Microbiology at NDSU: Gram Stain

Microbiology at NDSU: Gram Stain


Hello everyone. Today we are going to be doing the Gram stain, which is a differential stain that tells us whether or not we have Gram-negative or a Gram-positive bacteria, and this is based off of their cell wall. Gram-positive bacteria have a thick peptidoglycan layer, and they should stain purple, and Gram-negative bacteria have an outer membrane and the thin peptidoglycan layer, and they will stain pink. So, what you want to do first is you want to find your stains so we should have crystal violet, iodine, ethyl alcohol, and safranin. You also want some water handy and you should have your bacterial smear. So on your slide you should have drawn a red circle and flipped it over and put the smear on the circled portion so that you can differentiate which side has been used. So what you’re going to do is you’re going to take this slide and you are going to put it on the wire rack in the sink, and so we’re going to start with our crystal violet. This goes on for one minute, and the crystal violet is our primary stain. Following the crystal violet, so after that one minute, we’ll rinse off the slide, and we will add the iodine. And the iodine is the mordant, and it is left on there for one minute and it will form a complex with a crystal violet and get stuck in the thick peptidoglycan layer of the Gram-positive bacteria. In the Gram-negative bacteria it’s forming this complex on that outer membrane which will be washed off with the ethyl alcohol, which is our next step. So following the one minute time frame for the iodine, we’ll rinse it off, and now we’re going to take our slide and we are going to hold it at a 45-degree angle. We are going to apply the alcohol, which is ethyl alcohol, for 5 to 10 seconds. Now we’re going to rinse off the slide and we’ll add our counterstain, which is our safranin, and this is going to go on there for 30 seconds, and this will stain that Gram-negative cell, that’s currently colorless, pink. So after the 30 seconds, we’ll rinse it off. Now our slide is ready to go into our Bibulous paper and we dry it off. And we can visualize it now under the microscope.

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