Blog #6 2/26/24-3/1/24

 Introduction 

The following week, I made some media and did gram-staining on D. caeni and D. sonorensis. My classmates Emme and Bryan did a presentation discussing different chemical tests and their purposes. The plan is to use some chemical tests to characterize Deinococcus Sonorensis. We also had a discussion where we learned the difference between Rt-PCR, q-PCR, and Rt-qPCR. 


Methods

For the gram staining, I began by getting the bacteria (sonorensis and Caeni) on two separate microscope slides. First, you turn on the flame and sterilize the loop. Then after letting the loop cool, slightly lift the lid of your plate and scoop some of the bacteria with the loop. After spreading the bacteria on the slide finally heat fix it by passing the slide over the flame. Next, you have to complete the steps of the gram stain. You add a couple of drops of crystal violet on top of the bacteria, and after a minute you wash the slide with deionized water. Repeat this step with iodine. The third step is a quick wash with alcohol, then quickly after washing it off with deionized water. Finally, add drops of safranine on top of the bacteria, leave it for a minute then wash it off with deionized water. 



Emme and Bryan gave a presentation on the chemical characteristic tests, some of which we will perform on D. Sonorensis.--

  • Sugar fermentation test: there is a lactose fermentation test, which is used to determine if a bacteria can ferment lactose as a carbon source. The Sucrose fermentation test determines if the bacteria can ferment Sucrose as a carbon source. For both tests the media needed is phenol red base broth. The phenol Red is a Ph indicator, if there is a color change to yellow

  • MRVP test: this test determines what pathway is used to ferment glucose. The two pathway option is the mixed acid pathway or butanediol end product pathway. 

  • Enzyme testing: 1. urease test- a chemical test that determines if The bacteria can use urea As a source of carbon and growth 2. Starch test- is a chemical test that determines if The bacteria can digest starch with the enzyme amylase. 3. Citrate digestion test- this test determines if the bacteria has the enzyme citrate. 4. Oxidase test- determines if oxidase cytochrome is present and functional. 

  • SIM: The SIM test is used to test for sulfur, indolent, and motility. 

  • stainings: Gram stain determines if the bacteria is gram-positive or Negative. There is also the sporulation staining that determines if The bacteria can form spores. 


PCR discussion 

PCR or real-time PCR (or quantitative PCR) is a laboratory technique that is used To make copies of a specific section of DNA sequence. There are three types of PCR 

  • Rt-PCR is reverse transcript (the first Step of RtQ-qPCR) the process starts with isolated RNA, the the primers that were added come in to create a cDNA strand using the RNA as a template. The next step is amplification where several copies are made of the cDNA sequence.

  • Q-PCR process begins with an isolated double strand of DNA. Then the strand is heated up and the strand is denatured. Then dyes are added, and primers are added. The primer creates a complementary strand which the dyes will bind to.

  • RT-QPCR process begins as a strand of isolated RNA. Primers are then introduced to create a complementary strand of cDNA. Then DNA polymerase created a complementary DNA strand. The dyes bind with double strands. The cycle repeat creates several copies. 


Results & Conclusion

The gram stain for D. sonorensis did not turn out right, I might have left the ethanol on too long. D. sonorensis is a gram-positive bacteria but my stain appeared gram-negative. Next time I do a gram stain I'll be more careful with the time left on each stain. The PCR discussion was really interesting, the process overall reminded me of the central dogma of biology. The processes are similar and probably the PCR machine was created to mimic the same process. The presentation by Emme and Brian was informative and well-presented. 

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