As our 6th week of lab rolled around, we were getting closer and closer to being able to identify this unknown bacteria. Our 6th week focused on discovering our bacteria's oxygen requirements. This week we also said a fond farewell to our environmental bacteria. It had served its purpose in giving us another type of bacteria to experiment our staining skills with, but now it was time to focus on our unknown.

     First, we did the Nitrate Reduction Test. The purpose of this test was to determine if our bacteria was able to reduce nitrate ions to either nitrite ions or to nitrogen gas.
     In anaerobic respiration, bacteria add electrons that have been passed along the electron transport chain to an inorganic substance that is not oxygen. One such final electron acceptor is the nitrate ion. The enzyme complex nitrate reductase facilitates this reduction of nitrate ion. For some bacteria to do this, nitrate ions are reduced to nitrite ions.
     Other bacteria that use nitrate reduction during anaerobic conditions for energy production are able to reduce nitrate completely to molecular nitrogen. This is called denitrification.
     To detect nitrite ions that are products of nitrate reduction by bacteria inoculated in nitrate broth, two reagents are added: sulfanilic acid and dimethyl-1-naphthylamine. If nitrite is present, the medium turns pink or red. This is a positive test for nitrate reduction.
     But the absence of a color change cannot be interpreted as a negative test. Either the nitrate ions were not reduced to nitrite ions (a true negative test) or the nitrate ions were completely reduced to molecular nitrogen (a positive test). To distinguish between these two possibilities, a small amount of zinc is added to the tube, which already contains the two reagents. Zinc will reduce nitrate ions to nitrite ions. If the bacteria did not reduce nitrate ions, zinc will do this. Therefore the appearance of a pink or red color after addition of zinc is interpreted as a negative result. No color means the test is positive for nitrate reduction. No nitrate ions remain in the brother because they were completely reduced to molecular nitrogen.

Our unknown bacteria tested positive in the first step; turning a red/pink color. Therefore, nitrite ions are present, and our bacteria can reduce nitrate ions to nitrite ions.

     Next, we preformed the Oxidase Test. This test is to determine if bacteria have cytochrome oxidase, a participant in electron transport during respiration. When cytochrome oxidase adds electrons to the oxidase reagent, the reduced for turns dark blue to purple within seconds.

As you can see, our bacteria tested positive.

     Next we performed the Catalase Test. The purpose of this test was to detect the presence of catalase, an enzyme that degrades hydrogen peroxide.
     In aerobic respiration, hydrogen peroxide is a reactive intermediate that forms as oxygen is reduced. H2O2 damages DNA and cell membranes and alters the active sites of some enzymes. Bacteria that tolerate oxygen or require the gas for metabolism use the enzyme catalase to quickly break down H2O2 into water and O2.
     When drops of a dilute solution of H2O2 are added onto bacteria containing catalase, bubbles of O2 rapidly appear. These bubbles denote a positive test.

Our bacteria tested positive. 

We also discovered our bacteria to be obligate aerobic.