Observing Gram Stains: One of the Most Important Microbiological Observation Techniques Using a Biological Microscope

Microbiology is the study of microorganisms. These organisms are too small for the naked eye to see, much less observe in detail. That is why there are various optical devices such as the biological microscope for better observation. In a while, you’ll be learning how to perform one of the most important observation techniques using a biological microscope – performing and interpreting Gram stains.

What Are Gram Stains?
This technique has been in use for more than a century already, and it is considered one of the best ways to identify microbes. Microorganisms are labeled as either Gram positive or Gram negative, depending on their membrane structures.

How to Make Gram Stains

When you have a heat-fixed slide ready, pour an adequate amount of crystal violet on it. After a half a minute, rinse it off with water while holding the slide in place with forceps. This will make Gram positive specimens more distinct. Afterwards, pour the same amount of Lugol’s Iodine and repeat the process. You’ll now see Gram negative specimens stained as well.

The next step is the most crucial part of the procedure: decolorization. While microbiologists tend to frequently use acetone, it’s better for beginners or students to stick with 95% ethanol for the time being. Ethanol will not show a terribly compromised sample even if small mistakes have been committed during decolorization.

Use forceps to hold the slide at a diagonal or slightly leaning angle once more. Pour an adequate amount of your preferred decolorizing liquid after letting the slide stay in that position for three seconds. Rinse immediately with tap water and afterwards, you’ll see Gram positive appear purple while Gram negative microorganisms appear colorless.

The last step is definitely easier. Now that you’ve decolorized the sample properly, you can finally counterstain the sample to make Gram positive and Gram negative microorganisms truly distinguishable from each other. Pour an adequate amount of saffranin on the slide. Again, wait for half a minute before rinsing it with tap water. By now, you should be seeing Gram positive microorganisms appearing violet or purple while Gram negative microorganisms will appear red because of the dye.

Preparation and Use of Biological Microscopes for Gram Stains
Now that you have your Gram stains prepared, use blotting or lens paper to gently blot the stained side. Don’t try rubbing it dry because you could end up removing the microorganism from the slide.

Place the stained slide on the stage and lock it in place with the stage clips. Observe at the lowest power first. Grab a grease pencil and draw a line to help you focus on the microorganism.

Slowly increase magnification, making sure that your focus is still centered at the magnification. Trying to identify the microorganism’s species at any magnification lower than 100x is moot because you still aren’t in the position to view the microbe in complete detail.

Place a maximum of two drops of immersion oil on your objectives when it’s time to view the microbe at 100x magnification. Once you reach this level, you can’t return to using lower magnification as the oil could spread to other objectives and impair your view. If you need to reduce magnification, your safest bet is to use your biological microscope’s lowest magnification at 4 or 10x.

Observing and Interpreting Gram Stains with a Biological Microscope
Before you start observing and analyzing your specimen, take a mental step back and review the whole process. Did you do everything correctly? The smallest mistake could affect the quality of your Gram stain and result to a flawed sample.

If you feel you’ve done everything correctly, it’s now time to identify whether the microorganism is Gram positive or Gram negative. If it’s positive, you’ll see mostly purple specks while Gram negative species appear mostly red. Of course, be sure to view the microorganism at all angles since dense areas of your slide typically appear purple even with Gram negative species. Also, try to observe the microorganisms when they’re separate from each other.

Consider the size of the microbe then recheck or compare with referential guides to ascertain that you’ve got the right microorganisms under your biological microscope.

Lastly, consider its morphology. Again, check your references to validate your observations. Memorizing data is good, but it’s still better to double check with reference materials.

Of course, Gram stains are not the only microbiological observation techniques you’ll need to learn when using a biological microscope. There are definitely more, but starting with Gram stains are good for the time being.