I read somewhere (I can't remember exactly where) that every medical student should be familiar with the basic techniques of microbiology, like Gram staining and culture of bacteria on agar. We had practical sessions recently where these techniques were demonstrated before we did it for ourselves. Despite following the steps in the handout and thinking that I was doing everything the same way that I had seen it being done, the results were not always very satisfactory. The nagging fact with microbiology is the time it sometimes takes to yield results. When you come to realise that something went wrong a day or two later when you look at the results, it is usually too late to repeat the experiment. Here I am more precisely referring to the culture of bacteria on agar (streak or dilution culture). Despite the fact that I watched how the wire loop was being handled and rubbed on the agar, when I did it, it didn't turn out the way I expected. It looked fairly easy, touch the loop to a single colony of bacteria growing on a plate (here I should also point out that when you are doing a single colony culture and are told to touch only one colony with the loop, just do it. Don't worry about it not being enough), and streak it on the fresh agar plate where the bacteria is to be cultured. Well, when I did it, the wire loop cut through the agar! You may wonder whether I not only clumsy and that it can't be that difficult. You may be right, but when your turn comes, you'd better pay attention to the details: the angle at which the loop is applied onto the surface of the agar (it should be flat). Coming to the staining techniques, it's even easier for things to go wrong. The first time I stained a plate with both Gram positive and negative cocci, they all looked the same colour under the microscope. You should not be afraid to copiously rinse the slide when required (with water or alcohol/acetone). I had this feeling that I was going to wash the whole stuff away. But if you've fixed the slide properly in the first, there's no way that the smear will get washed away. So see to it that your slide is properly washed before counterstaining. In case at first you see no difference between Gram positive and negative bacteria under the microscope (as was the case with me) the trick is to move the slide to change the field you are viewing. There should be an area where the bacteria are distinguishable somewhere on the slide. Keep looking.
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