So you want to do a Gram Stain?
I took some pictures while I was doing a gram stain today. The end result was kinda garbage, I had a bad sample, and I honestly can't tell how bad my technique was, just because I got a little impatient trying to heat fix the slides. Explanation of what this process is for at the end.
Anyhow, first, you want to get your slides ready. There's a few ways this can happen but we're probably here for pictures and scrolled back up to understand what the heck they are, right? You wait for your lil drop of bacteria to dry and then pass it over a flame to make it "stick".
Second, take your first stain (crystal violet) and cover the cells. Let soak. You should have already had your stopwatch/ timer running because you know you can't be trusted to count past 20 seconds.
Rinse and proceed to three. Your textbook has some noise about "letting the water roll off your finger onto the slide". Just don't jet clean the spot with cells. You're not dealing with the cure for cancer, you can afford SOME recklessness. Nbd.
Three, you'll do the same with an agent that will help your cells hold the dye (Gram's Iodine)
This is where the process starts to be ever so slightly annoying because you've got water leftover on the slide and it surface tension is a little deletion mutation in the ass, but not really a big deal (but maybe it is because these slides were garbage) anyhow we power through to four.
Four, we're actually removing dye. We used the iodine so we don't remove all of it, but we're gonna rinse it with alcohol, just like a suburban white lady having a "rough morning". A quick splash. This is area number two (three if you count the water droplets that i've never had issues with before) where I could have gone wrong. Again, prooobably just the culture but ehh. Anyway, rinse em off.
Yes, it's one of those bottles that you squeeze upright, they're really fun to use honestly, but if you've got big gay hands like me, you'll probably spill if you underestimate your absolute power when you pick it up.
You're rinsing the alcohol off as soon as you're done with the few moments of contact.
Step five is to add one more DIFFERENT stain (safranin red) to replace the dye we removed. Sounds backwards, but it makes sense, I swear.
Admire briefly that it looks like blood and then remember you've got a blood agar plate sitting about 1.5 feet away from you. This is science. It's badass, but let the actual edgy things be edgy. (Although admittedly, dyeing things with blood sounds cool)
Last, you're gonna pretend you have the willpower to wait for it to dry but inevitably dab it with a paper towel because you turned off your bunsen already, and you wouldn't be able to use it anyway because you've already not only heat fixed, but also stained your slides. Banging them on the lab bench only slightly improves the drying time.
The purpose of this is to differentiate between cells with a thicker or thinner layer of a particular molecule in the structure of the cell wall. Since they both absorb dye and lose it at similar rates, if you wash all of the dye out of the thin layer, and stop exactly then, there should still be dye in the thick layer cell. This is important because we can target the cells based on the cell wall. If you want more info, it can get complex, and i'm sleepy, so you'll have to deal with any major burning questions or holes i've left by consulting google. Here's a pretty picture and terrible example of the end results.
There are other ways to test for gram negative or positive cells which i'll be doing as i'm trying to ID these for class. I'll reblog with results and hopefully better pictures. There were just a lot of splotches and awful spots on the slides, and honestly this was one of the best spots. It looks misleading because depending on where you looked on the slide, the cells were different colors, and they're pretty badly stacked/ clumped in this imo.
Gravity will also not improve the drying time so that the water will not interfere with your oil immersion lens usage. You are an impatient bastard. Just go get the paper towels already.