 15 drops of Barritt's reagent A (α-naphthol) and 5 drops of Barritt's reagent B (KOH) to the tube. We tapped the bottom of the tube vigorously and shook the tube so that oxygen in the air could aerate the medium. Even after 30 minutes there was no pink-red color forming at the top of the broth (which would indicate a positive test). This negative test result indicates that our bacteria does not ferment glucose via butanediol fermentation.

Barritt's reagent A (α-naphthol) and Barritt's reagent B (KOH)

Near the end of the lab our professor took our streak plate and poured hydrogen peroxide over it. Lots of bubbles began to form for several minutes. This is due to the enzyme catalase our bacteria produces due to being an aerobic bacteria.

Hydrogen Peroxide Filled Plate with Foam