Reactions: Catalase Test & Triple Sugar Iron Test

Earlier in the week, our microbe underwent a Catalase Test to determine whether or not there was activity or a reaction. The catalase test is used to determine whether or not there is enzyme catalase in the tested bacteria. Catalase is produced in situations of oxidative stress where it will facilitate cellular detoxification and when it correlates with pathogenicity in bacteria (lab handout). Some microbes might have evolved to have a catalase activity as a response to the oxidative damage of hydrogen peroxide to keep them from being killed off. In our catalase test of our soil microbe, we dropped 1 drop of 3% H₂O₂ onto our bacteria and waited for a reaction. Our soil microbe reacted to the H₂O₂ , leading us to believe that our bacterium resulted in a positive reaction. We compared our slides with a positive control, Staphylococcus epidermis as well as to a negative control, E. faecalis. Below is a picture of the reaction of our soil microbe in the catalase test. Our reaction was not large, however, it reacted to the H₂O₂, leading us to believe there is the catalase enzyme in our bacterium.

Catalase Reaction

We also performed a Triple Sugar Iron Test (TSI) on our soil bacterium to determine carbohydrate fermentation and hydrogen sulfide production. This test differentiates bacteria according to how they ferment lactose, glucose, and sucrose. Bacteria can metabolize carbohydrates in two ways: aerobically or fermentatively. This test will helpE. coli, B. megaterium, P. areuginosa, and P. vulgaris.

us decide according to the resulting color how our soil microbe metabolizes carbohydrates. The agar in the tube is defined as a differential medium and will select for carbohydrate fermentation and hydrogen sulfide production. Four controls were tested against our soil microbe:

Bacterium	Tube Reaction	Reaction Color
E. coli	Acid/Acid	Yellow
B. megaterium	Acid/No Change	Pink on Bottom
P. areuginosa	Alkaline/Alkaline	Dark
P. vulgaris	Acid/Acid + H2S	Yellow over Black
??? Our Soil Microbe ???

Below are pictures indicating the result of our Triple Sugar Iron Test for our controls and for our unknown soil microbe. The controls behaved as expected according to observations of the tubes with the TSI agar and from the lab handout. Our tube with our unknown bacterium in it had a pink bottom with a little yellow on the slant. The control most similar to our unknown bacterium is B. megaterium. There was no evidence of hydrogen sulfide production, as there was no observed blackening on the butt of the tube. The butt of our tube was pink, leaving us to believe that there was no yellow or black coloration and no hydrogen sulfide production. According to our initial observation after 24 hours at 37°C, our soil microbe is a glucose fermenter. Our tube reaction is alkaline over acidic (K/A), meaning that our bacteria can only metabolize glucose. Both aerobic and anaerobic metabolism can be used to produce ATP and pyruvate. Glucose is consumed by our bacterium around 18 hours and the amino acids were used as an energy source on the slant in the form or aerobic metabolism. The butt stays acidic due to the stable acid end-products of the Embden=Meyerhof-Parnas pathway that metabolizes glucose.

B. megaterium

E. coli

P. areuginosa

P. vulgaris

Unknown Soil Microbe

Unknown Soil Microbe

Unknown Soil Microbe

According to the dichotomous key, we can begin eliminating ideas and narrowing down our guesses on what our bacteria might be. Last week, our acid-fast stain left us confused in which direction to take our initial hypothesis on what our bacterium could be. This week, with further testing, we can begin to eliminate a few options and narrow our ideas. We found that our bacterium was catalase positive, and both aerobic and anaerobic. This narrows down our options to Actinomyces spp., and Peptococcus spp. However, further tests need to be done in order to solidify our observations. More tests will be needed and may lead us to reevaluate our hypothesis. But, as of right now, our tests have shown us that our bacterium could be either Actinomyces spp. or Peptococcus spp.