Talk:Biology 210 at AU:Lab Members

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2/5/16 Notebook entry 3: Microbiology and identifying Bacteria

Purpose:the Characterization of different species of bacteria by observing the motility, gram stain, colony morphology and sequencing of the 16s ribosomal subunit gene.

Materials and methods:

  Before starting the procedures an observation was made of the Hay Infusion cultures plates.the cultures without the antibiotic grew noticeably. the blood agar plat had growth with clear indications that halos were formed, meaning that the bacteria ate all the blood in certain areas leaving wholes or halos in the plate where parts of the blood agar are missing. The smell also changed and was a potent odor similar to spoiled food. 

.First, plates were arranged in order of serial dilutions from least diluted to most. . all bacteria colonies in each plate were counted and recorded. . Once accounted for the number of colonies were converted to colonies over mL. .Then Bacteria cultures were observed with microscopes. . Wet mount and Stain Gram were observed with the microscope. Wet: sterilize loop over flame

    scrape tiny amount of growth from surface of agar.
   Mix it into a drop of water.
    place cover slip over drop.
    observe with 4x and 10x
    observed cell shape and size.

Gram Stain: sterilize loop over flame

       scrape tiny amount of growth from surface of agar.
        Mix it into a drop of water
       circle the area underneath the sample with red wax pencil
       heat fix the air dried slide by passing it through a flame 3xtimes with bacteria side up
       with staining tray cover slide  with crystal violet for 1minute.
       risne slide off with filtered water
       cover slide with gram iodine mordant for a minute
       decolorize by flooding the smear with 95% alcohol for 10-20 seconds.
       cover the smear with safranin stain for 20-30 seconds.
       rinse off with water
       dry excess water carefully with kimswipe, then allow air dry 
       No cover slip needed 
       observe sample at low magnification with 40x and 100x.
PCR: label 2 PCR tubes with own identification 
     add 20ul of primer/water mixture into each tube, mix to dissolve PCR bead   
      using sterile toothpick pick small amount of a bacterial colony on one of the plates
      place toothpick into PCR tube and twist for 5 seconds
      repeat last two steps for the other tube.

Data and observation: There were only a few bacteria that were sited under the microscope. Conclusions and Future directions: if the experiment was redone, there would have been more bacteria to look at on the slides instead of just two organisms. KKM.*Keanan K. McDaniel 23:56, 4 February 2016 (EST):