Hey everybody, Dr. O here. In this video I want to talk about smear preparation. So a smear is a thin preparation of microorganisms on a slide.
So the reason that we make smears is so that we can stain them to help identify which type of organisms we're looking at and to run differential or what I like to call diagnostic stains to know if an organism that we're looking at is dangerous or what family it belongs to. So what you're looking at here is the most powerful weapon that you have in your arsenal as a microbiology student. It is the wire loop.
The wire loop is one of the few things that we can do in a microbiology lab where we know that we can sterilize it. So this is the wire loop. Just FYI, if you took that little circle off the end and just left the piece of wire, that would be called a needle. We use that for some of the stabbing techniques that we do, but the wire loop is something you'll be using almost every week in a microbiology lab. So in order to prepare smears, the first thing we're going to do is we're going to flame.
So we're going to flame this wire loop, as you can see here, using a Bunsen burner. You can use a Bunsen burner or an incinerator. Now personally, I'm using Open Education Resource Images, but I like to flame my wire loop with my hand underneath it. But that doesn't matter. I'll show you that in the lab if you're my student.
So you're going to flame this entire wire and the loop at the end of it until it's red hot. So that's how we have now sterilized this wire loop. And that means that when we...
When we use our aseptic techniques, we're going to transfer pure cultures of organisms on a sterile wire loop. So we know that we're actually working with the organisms that we want to work with and we want to test. All right, so we take our wire loop, we flame it. Then you're going to let it cool.
See, I count to 10 Mississippi and then I start transferring microorganisms. Now, you'll know if you didn't wait long enough when you hear a sizzle when you try to use this in a liquid broth. So make sure, and if that happens, just start over, re-sterilize and count to maybe 13. So we're going to flame the wire loop, we're going to let it cool, and then we're going to add two loopfuls of organisms onto our slide. So you see a slide here that has a couple loopfuls of organisms on it.
If you're starting from a liquid culture like a nutrient broth tube, then you just transfer two loops of organisms to the center of the slide and then spread it out. So that's pretty simple, just using your regular aseptic technique. If you're starting with solid organisms from a plate or a slant, then you're going to actually add two loopholes of water first and then add a clump of the solid bacteria and break it up here.
So we've sterilized our loop, we've done our transfers, we now have two loopholes of organisms on our slide. Make sure you spread it nice and thin. You want to make sure it's a thin preparation of bacteria. Then the key here is to let it air dry. Super super important that it air dries because after the air drying process we will heat fix the slides.
and if they're still wet when you do that, The chances that they actually will attach and stick to the slide and stay there when you cover them in stain and alcohol, etc., is slim to none. So if you don't wait an extra couple minutes here and you rush to do this, then you're going to ruin your slide and not be able to actually see anything when you stain it anyhow. The other big problem is if it's still wet, when you heat fix the slide, that water will actually destroy cells.
So yes, we do want to kill the cells when we heat fix them, but you don't want to destroy them because if it changes their structure, or it destroys their cell wall so they can't hold stain, we actually can't learn anything from our staining techniques. So super important to let them air dry. I like to kind of hold them up to the light, hold them at different angles, make sure they're dry before you heat fix them.
And then we have heat fixing. So here's an example of using an incinerator to heat fix. There are trays you can put on these and just leave them sitting or you can hold it like this.
Incinerators work just fine, but they take a while. This will generally take several minutes to heat fix a slide this way. We primarily will use the Bunsen burner and when we heat fix slides now now how I do it and how you do it be different but I like to just hold the slide and then just just gently pass it through the flame of the Bunsen burner until I can feel the the slide get warm I would have a glove a glove on that hand or else it would get super warm But I don't generally have students do that So I'll have you take a clothespin and just pass the smear through through the through the flame of the Bunsen burner six six times six one second passes and is generally about perfect so we've transferred our organisms we've let them air dry we've now heat fixed them and now you're ready to stain them so i just put a picture an example here of what a what a grand gram stained organism will look like so now you have your smear now you can go and use your different staining techniques to to see what you need to see all right have a wonderful day be Be blessed.