In this video we are going to look at the negative stain technique. Now the reason we use the negative stain technique is because it gives us a slide where the bacterial cell is very clear and distinct. Now you will recall when you do a bacterial smear you have to heat fix the slide. Anytime you use heat it distorts the cells.
With the negative stain technique we don't use heat. So the cells come out more natural and you get a much better image. Now the reason we call it the negative stain technique is because we are applying a stain that carries a negative charge. Now you will recall that bacterial cells have a negative charge too.
So the cells actually repel the stain. So we are really staining the background. So what you get is a white cell against a dark background.
You get really nice contrast. So let's look at the materials you're going to need to do this technique. You'll need a bottle of negatively charged stain. I'm using Nigresin. You'll need a bacterial culture.
An inoculating loop, a staining tray, two microscope slides, a heat source to sterilize your loop, and finally a slide warmer. So let's look at the technique itself. First what you want to do is take one of your slides and put a drop, a small drop of nigrasin near one end. Now don't get it on your fingers, so I go about, oh, probably a half inch from the end of the slide, and I put a little drop of nigrasin. Next.
We're going to get a culture of bacteria, and we're going to stir them into the drop of dye. Let me flame my lube to sterilize it. I'll let this cool. I'll go in and get me a nice sample of bacteria. And I'm going to stir these into the dye drop.
But when I stir it into the dye drop, I'm just going to stir it side to side. So I'm moving the drop laterally as I'm mixing the bacteria into it. from your finger, you don't want to get the bacteria all over you.
Now immediately flame and cool your loop to destroy the excess bacteria. And now you're ready to spread out the dye. So in this part you take your other slide, your spreader slide, and you put it on your base slide at about a 90 degree angle like this.
Then lean it towards the die drop, towards your fingers, and pull it up so you're pulling it into the die drop. And the die will grab onto the slide. And then you push it away from your fingers and that will spread. the dye out. You just keep spreading it out, spreading it out, and then as you get towards the end of the slide, just roll the spreader slide off.
Now this is contaminated with bacteria, so you want to immediately drop this into a biohazard bin or into a beaker full of bleach and water. Now we're going to take this and we're going to put it on our slide warmer. Now as the water evaporates, the dye actually bonds the bacterial cells to the slide.
And you end up with your finished product. Now let me put this on this bibulous paper so you can see what the final product should look like. There you have it.
Now a few things you want to avoid. Make sure that when you do this technique you do not put too much nigricin on the slide because otherwise you'll be waiting forever for it to evaporate. Also, make sure you don't get the slide on your fingers because it's loaded with bacteria. So that's pretty much the technique and let me show you an image of what the bacterial cells will look like in a negative stain technique.