5 Lab 5: Staining
Lab Objectives
After completing this lab, the student should be able to:
- Use Aseptic Technique to prepare smears for staining.
- Examine smears to determine bacterial morphology, arrangement, and Gram reaction.
- Determine if a cell is positively or negatively charged and if a stain is direct, indirect, simple, or differential.
- Classify stained cells based on their cell structure as either Gram positive or Gram negative.
- Apply the Gram reaction to cell wall structure.
Introduction
Staining is a key skill in microscopy work because it helps to create contrast between cells and their background. If you have previously viewed stained slides, you are already aware of the contrast difference made by staining techniques. This lab will review how to create smears, stain smears, and explain simple and differential bacterial staining techniques.
Smears: Bacterial smears are created by applying cells to a microscope slide. If the cells come from a liquid preparation (e.g., a broth culture) then the cells are directly applied to the slide. However, if the cells come from a solid culture (e.g., a slant or plate), then a small drop of water is applied to the slide first then the cells are mixed into the drop of water. The reason why cells must be in a liquid is to allow for spreading out the cells into a thin film termed a smear. This reduced the number of cells in a physical space so that as the smear dries, cells are more evenly distributed and are less likely to trap stains. It is also important to ensure that cells have sufficient space between them so that cells can be easily seen to determine morphology and arrangement which becomes important in identification methods.
Before staining, cells must be “fixed” to adhere them to the glass microscope slide. Fixing slides can be accomplished in two ways: either through the use of strong fixatives (chemicals) such as alcohol or acetone in the case of human blood cells or by heat. Our lab will use heat fixing as the method of choice. To heat fix a smear, you will simply run the slide through the top portion of the Bunsen burner flame twice.
Types of Stains: Stains are classified based on charge of the chromophore (colored ion). Because the overall net charge of a bacterial cell is negative, positively charged chromophores are attracted to the bacterial cells and will directly stain the cells – thus direct stains are stains that contain positively charged chromophores. Negative stains, on the other hand, use negatively charged chromophores that are repelled by the bacterial cell and stain the slide (“background”) so that the bacterial cells appear colorless on a dark background.
Negative stains do not require heat fixing so negatives stains are used when one wants to observe living cells for motility. Because negative stains are not heat fixed, the cells will appear crisper thus determining morphology and arrangement can be a bit easier than with direct stains.
So when would a direct stai