S-STEM Scholar Alex Hugenberg-Cox Blog

      This week we started work on the poster as well as were able to finish the sporulation tests. For the sporulation tests we used the two sets of plates that had sat out through spring break in order to truly starve the cells in order to force them to produce spores if they are able. The procedure for a sporulation test is very fundamentally similar to gram staining, using malachite green to distinct vegetative cells from spores.  Sporulation Procedure 1. Bring 250mL of water in a beaker to a slight boil using a hot plate. 2. Use a wax crayon to draw a circle onto six slides.  3. Label two slides each of the positive control , negative control, and tested bacteria (+B. subtilis, - S. epi, ? D. sonorensis.) as well as the date in order to separate the two sets 4. Spread each sample within notated circle and use Bunsen burner to heat fix onto slide.  5. Attach clothing pins on either end of the slide and rest upon the top of the beaker, allowing the steam to hit the sample.

      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 in to  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

    This week we looked at the biochemical tests we inoculated last week. The lactose, sucrose, and citrate plates were all color changes while we had to add iodine to the starch plates and perform a procedure to see the MRVP results.      Deinococcus sonorensis was positive for lactose fermentation, inconclusive for sucrose fermentation, negative for the MR test and inconclusive for the VP test, positive for the starch test and inconclusive for the citrate test. The sucrose, VP and citrate tests all showed no difference between the negative control, positive control and test and will need to be redone. All of f the TGY plates grew, with the 1/2 TGY plate having a smaller amount of growth and the double TGY plate thriving. However the only R2A plate that had growth was the regular recipe.  Methyl red test  1. Transfer 2.5 ml of culture into a new sterile culture tube. 2. Add 5 drops of the methyl red reagent.  3. Compare the test organism to the control cultures to immediately interpre