We’re going to start looking at what’s important in experience, 2 and basically experience 2 says how do we see bacteria? We talk about staining when we look at the size of things that we’re going to deal with. You can see over here all sorts of things that go from the tiny little atom.
On the left hand, side all the way up to great big tree depending on what it is.
We need different ways of being able to observe it.
This uses different types of microscopes.
We’re, going to use a compound light microscope.
That really, for our point of view, goes from about a micron up, and you can see in here that we’ve got some photographs of some different materials that were all taken with a microscope.
So we need to be able to differentiate things because of the size they’re at.
Why do we stain bacteria? Well, we’ve got some reasons here.
It says bacteria cells are very small, 1 to 10 microns.
They are clear or transparent as our most microbiological specimens, so we apply stain, which is really a colored dye to improve contrast and increase visibility that’s, why we apply stains and a very easy way to look at it in the atlas it says, microbes are Too small to be seen by the unaided eye and because cytoplasm is essentially transparent, viewing cells with standard like microscope is difficult without stains to provide contrast once stained, we can look at things like cell morphology and size and arrangement, and other things like that. Now let’s look at some prokaryotic shapes.
There are a few basic shapes that we always see caucus the pluralist cosi is a round shape.
It’s, basically one to one.
They look like these little tiny spheres, all next to each other.
We have the coco bacillus, which is about twice as long as it is wide.
We have the bacillus, the plural is bacilli, which is about three times or more longer than it is wide.
And if you look at bacilli, they often look like these little tubular shapes.
We have the vibrio, which is kind of a comma shape, not really regular, but rather it tapers.
To one end, we have what is called the spirulum.
The spirulum is a curved shape where it’s a kind of bacillus that has been twisted or wrapped around. The spirulum often has a corkscrew like shape and polar flagella.
These are not flexible.
The spirochete is elongate and thin.
It’s kind of twisted on an axis so that it gives it more of a stretched out elongate spring kind of look.
These are very flexible and we have pleomorphic and pleomorphic says that the shape varies.
One of the things we often talk about is cellular arrangement.
In this particular case.
If you look at a caucus, a caucus can divide on one of three different planes.
You call it an xyz axis: if you get single division, we get single ones like that.
You can have single ones where they kind of hold together, and we call that a diplococcus or a diplococci. You can have them where they form together in chains, and we call that a streptococcus or a streptococci.
You could have them where they divide in two planes, but they hold together, in which case we’d, call it a tetrad or three planes and hold together, and we call it a sarcina, and this is kind of a packet of eight and you can get These in eights and sixteens going up like that or you can get them dividing in three planes where the three planes really aren’t at right angles to each other, then we would call that a staphylococcus with bacilli, since they’re elongate.
You can get the single bacillus, you can get a diplobacillus, you can get a streptobacillus where they divide down like that.
But again you won’t get much different than that because they’re only dividing kind of like links on a sausage.
We have a palisade in the palisade is where they kind of go up and they stack and they kind of stay together that’s kind of difficult to see.
Unless you’re very careful when you make the preparation of these one of the things we deal with is preparation of a bacterial smear.
Now watch the video on that, because it really shows you how to do it.
This is going to give you a overview.
It’s get a microscope slide and clean it.
You transfer bacteria using aseptic technique. You light the bunsen burner, you flame, the loop, you let it cool down a little bit.
You remove the material out of the culture, you smear it on a drop of water.
On the slide you allow it to air dry, then you heat fix it, draw it through the long way three times and you’re going to get your slide that you can use.
You make the smear you, let it air dry, you heat fix it, and we show you all that in that video.
Your slide should look something like this.
In the end, simple staining, when we do simple staining, we add only one dye to the specimen.
We flood the slide with dye, we let it sit for 30 to 60 seconds, we rinse it, we blot dry, and then you can see it.
We also have differential, stains and differential stains, add more than one color, and that gives you differentiation based on some feature of the cell, as this includes acid, fast stain, which will show up mycobacterium.
The waxy wall on the mycobacterium really does not take up the gram stain, even though these should be gram.
Positive. The acid fast procedure allows them to take up stain.
We have an endospore stain which shows you the endospores on the inside, and you see these little openings in there.
That is really where the stain was not taken up and since the stain was not taken up, you get a look like this.
We’ve got capsule stain and on the capsule stain it stains everything else, but what you want to see by staining everything else.
What you want to see, which is clear, is going to show up and we’ve got a flagellus stain in the flagella.
Stain basically makes the flagellus stand out by adding thickness to it, and that way we could see what the flagella look like.
We also do the gram stain gram stain that says.
There are four major steps.
We have the primary stain, which is crystal violet.
We have a mordant fixer, iodine potassium iodide, which is a material we call grams iodine and that basically is going to complex the crystal violet to keep it in place. Our decolorizing agent is ninety five percent alcohol.
The counter stain is safer way.
It works is like this.
We’ve got two different cell walls on these.
Now you can see on both of these.
You have a cell membrane down at the bottom, then, on top of that, you’ve got a layer of peptidoglycan the layer of peptidoglycan.
In the bottom picture is thin: it’s thick on the top picture.
You add the crystal violet to it.
The top picture gets flooded with crystal violet.
It gets held in place on the bottom picture. It’s going to get a little in there.
It’s not going to get a whole lot because you’ve got another membrane.
On top of it, then you add your iodine.
It’s going to complex it into position.
You decolorize, with, alcohol and when.
You.
Do that it’s going.
To destroy, the? Outer? Lps? Layer? On? The? Bottom? One? Which? Is? Going to, allow the? Stain to? Leak out, and on? The top, one it? Still remains stained, then you go and you add the counter stain, which is pink and pink on purple leaves purple pink on clear, leaves pink, so gram positive is purple.
Gram, negative is pink general run through one minute, one minute less than 30 seconds.
One minute watch the video and it shows you exactly how to do this. One of the things we often look at is on these four major steps.
What color do things have it says if you start out with the cells on the slide, you’ve got.
No color to it, you add the primary stain it stains, everything purple, you add the grams iodine, it leaves everything purple, you add the alcohol to decolorize it and it’s going to take the color out of the gram negative bacteria, but not out of the Gram positive and then, when you add the counter stain the pink on the purple, is going to not change color, the pink on the clear is going to give it a pink color.
If we have any problems with this – and this is one of those things that sometimes happens – we want to deal with a culture that ideally is between 24 and 48 hours.
Sometimes that’s a little bit difficult, because sometimes the culture is a little older, and if you do that, you may not get a good result.
If you heat fix it for too long, you will actually cook the bacteria and that’s going to leave them come out as a gram negative too much alcohol, which is very easy to do.
We call that over decolorization over to colorization it’s going to leave it as a gram, negative bacteria if the reagents are too old, that can be a problem as well as can, if you don’t, add the iodine.
So when you do this, you have to make sure that you do everything in the proper procedure in the proper way in order to be able to get the best result out of it.
So when we look at bacteria and staining bacteria, they’re small, they’re transparent.
You know we have to add the stain to improve contrast, so that we can visualize many different ways of doing this. This is, in a nutshell, as a couple watch the videos, because the videos really show you how to do the procedures.
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