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.
Article Summary: 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.
How to Prepare & Heat Fix a Bacterial Smear for Staining
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Bacterial smear that has been heat fixed and stained.
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.
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 is 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 known + & - bacterial 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.
The following are examples of bacterial controls that could be used in differential stains: