Before bacteria can be viewed through a microscope, a bacterial smear must first be created and then heat fixed. Heat fixing kills the microbes and firmly attaches them to the slide, so that the sample is not washed away during the staining procedure.
* Simple and Differential Stains *
Because individual bacteria are essentially colorless, it is necessary to apply a single stain, or series of dyes, in order to see them. A simple stain is the application of just one dye, and provides information limited to the shape and cell arrangement of the bacteria being viewed. Differential stains, such as the Gram, Acid-fast and Endospore stains, employ a series of dyes that help to distinguish groups of bacteria based on a specific chemical or physical attribute of the bacterial cell.
Once stained, the individual bacteria within the smear can be viewed using a compound light microscope, at a total magnification of 1000X. This high level of magnification is achieved through the use of an oil immersion objective lens.
* The Compound Light Microscope *
A microscope is considered compound when it has two sent of lenses; the ocular lenses and objective lenses. The ocular is the lens nearest the eye of the observer. The objectives are the lenses nearest the stage of the scope.
Most compound microscopes have either three or four objective lenses. If three, they most often include a:
* scanning objective, which magnifies objects 4X actual size
* low power objective, which magnifies objects 10X actual size
* high dry objective lens, which magnifies objects 40X actual size
Some compound microscopes also have an oil immersion objective lens, which has a magnification power of 100X. The magnification of the objective lenses is multiplied by the 10X magnification of the ocular lens, resulting, for the oil immersion objective, in a total 1000X increase in the apparent size of the object being viewed.
The oil immersion lens is required for viewing individual bacteria. In order to clearly see an object with this lens, immersion oil must be placed directly on the sample being viewed. The oil increases image clarity, or resolution.
* Steps to View Bacteria under Oil Immersion *
1. Place the slide in the mechanical stage apparatus. Do not use a coverslip.
2. Focus the 10X low power objective lens on the smear and examine.
3. Rotate the nosepiece to view the sample using the 40X, high power objective lens and examine.
4. Turn nosepiece so that sample is positioned half-way between the 40X and 100X objective lenses.
5. Cover the 40X lens with a finger cot to prevent it from coming in contact with the oil.
6. Place a drop of immersion oil directly on the sample.
7. Turn nosepiece until 100X objective lens snaps into position.
8. Examine the lens; it should almost touch the slide and the oil should fill the space between the slide and the lens.
9. While looking through the eyepiece, use only the fine adjustment knob to move the lens up and down in tiny increments; this action should bring the bacteria into focus. If the sample is not visible after adjustment of the fine focus, try adjusting the lighting. If this fails, switch back to the 10X objective lens, refocus, and then try the oil immersion lens again.
* Sources *
Schauer Cynthia (2007) Lab Manual to Microbiology for the Health Sciences, Kalamazoo Valley Community College.
Bauman, R. (2005) Microbiology. Pearson Benjamin Cummings.