Oxygen Requirements {Lab 7 - May 20, 2015}

      As you may guess from the title, the next set of experiments Clare and I ran were to discover if our bacterium used oxygen or not. There were four tests we used: an anaerobic chamber and Thioglycolate, Catalase, and oxidase test.

Materials:

·       inoculating loop

·       Bunsen burner

·       ager plate and slant

·       filter paper

·       oxidase reagent

·       3% H₂O₂

·       Anaerobic chamber

Procedure:

     First we had to prepare fresh samples of our unknown for the test. Using our aseptic techniques we inoculated an agar slant for our oxidase test and stored it in the 37⁰ incubator. Next, we inoculated an agar plate with a pure culture from our unknown for the catalase testing, also storing the sample in the 37⁰ incubator. After that, we need to inoculate a tube of Thioglycolate with our unknown and store it with the other samples. The last preparation needed was to inoculate an agar plate for the anaerobic chamber. 

     After 24 hours, we could finish the tests to see what the oxygen requirements for our unknown were. If the unknown didn’t use oxygen at all, it would be an Obligate Anaerobic, but if it needed oxygen, it would be Obligate Aerobic. However, these are not the only two options. If the bacteria grows better with oxygen but can grow in anaerobic conditions as well then it is a Facultative Anaerobe. 

The next day we looked at our Thioglycolate. The Thioglycolate is a gel that helps us see clearly were the bacteria grow, near the oxygen or not. Our unknown was growing on the top and was stretched down a little in to the tube. This would indicate that our unknown is a Facultative Anaerobe. 

Facultative Anaerobe Growth

      Turning to the Anaerobic chamber, we looked for data to support this. The Anaerobic chamber is used to create an environment with no oxygen. We did this by placing GasPak envelopes that react with the oxygen to form water. An indicator strip was also placed inside so that the oxygen level could be known (blue with oxygen, white without). The class put in our samples and screwed the clamp on. However, the next day, the indicator was still blue signaling that there was still oxygen left inside. Even so, Clare and I could still make some observations. Our culture had some growth an environment that had oxygen that was being taken out somewhat. This could also be an indicator that our bacterium is a Facultative Anaerobe.

Our Unknown Bacteria -
from anaerobic chamber 

The Catalase test was next. Taking the Agar plate with a fresh pure culture of our bacterium we poured H₂O₂ on it. An organism that uses Aerobic respiration like ourselves has the ability of producing H₂O₂ which is harmful. So we have an enzyme, Catalase to break the H₂O₂ down. If our unknown is indeed a Facultative Anaerobe, it will have Catalase, and we will see bubbling when we pour the H₂O₂ on it. And yes, we did observe bubbles indicating the presence of Catalase.

Bubbling indicating catalase

The only thing left was the Oxidase test. Cytochrome oxidase is an enzyme used in electron transport during respiration.  For this test, we wetted a piece of filter paper with oxidase reagent that will turn blue when our unknown is added if it has Oxidase. However, it did not turn blue when we place bacterium from our fresh sample from the agar slant onto the reagent. 

Conclusion/application:

     Now we know our unknown is a Facultative Anaerobe, meaning it grows with oxygen and will grown somewhat without. Also, it does not use oxidase in its Aerobic respiration, but it does have Catalase. Knowing whether a bacteria is aerobic or anaerobic will determine the plan of action that a doctor will take against the bacteria. For example, propionibacteria that causes acne is an obligate anaerobe. So some treatments include putting the patient in an oxygen rich environment to kill the bacteria.