Completing a Gram Stain on Deinococcus radiodurans in MHA Media

    This week during lab we completed a gram stain on Deinococcus radiodurans after it was grown in MHA (mueller-hinton) media. Deinococcus radiodurans is an extremophile bacteria that we are growing in our lab. We are trying to grow Deinococcus radiodurans for 24 hours, in a media that we've never had in before, to see if it's successful. 

    To complete my gram stain, I first started by drawing a wax circle on my slide for and an indicator in the top right corner, to ensure my slide wouldn't be upside down at any time. I then ignited my Bunsen burner then sterilized my loop with the flame. After allowing the loop to cool, I unscrewed my tube containing the D. rad in MHA media, ran the top of the tube over the flame, collected my culture, ran the mouth of my tube over the flame once again, before finally screwing my tube shut. After closing tube, I smeared my culture within the wax circle on my slide. I waited about 2 minutes for it to fully dry before I heat fixed my slide (running it over the flame 6 times). We then continued with the staining process of my slide. I flooded my slide with crystal violet dye and let it sit for 60 seconds. The crystal violet will adhere to the cell membrane of a gram positive or a gram negative cell. I then rinsed my slide with DI water, placing it at a 45 degree angle, making sure I am not directly spraying onto the culture site. After rinsing off crystal violet, I then added grams iodine for 60 seconds. Grams iodine is used for finding gram positive bacteria. I did the same rinsing process for the gram iodine as I did for the crystal violet. After the gram iodine, I rinsed my site with ethanol alcohol. I did this by doing a similar process to rinsing the slide with DI water. Placing my slide at a 45 degree angle, making sure I am not directly dropping the ethanol onto the culture site. After ethanol comes the actual rinsing process with the DI water. The last dye used is the safranin dye. You flood the plane with safranin, let sit for 45 seconds, then do a final DI rinse like the ones done previously. Once you finished your gram stain, you are to blot the slide gently with the blot book, then you are able to look at it under the microscope. 

    Once I found the cells under the microscope, we observed the D. rad did very well in this media. It was even described to have excelerated growth in 24 hours compared to other medias that D. rad has grown in, in the past.