All's fair in love and bacteria

The remaining differential test results are in and my unknown bacterium is, Staphylococcus epidermidis... or, well, maybe not...

In my last post I mentioned that there were two more tests that I was conducting. The first of these was a test referred to as the glucose O/F medium. This test would indicate wether the bacteria was capable of metabolizing carbohydrates (glucose) through oxidation or fermentation. Two test tubes of the glucose media and bromthymol blue (a pH indicator) were inoculated with samples of the bacteria. To one of these tubes a layer of mineral oil was added to create an anaerobic environment. The theory here is that a bacteria that ferments will exhibit growth in the anaerobic tube with the mineral oil, and the oxidizer will grow in the open or aerobic tube. The metabolic processes will result in the production of an acid that will change the pH indicator from dark blue to yellow. If yellow is seen in the open tube, the bacteria is an oxidizer. If the yellow is seen in the closed tube it is a fermentor.

These are the tubes after inoculation, and before incubation. Notice the layer of mineral oil on top of the media in the red capped tube.

This picture is after the incubation period as seen with backlighting. There was no perceivable difference under normal lighting. The aerobic tube on the left showed the faintest bit of a haze in the center of the tube. The anaerobic tube on the right does show what appears to be a colony growth and a yellow color change in the center of the tube. I have conducted this test in a previous experiment with a different bacteria that was a fermentor and the results were much more conclusive: the tube showed a large amount of color change. I interpreted the current result as being a weak positive for fermentation due to the apparent colony growth in the anaerobic tube. My thoughts were that maybe it was a slow grower and thus the minimal results.

I then analyzed the results of a mannitol fermentation broth. This test determines wether the fermentation of glucose results in the production of an aqueous acid or a gas. It was inconclusive on both counts. This stumped me as I believed the glucose O/F test indicated that this was a fermentor. It should have produced in the mannitol test as well, and would have been Staphylococcus epidermidis. 

I then consulted with Josh. He informed me that the glucose O/F test has been problematic in the past. My unknown should have produced in the aerobic tube and not the anaerobic tube. He did not elaborate on the reasons why the test had shown problems in the past. I suspect that the reasons are as follows. In the aerobic tube, the inoculation method required a "stab" with an inoculation needle through the gel like media. This may have created a situation that actually made it anaerobic. When the inoculation needle is withdrawn from this gel media it is plausible that the media could have "resealed" itself (the media may have been more fluid than it should have been) and not allowed oxygen to the sample once inoculated so it did not have the air needed for oxidation to occur. In the anaerobic tube, it is conceivable that the opposite occurred. Movement of the needle during inoculation could have created a void that filled with air and then was trapped in by the mineral oil. This small amount of air could have allowed some oxidation to occur and show the mild results I saw. At this point this is all speculation on my part and I need to dig deeper.

So, had the glucose O/F test showed the results it should have I would have correctly identified my unknown bacteria as Micrococcus luteus.