Observing Bacteria Under The Light Microscope

Can one see bacteria using a compound microscope? The answer is a careful “yes, but”. Generally speaking, it is theoretically and practically possible to see living and unstained bacteria with compound light microscopes, including those microscopes which are used for educational purposes in schools. There are several issues to consider, however.

Why bacteria are difficult to see

Bacteria are difficult to see with a bright-field compound microscope for several reasons:

• They are small: In order to see their shape, it is necessary to use a magnification of about 400x to 1000x. The optics must be good in order to resolve them properly at this magnification.
• Difficult to focus: At a high magnification, the bacterial cells will float in and out of focus, especially if the layer of water between the cover glass and the slide is too thick.
• They are transparent: Bacteria will show their color only if they are present in a colony. Individual cells present on the slide are clear. Regular bright-field optics will only show the bacteria if one closes the condenser iris diaphragm. This is due to the difference in the refractive index between the water and the bacterial cells.
• Difficult to recognize: An untrained eye may have problems differentiating bacteria from small dust and dirt which is present on the slide. Some bacteria also form clumps and therefore it is difficult to see the individual cells.

Research organizations and advanced amateurs use phase contrast optics to see bacteria. This system converts the differences of the refractive index of the bacteria into brightness. The transparent bacteria can then be seen dark on bright background. In bright-field, closing the condenser iris diaphragm will also make the bacteria appear darker, but at the same time one also introduces artifacts (“fringes”) around the individual cells. This is because of the diffraction of light. One possibility is to stain the bacteria, but in this case the fixing and staining process may introduce other artifacts, and it kills the bacteria.

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How to observe bacteria

Most people do not have a phase contrast microscope, which makes it much easier to see bacteria. It is still possible to see them, if your microscope has a condenser. Here are some suggestions.

• Use the 40x objective: With the 10x eyepiece you get 400x total magnification. This is enough to see them. Of course you have to start focusing with the 4x and 10x objective first and then work your way upwards.
• Completely close the condenser: This will increase contrast and depth of field. If your microscope does not have a condenser, then you might not be able to see them. In this case I suggest that you buy some ready-made slides with stained bacteria.
• Use very little water: When making the slide, use very little water and make sure that there are no larger particles under the cover glass. There must be a thin film of water beneath the cover glass. Otherwise the bacteria can float vertically and go out of focus. The cover glass should not float on the water. As a matter of fact, it is better if the water is so little that it is not even able to spread beneath the whole cover glass.
• Keep bacterial density low: If there are too many bacteria in the sample, then they will overlap and you will only see a clump.

What is a safe source of bacteria?

For recreational or educational purposes, one should never use spoiled food or (heaven forbid!) use bacteria obtained from the human body and grown on agar plates. The risks involved are simply not worth it, especially when working with students. Other sources, such as soil or humus have other disadvantages. The impurities make it difficult to keep bacteria from other particles apart, especially if one uses bright-field optics. Ideally you would like to have only bacteria, without many other things. I recommend the following:

• Yogurt: You can make own yogurt and then put a sample under the microscope. It should be possible to see small circular cells (cocci), which may also occur in pairs or chains. There are also many clumps in the yogurt and you have to look between these. Watch the video here.
• Cheese: It is also possible to scratch some bacterial cells off from certain kinds of cheese. Brevibacterium can be found on Limburger cheese, for example. One has to be aware that some cheeses use a combination of bacteria and fungi, however, and that the larger fungal cells may outweigh the bacteria. It is also possible to buy freeze-dried bacteria.
• Freeze dried: It is possible to buy freeze-dried bacteria for the purpose of making yogurt. Take a small amount of it and put it into water to dissolve before putting it under the microscope.

What is the easiest solution to see bacteria?

Commercial slide showing stained spiral-shaped bacteria.

The easiest and least complicated solution to view bacteria using light microscopes is to buy a prepared permanent slide. The bacteria are then stained and in a sufficiently high concentration. The image above shows bacteria of such a slide. This is not only safe, but becasue they are stained they can be easily seen also with microscopes that do not have very good optics.

Alternatives

If you just want to look at microorganisms and do not mind so much if they are bacteria or not, then I recommend baker’s yeast (Saccharomyces cerevisiae). These are not bacteria but also interesting to watch. Add some water and sugar and watch them divide after a few hours. It is also possible to get some fungus from various cheeses. These fungi can be eaten and are safe.

In summary, there are easier (and maybe also more interesting) specimens to observe than bacteria. I you want to see individual cells, then I do recommend that you start out with yeast suspensions. These eukaryotic cells are much larger and can be more easily identified.

Further Reading

• For pictures of bacteria in phase contrast read the following post: Bacteria in phase contrast
• Should you grow bacteria at home?
• Spiral-shaped bacteria can be seen here moving
• Bacteria growing in water