Day 6: 5/21

"Is that Kosher?" - Finding Out What Our Bacteria Eats

Today we followed up on our different tests of what the bacteria uses as food. 

In the Hydrolitic (Digestive) Enzymes we first tested with the starch agar plate that had been incubated.  After flooding the plate with Gram's Iodine and waiting 1 minute we concluded that the test was negative because there was no clouded area around the bacteria growth.

A positive result next to a negative one

For the Casein Hydrolysis test we just examined the plate after incubation. The test results were negative again because the area around the bacteria remained white during incubation. We placed this plate back in the incubator for further testing.

The Gelatin Hydrolysis test with the nutrient deep agar tube was examined and found to be negative, at least so far (Don't worry we are not losing hope) After tilting our tube there was no liquification and the gelatin was still in a solid state.  We also placed the gelatin tube back in the incubator to check tomorrow.

The Fat (Triglyceride) Test was with the blue tributryrin plate was negative.  Although there was discoloration around the whole bacteria sample there was no cloudiness around the grown bacteria.  (so close!) We also put the lipid plate back into the incubator for further growth.

For examining the DNA agar test plate we flooded the plate with 1N HCL. The result turned out negative for DNase because there was no clear are around the bacterial growth.

Still pushing on despite the negative feedback we checked the Triple Sugar Iron Agar Test (TSIA). the TSIA slant tube contains 1% lactose and .1% glucose.  When our known bacteria fermented in both of these sugars (and sucrose) it turned the slant yellow with a yellow butt (bottom). Therefore the result of our test was acid slant/acid butt with gas. (Finally a positive test!) We were relived to find our bacteria did eat something.

Onto the 4 tube tests of sucrose, lactose, glucose and maltose.  The results were all positive and gas was formed. Fermentation of lactose produced the least amount of gas out of the four and glucose and sucrose produced the most gas. The small tubes inside were filled with gas, and the yellow tubes signified a positive test.  This verified our TSIA test.

Finally the litmus milk tube was our last test.  The tube appeared lavender and it appeared to be a negative test. But we placed it in the innoculator to see if more bacterial growth would change the test.

We examined the air tight container with the plate of bacteria.  Unfortunately the jar was not air tight and again the strip was still blue.  We all concluded that the GasPak packages were all burnout (sucking up oxygen is hard work!)

Oxidase Test:

This respiration test was used to determine if the unknown bacteria have cytochrome oxidase which is a participant in electron transport chain.

For the oxidase test we used the following materials:

Oxidase reagent in a crushable ampule

Piece of filter paper

Pure culture of bacteria agar slant tube

Inoculating loop

Bunsen Burner

striker

In this experiment we used oxidase reagent N,N,N,N tetramethyl-p-phenylenediamine and crushed it inside the plastic dropper. Then we dropped the oxidase on tonto the round filter that was placed inside the lid of the Petri plate.  Using Aseptic technique we placed a sample of our bacteria on the area of the filter paper with the reagent. The result was negative because the bacteria did not turn a deep purple. Therefore our bacteria does not have cytochrome oxidase.

The Start of 5 Miscellaneous Tests: Inoculation

Next we inoculated a urea containing medium broth.  This test will determine the unknown bacteria's ability to hydrolyze urea. 

Then we inoculated a tube marked “T” for Tryptone for a protein test.

Continuing on we inoculated a nitrate broth tube to determine if the unknown bacteria is able to reduce nitrate ions to either nitrite ions or to nitrogen gas. 

Next was the MR-VP or Methyl Red Test (Mixed Fermentation). After inoculation and incubation we will determine the ability of the bacteria to ferment glucose via mixed acid fermentation. 

Last and but certainly not least we did a citric utilization test to determine if the bacteria can use citrate as its sole source of carbon and energy. We inoculated the Simmons citrate agar slant tube using a inoculating needle.

We placed all five tests in the incubator.

Follow ups:

Eleni has germy hands. (but we still love her)  Here is a picture of the hand washing test we did the first day of lab. After 7 days the bacteria growth has greatly increased.  Notice on the before hand washing portion of the plate the many different colored bacteria and the after only has slight cream spots (Phew! Hand washing does work)

And finally we examined the strep test and after several minutes Dr. P concluded that Theresa does not have strep (YAY!)