Carrying out a Gram stain --> video
Gram stain and results --> video

TITLE: PERFORMING A GRAM STAIN TO IDENTIFY BACTERIA

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PREPARING THE SLIDE
The Gram stain is quite useful in identifying different bacteria; it separate them into two main groups, those that are pink Gram negative and those that are purple Gram positive. In order to do the Gram stain what we need to do first of all is turn the Bunsen to the hottest part of the flame. Sterilise your bacterial loop until it is red hot, and then using that sterile bacteriological loop to take a loop full of distilled water. So I’m just taking a loop full and transferring it onto that slide, doing the same again and transferring onto the second slide, and then flaming your  loop again until it is red hot to sterilise it.

It is important to label your slides before you actually put any bacteria on them so I’m just writing Staph. aureus (Staphylococcus aureus) on the first one because we are going to be transferring Staph. aureus onto here, and E. coli (Escherichia coli) on the second one, because we are going to be transferring E. coli onto this one. Then using your straight wire this time, flaming it until it is red hot, and then using that, wait until it has cooled a little bit, again taking one colony of your bacterial culture, so just choosing one nice isolated colony there, and emulsifying that on the surface of your slide, and leaving it to air dry. Flaming your straight wire again because this time were are going to pick a different bacterium. E. coli this time. Picking a nice big colony, so that is a nice one there, got a little bit on the edge of the straight wire, and then transferring it, emulsifying it in that saline. And again flaming that straight wire until it is red hot, and what we are going to do is leave these to air dry for 5 to 10 minutes.

HEAT FIXING THE SLIDE
Once your slide has had 5-10 minutes to air dry it should like that (a round dry patch on the slide), you shouldn’t see any moisture on there at all, what you need to do then is then heat fix it and you can do this by passing it through the Bunsen flame, the hottest part, for about 2-3 seconds and then touching the back of your hand, and if it is a little bit to hot it has been heat-fixed. And once it has been heat-fixed, just pop it onto the staining rack ready for staining. And again with your second slide it should be nicely air dry no moisture on there, heat-fixing it in the Bunsen flame for a couple of seconds until it is a bit to hot to put onto the back of your hand, and that can go onto the staining rack ready for staining.

APPLYING THE STAINS
These are the Gram stains that we are going to be using crystal violet, 95% alcohol, iodine and saffronin. And if we start first by putting the crystal violet on making sure you cover the area where you have put the bacteria on the slide, and the same with the second slide. It is not crucial  how long you leave the crystal violet stain on for, but no more than 60 seconds, and then wash off with some tap water or distilled water or  tap water and what you should see is a stain looking like that (purple) because the bacteria have taken up the crystal violet stain.

The next stage is using your Lugol’s iodine and again similarly it is not crucial how long you leave it on for but make sure you cover the area where the bacteria are on the slide, and again washing off with your tap water in the bowl. So now what you’ve got on the slide is an iodine crystal violet complex.

Then next part where you add the 95% alcohol is the crucial bit because you don’t want to leave the alcohol sitting to long on the slide because what it will do is decolourise all the bacteria. So what you do is add your alcohol, and almost straight away washing it off with your water. So you don’t leave it on very long certainly for no longer than 15 seconds, and again adding your alcohol and washing straight off with your distilled water or tap water.

And your final counter stain is the saffronin and you can leave this on for about a minute, making sure you cover the area where the bacteria are, and once it has had its minute then again washing off your stain. The next stage is you’ll need to blot dry your slide, so using some blotting paper, take your slide, get rid of any excess, and then pop it onto your blotting paper, and then using a second sheet popping it on top and making sure you have blotted it dry ready for when you add your oil. And do the same with the second one, getting rid of any excess, popping it onto the blotting paper a different area to the first one and again blotting it dry with your figures and leaving it to air dry for a few minutes to make sure all the moisture evaporates.

VIEWING THE SLIDE
Once we have stained our slide the next step is to put a drop of immersion oil onto the area were we have got the bacteria, so just one drop should be sufficient, (put an extra drop on there), and then we are going to view it firstly on low power. So if I take the slide now and pop it onto the microscope stage, and just slide it in so it is fitting snugly, so it is flat, and then just making sure the that condenser is not to far up and not to bright, roughly about there. And then I’m just going to focus firstly on low power which is this lens here, the one with the yellow ring on it, until we can get something in focus. And once we have got something in focus then switching to the high power oil immersion, which actually says “oil” on it and usually has a white ring around it, and because we have already focused on low power we shouldn’t need to do much focusing now on high power. So using the fine focus which is this one on the outside just focusing that until you can see the bacteria nice and clearly under the microscope. And then you should be able to see whether they are pink Gram negative or purple Gram positive.

So just having a look at that Gram stain again, we are going to have a look on low power, so as we see it is not in focus, so using your focusing ring, focus it until you can see something in focus relatively clearly and there you can see the bacteria in the background staining a dark greyish colour. And then what I’m going to do now is switch to the high power oil immersion lens and then re-focus it using the fine focus this time hopefully to see the same bacteria but hopefully at a much higher magnification. (Turns the light up a bit). You can see now those purple cocci which were the Staph. aureus which we originally put onto the slide, in clumps, so that is high power under oil immersion.


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