Biochemical Tests and RNA isolation

     This week we started a second spore test as well as performed both the oxidase and oxygen requirement tests and made/inoculated urease plates. We also performed another RNA isolation. 

    Starting another spore test was simple as we only needed to inoculate it, let it incubate for a few days, then leave it to rest with the other spore test. The oxidase test was simple as we only needed to add a few drops of a specialized reagent and look for a color change. For the oxygen requirement test we needed to inoculate our sonorensis and controls into thioglycollate agar tubes. After they incubated we would be able to tell the oxygen requirement based on the location and distribution of the bacteria in these tubes. Then we can categorize it as an obligate aerobe, microaerophile, facultative anaerobe, aerotolerant anaerobe, or obligate anaerobe. For the urease plates we first needed to make a concentrated urea solution and a solution of only agar and water. After autoclaving the agar, remelting it then adding 50ml of the urea solution to 450ml of the agar and pouring the plates. 

    D. sonorensis ended up being an aerobe and oxidase positive, and the urease test ended up negative. While it initially looked inconclusive, after letting it incubate for two additional days you could see the clear difference in positives and negitives. 

Oxidase test

Urease test, positive, negative and sono


    We also did another RNA isolation on E.coli to stockpile RNA for the final qPCR run however the organic contaminates were once again high, and some of the results for the ng/μl were to low to risk doing an ethanol precipitate. At this point it is very frustrating. I feel as its something with the technique but I'm doing that the same as I have been for the last year and a half, plus Chad has verified that my technique is good. At this point have changed kits, pipette tips and tubes so i am starting to suspect the pipettes. However the last isolation went well but at the end i ran out of tubes so before this isolation we relaced them with ones from another kit. I would like to order new tubes but until i know for sure what is causing these issues i don't want to waste resources on my RNA anxiety. 


    

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