In order to view individual bacteria through a light microscope, a bacterial smear must be attached to a slide and then stained. Here is the procedure.
First, use a wax pencil to draw a circle on the microscope slide to separate each type of bacteria that is going to be sampled. If a simple stain of only one type of bacteria, or single mixed sample of bacteria is being prepared, no separation is required.
* Preparing a Bacterial Smear *
In order to be able to clearly see individual bacteria, a sample of a bacterial colony must be mixed into water or physiological saline. This helps to evenly spread out the bacterial sample.
Placing the Bacterial Sample on the Slide:
* Place a drop of water into the wax circle that has been created on the slide.
* Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony.
* Gently mix the bacteria into the water drop.
* Heat Fixing the Bacterial Sample *
Before staining, the sample must be heat fixed. This process accomplishes three things. It functions to:
kill the bacteria
firmly affix the smear to the microscope slide
allow the sample to more readily take up the stain
In order to heat fix a bacterial smear, it is necessary to first let the bacterial sample air dry. Then either place the slide in the slide holder of a microincinerator, or pass the dried slide through the flame of a Bunsen burner 3 or 4 times, smear side facing up. Once the slide is heat fixed, it can then be stained.
* Staining Bacteria *
There are many staining techniques that can be used to view bacteria. Some are considered simple stains, a staining process in which only one dye is used. The use of differential staining procedures is more complex. Differential protocols use a series of dyes to distinguish different types of bacteria based on some chemical or structural attribute of the cell.
Differential staining procedures are often used for general identification of bacteria, but do not allow for identification of the exact species. Examples of differential stains include the Gram stain, Acid-fast stain and Endospore stain. When doing a differential stain it is best to use controls in addition to the unknown bacteria that is being identified. Controls allow for comparison of the unknown to a sample of bacteria that tests positive for that stain and one that tests negative for the stain.
* Sources *
Schauer Cynthia (2007) Lab Manual to Microbiology for the Health Sciences, Kalamazoo Valley Community College.
Bauman, R. (2005) Microbiology. Pearson Benjamin Cummings.
