SO...WHAT IS IT??? -Identifying our unknown bacteria!

After a semester with our unknown bacteria, we have studied its physical characteristics, how it grows, how it moves, how it reacts in different conditions, and what it uses for energy. All the results of these tests have given us clues as to our mysterious unknown. Using the flow chart provided for us, we were able to FINALLY identify it!

These were the determining characteristics when using the flow chart:

• Gram positive--narrowing it down to Corynebacterium spp., Bacillus spp., and Lactobacillus spp.
• Spore-forming--narrowing it down to Bacillus spp.
• Positive mannitol test--narrowing it down to the species B. megaterium and B. subtilis
• Positive Voges-Proskauer (VP) test-- Bacillus subtilis!!!!

Now that we know what our bacteria is, we decided to do a little research on Bacillus subtilis. Here are some interesting facts that we found:

• naturally-occuring
• widespread
• used for its natural fungicidal properties to prevent crop disease
• Used in commercial Japanese and Korean food production
• Can convert explosives into nitrogen, carbon dioxide, and water
• Used as an immunostimulatory agent in alternative medicine

References:

• http://epa.gov/biotech_rule/pubs/fra/fra009.htm
• http://en.wikipedia.org/wiki/Bacillus_subtilis

Cow Antigens & ELISA Test

Testing the purity of bovine extract and albumin. The Petri dish is dived into three sections and we begin by making four holes in the agar for each section with a pipette.  Then we fill the holes with the substance indicated in the work sheet and checked the results next lab. In section one we added green dye in one hole, red dye in the second, Barium Chloride in the third and Potassium Sulfate in the fourth. The second section got Bovine Albumin to the first hole, Goat Anti-horse Albumin to the second, Goat Anti-bovine albumin to the third and Goat Anti-swine Albumin to the fourth. The third section got Goat Anti-horse Albumin, Goat Anti-bovine Albumin and Goat Anti-swine Albumin and Hamburger extract. Our results were a little confusing because the agar did not change colors like the other groups, but I don’t think there was any contamination that we could see.

Cow Antigen Results

The ELISA was to determine if Patient 3 and/or 7 have HIV.  We marked a plus sign on the wells to make a positive control sample, and used a negative sign on the wells for negative control samples, “3” wells for Patient 3’s results, and “7” wells for Patient 7’s results.  The results were that Patient 3 does not have an HIV infection, but Patient 7 does.

ELISA Results

UV Light

The UV wavelength has the unique property of being able to kill microorganism. This really cool feature is being studied scientists and in some hospitals they use UV lights as a sterilization method to clean hospital rooms after a sick patient leaves. However, UV light is also known to cause mutations, and so in a hospital setting only the longer UV Light C wavelength us used just in case someone was to walk in the room by accident.

UV Light bulb and Petri dishes undergoing sterilization.

In Lab we decided to see if this was true and if UV light would kill or retard the growth or our bacterias. Everyone made a carpet spread of bacteria on a Petri dish. Since UV light is blocked by any physical barrier except air, to conduct the experiment we simply covered half the Petri dish with paper, and exposed each dish to one minute of UV light. Oh, and remember to take the glass lid of the Petri dish because that will block UV light too! Next lab we checked the dishes and found that there was still bacterial growth on the UV treated side of the plates, but it did seem to be a little less growth then the non-UV light treated side. We thought that maybe the experiment did not work very well because we set the wave length on too low a setting. No one checked the setting before we began.

Making Yogurt!!!

Today we get to apply what we learned about microbes to the real world - or rather the kitchen -- because we are going to make yogurt and Kefir!

The process was really simple. We start out with whole milk and boil it in the microwave to kill any pathogens. Then we filled two insulated cups and added a spoonful of yogurt to one and kefir to the other, mixed well, and let sit over night in a warm incubator. Another method would be to leave it in a stove with the light one. The light will provide just enough heat for the microbes to grow. Next the cups had turned from liquid to a solid cup of yogurt and a creamy cup of kefir! Most the students that tried them agreed it was good but a little sour. Just add a little sugar and berries and you have you're own homemade yogurt!

Sweet way to mix yogurt : )

Finished yogurt & kefire ready to eat!

Disclaimer: If you try this at home and get sick it's not my fault : )

Oxidase Test

Performing an oxidase tests lets us see if our bacteria have cytochrome oxidase, as participant in electron transport during respiration. To check, we simply take the old unknown bacterial plate we used to the antibiotic test and a vial of oxidase. It it turns blue right away the test is positive, but if it does not turn blue or turns blue after 30 seconds then the test is negative. Our plate only turned a little blue after a few minutes indicating that our unknown bacteria is NEGATIVE for Oxidase.

Negative Oxidase Results

We also added hydrogen peroxide to the plate to see if it would react and our bacteria fuzzed a lot! This means our bacteria can use aerobic respiration.

Results of Protien-Indole, Citrate, Nitrate, and Urea.

Protien-Indole Results: Negative. When we added the Kovak's solution after 48 hours, the broth did not turn red. Therefore our bacteria does not have the ability to use tryptophan as an engergy source.

Protien-Indole Restults: Negative

Citrate Results: Negative. Our slant had a tinge of blue on top, but the rest of the tube remained green. Therefore our bacteria does not use citrate.


Protien-Indole Results: Negative


Nitrate Results: Positive. When we added the A&B solution nothing happened. Then we added the zinc dust nothing happened either. If no change occures when the zinc dust is added after ten minutes, then the test is positive for nitrate reduction and nitrite ions are present.

Nitrate Results: Positive

Urea Restults: Negative. Our bacteria does not have the enzyme that breaks down urea because the test tube turned yellow. If the tube remained red it would indicate a positive result.

Urea Restults: Negative

Reading the Results of our Antibiotics Test

Upon retrieving our antibiotic test from the incubator, we saw the following...

We measured the diameter of the clearing around each of the antibiotics used to determine the sensitivity of our bacteria to each antibiotic. We found...

• Penicillin: 27mm--sensitive
• Vancomycin: 22 mm--sensitive
• tetracyclin: 25 mm--sensitive
• Erythrocyclin: 31 mm--sensitive
• Chloromphenicol: 30 mm--sensitive
• Neomycin: 20 mm--sensitive

In conclusion, our bacteria is VERY sensitive to a lot of antibiotics...yet another distinguishing characteristic!