CATALASE ACTIVITY

For our sixth lab Dylan and I tested our unknown microbe to see if it had any catalase activity. What is a catalase you may ask? A catalase is an enzyme that is found in the majority of living organisms that are exposed to oxygen. The enzyme catalyzes the decomposition reaction of hydrogen peroxide into the separate elements, water and oxygen.

This photo was taken from the Birmingham-Southern 

Microbiology Lab Manual for Week Six of BI 304    

In order to determine if our unknown had catalase activity or not, a very small amount of our microbe was placed on a sterile microscope slide. Once the microbe sample was on the slide one drop of 3% H₂O₂  was dropped onto the sample, and then the slide was sealed in a petri dish to eliminate contamination of aerosols. If the slide had immediate formation of bubbles then the result of the catalase test was positive, but if there was no bubble formation then the test was negative. This reaction can be seen in the picture to the right.

This test is done to differentiate catalase-positive Micrococcaceae from catalase-negative Streptococcaceae. Upon testing our microbe it was observed that there was no formation of bubbles, which means it was a negative catalase test or a catalase-negative Streptococcaceae. Additional characteristics that can be determined from a catalase activity test are determining if a bacteria is gram-positive or gram-negative and differentiating between aerobic and obligate anaerobic bacteria. 

The reason that some microbes have evolved to have catalase activity is because of how harmful hydrogen peroxide can be when produced by metabolic processes. In order for the organisms that produce hydrogen peroxide as a byproduct of these metabolic processes to be safe from its effects they catalyze the compound into safer substances (water and oxygen). Many organisms must rely on “defense mechanisms that allow them to repair or escape the oxidative damages of H₂O₂" in order to survive.

CARBOHYDRATE METABOLISM
             The next step in determining the species of our unknown soil microbe was to run a Triple Sugar Iron Test. A TSI Test is a tube of slanted agar medium that is used in order to determine if the production of hydrogen sulfide is present and to see is there is fermentation of carbohydrates. Three different substances can be used in order to differentiate fermentation and those are, sucrose, lactose, and glucose. TSI tests are also very useful for determining different types of gram‐negative bacteria.  
             The tube to the right shows what a TSI slant looks like before a sample has been placed on the medium. The red color of the medium comes from the pH indicator (phenol red). The slant is made up of an aerobic (oxygen) section which is placed near the top of the tube, while the bottom of the tube, also known as the butt, is anaerobic (no oxygen).
             Once the microbe sample has been placed on the slant a variety of results can appear; 1.)Glucose Fermenters, 2.)Glucose, Sucrose, and Lactose Non-fermenters 3.) Glucose, Sucrose and/or Lactose Fermenters 4.)Glucose Fermenter and Hydrogen Sulfide Producer, and 5.)Glucose, Lactose and/or Sucrose Fermenter and Hydrogen Sulfide Producer. There could also be there two cases of Gas Producers and Glucose Non‐fermenter Hydrogen Sulfide Producers. The pictures below show what the TSI slants would look like if any of the five results listed about were to happen.

Far Left: Glucose Fermenter
Second from Left:Glucose, Sucrose and/or Lactose       Fermenter
Second from Right:Glucose Fermenter and Hydrogen Sulfide Producer
Far Right:Glucose, Lactose and/or Sucrose Fermenter and Hydrogen Sulfide Producer.
Not Pictured: Glucose, Sucrose, and Lactose Non-fermenters



             The controls used for the TSI portion of the experiment were E. coli (A/A), B. megaterium (A/NC), P. areginosa (K/K) and P. vulgaris (A/A + H2S). A/A stands for acid over acid, which means that glucose, lactose and/or sucrose have been metabolized. A/NC stands for   acid over no change. K/K stands for alkaline over alkaline, which indicates that all three of the sugars have been metabolized. A/A + H2S stands for acid over acid with the production of a black precipitate at the butt of the tube. 
           The following pictures show the four control slants and then our unknown microbe slant after the butt had been punctured and the slant had been spread across the media.

Left: P. vulgaris (A/A + H2S), middle: P. areginosa (K/K), and right: B. megaterium (A/A).

Left: our unknown (?/?) and right: E. coli (A/A).

           Did our slants react in the way we expected them to? For the majority yes, except for P. vulgaris. P. vulgaris is (A/A + H2S) which means that it should've turned yellow in the early hours of the experiment, and then result in a sold black substance at the butt of the tube. This black substance that was supposed to be seen is the precipitate of the hydrogen peroxide, but it was not observed when the slant was checked on after incubation. As for the rest of the slants they all showed expected results. The B. megaterium metabolized glucose very quickly in the beginning stage of the experiment, which kept it the red color of the slant; ammonia was then released in the anaerobic butt of the slant which caused the medium to turn yellow. P. areginosa also followed the expected results by going from pink to red, which indicates that all three of the sugars were metabolized. 

         For our unknown the color of the media near the top of the tube was a yellow-orange color, and so was the butt of the slant. It was also determined, after observation, that our unknown has no evidence of hydrogen sulfide formation due to the lack of black substance in the butt. So we have come to the conclusion that our unknown must be very similar to. E. coli. They looked very similar after incubation, which leads us to believe that our unknown is also a Glucose, Lactose and/or Sucrose Fermenter just like E.coli. They both produced expected results by turning a yellow-orange color. This change symbolizes the fast metabolism of glucose, lactose and/or sucrose. 

SLOWLY DISCOVERING OUR DIRT 
         Thus far we have conducted five experiments to further our knowledge of our unknown soil microbe. We have done a Gram stain to determine if our microbe is gram positive or gram negative, we have tested to see if our microbe was acid or non-acid fast, we have tested for catalase activity, and most recently have tested to see if our microbe is aerobic or aerobic.
          We have determined so far, that our bacteria is neither gram positive or gram negative, but rather a bacteria called endospore-forming bacteria, which is a mix of bacilli and cocci shaped bacteria. We have also determined that our bacteria is non-acid fast due to the color it appeared after staining. There was also no sign of bubble formation during the catalase test which means that our bacteria has no catalase activity. Finally, we tested to see if the bacteria was an aerobic or anaerobic bacteria, and the results stated that there was activity of both types of metabolism. 

STAYED TUNED NEXT WEEK FOR DYLAN'S NEXT INSTALLMENT OF D^4 WHEN WE DIG FURTHER INTO FIGURING OUT THE CLASSIFICATION OF OUR UNKNOWN MICROBE!!!