3/25-3/29: Biochemical Tests on D. Metalli

 On Monday we started by Gram Staining our starter plate for D. Metalli, the species was observed to be Gram positive and rod shaped, our Gram Staining was consistent with these observations as we had a Gram positive, rod shaped, bacteria. After we had Gram stained we then proceeded to streak all of our experimental plates. These plates included R2A and TGY plates for the catalase and oxidase tests, then we had streaked the Starch TGY plate for the Starch hydrolysis tests, and finally for the D. Metalli plates we streaked 2 experimental Urease plates, as well as inoculated the SIM Test tubes, which we had 2 experimental tubes. Then we had streaked our control plates/tubes with our control bacteria. These bacteria consisted of B. Subtilus, S. Epidermis, E. Coli, and C. Fruendii. Then, once all of our experimental plates/tubes and our control plates/tubes were inoculated we had put the inoculated media into the incubator for the bacteria to grow. For our experimental plates/tubes we had put them in the 30 C incubator since D. Metalli grows best at 28-30 C. For our control plates/tubes, we had put those in the 37 C incubator since a majority of the bacteria grew best at 37 C. 


D. Metalli Gram Negative 

On Wednesday we had enough growth to start our tests. In reality, the control plates had grown after only 24 hours after being inoculated so we had taken those plates out on Tuesday such that the bacteria did not overgrow. After another 24 hours our experimental plates had the perfect amount of growth to conduct the biochemical tests, which were pretty straight forward for the most part. Most of the tests consisted of just adding a couple drops of the appropriate reagent and waiting for around a minute to observe any reaction that occurred. For the catalase test, the test consisted of dropping a few drops of 3% Hydrogen Peroxide onto a culture and observing if any bubbles form, if bubbles form then the test is positive and the bacteria produces O2 gas. For the oxidase, you add a few drops of Korvac’s Reagent, which is just an oxidizing agent which will turn the culture blue within a minute of being added. For the starch test, we add a couple drops of IKI solution to the plate and if a halo forms around the bacteria then the bacteria can break down starch. For these tests; oxidase, catalase, and starch hydrolysis D. Metalli had a positive result. For the SIM tests, which include if it reduces sulfur into H2S, the motility test to test if the bacteria is flagellated, and the indole test to test if indole is formed after adding Korvac’s reagent, D. Metalli was observed to be negative for all of these tests. 

D. Metalli Oxidase Positive

One things that stood out to us was that our negative control plates for the oxidase test ended up turning blue which we took as meaning that it was oxidase positive, we were worried then that either our bacteria was contaminated or that there was an issue with the reagent. We considered that it was an issue with the reagent since we tried the reagent on a plain R2A plate and it still ended up turning blue. As it turns out this was not an error with the reagent or the bacteria, rather a misunderstanding of the reagent itself. It turns out that the reagent will react with oxidase positive bacteria in under 1 minute and after that it will react with anything after around 2 minutes or longer. Since our negative control plates turned blue around 5 minutes later it means that the test was correct in that the bacteria is oxidase negative, but the reagent is just reacting to other chemicals around them because of the reactivity of the oxidizing agent. 

Once we learned this we were confident that our tests were accurate and that there were no underlying issues with the bacteria or the tests. Another thing that stood out to us, is that the paper on D. Metalli that we have been referencing for data, says that D. Metalli is starch hydrolysis negative, however this is not what we observed, on our plates it is clear that the bacteria form a halo around the black bacteria, meaning it can metabolize starch. This was just an interesting observation that we found since it outright contradicts the currently known information we have on this species of Deinococcus. 

D. Metalli Starch Positive
Starch Test Negative (Left) and Positive (Right) Control

Source: 

Feng, Guang-Da, et al. “Deinococcus Metalli Sp. Nov., Isolated from an Abandoned Lead-Zinc Mine.” Microbiologyresearch.Org, Microbiology Society, 1 Oct. 2015, www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijsem.0.000439. 





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