Gram Staining on Deinococcus Aquaticus

Introduction

This week in lab consisted of many plasmid isolations from various cultures of Deinococcus Aquaticus grown in TGY media. The concentrations of these samples have not been excellent, and genomic crossover is suspected. When observing a culture, precipitate was noticed and contamination had become a concern. Due to this, a gram stain was done on this culture. 

Gram staining is a procedure used to test for infection or simply to stain bacteria. Bacteria can either be categorized as gram-positive or gram-negative in this test. After a series of specific stains, gram-positive cells will be stained red or pink; gram-negative cells will be stained blue or purple. These stains differentiate bacteria by the different aspects of their cell walls. For example, gram-postitive cells have many layers of peptidoglycan, whereas gram-negative cells have a much thinner layer of peptidoglycan; this makes certain stains only stick to gram-positive cells.





Procedure

To begin, a heat fixed smear must be prepared. to make a heat fixed smear, I began by grabbing our suspected infected Deinococcus Aquaticus. I then prepared a slide with a wax pen and sterilized an inoculation loop under a bunsen burner. After the inoculation loop had cooled, I uncovered our sample , obtained a loop of sample, covered sample, and smeared the loop circularly onto the prepared glass slide. After letting the sample completely dry on the slide, the slide must then be swiftly passed over an open flame 3 times. This allows bacteria to be fixed onto our slide, and better bind to our stains.

After the slide is prepared, a few drops of crystal violet must be added to the dried sample until fully covered. After 60 seconds, the slide should be indirectly rinsed with DI water to avoid losing sample. This stain will dye both bacteria blue or purple. Next, the same amount of Grams Iodine must be added to the slide until bacteria is covered. This will allow the crystal violet to further penetrate the peptidoglycan layers in gram-positive bacteria. After indirectly rinsing with DI water, bacteria should then be indirectly rinsed with ethanol for ~7 seconds and then with DI water immediately afterwards. This step will leave gram-negative bacteria colorless. Next, the same amount of safranine should be added to slide,  left for 60 seconds, and rinsed indirectly with DI water. This step will stain gram-negative bacteria red or pink. Lastly, the slide should be gently blotted with bibulous paper until dry. The slide should be ready to be looked at under a microscope ( 1000x). 





Results




Discussion

Though clearer in person, there were many clear signs of contamination in our Deinococcus Aquaticus culture. Both gram-positive and gram-negative bacteria of different shapes were noticed in our sample. Therefore, more Deinococcus Aquaticus was innoculated and grown in TGY. This was then placed in an incubator at 30°C. The contaminated sample was labeled and kept to be autoclaved. Therefore, a pause was placed on plasmid isolation, as this was our last sample of D.Aquaticus 

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