Topic 7: Preparation of Smears and Simple Staining

Most stains used are synthetic aniline (coal tar derivative) dyes derived from benzene

• Dyes are usually salts (a few are acids or bases) composed of charged colored ions. Colored ion is referred to as a $chromophore$$$chromophore$$

• Ex: methylene blue chloride ⇌ methylene blue + (chromophore) + Cl -

• If chromophore is a positive ion, the stain is considered a $basic stain$$$basic stain$$

• If chromophore is a negative ion, the stain is considered an @@acidic stain@@

Most bacteria are stained with a basic stain permeates the cell wall and adheres by weak ionic bonds to the negative charges of the bacterial cell

Staining procedures that use only one stain are called $simple stains$$$simple stains$$

• $Direct stain$$$Direct stain$$: simple stain that stains the bacteria

• $Negative stain$$$Negative stain$$: simple stain that stains the background, not the bacteria

• Simple stains can be used to determine cell morphology, size, and arrangement

Before bacteria can be stained, a thin film of bacterial cells, called a $smear$$$smear$$, must be placed on a slide.

• Made by spreading a bacterial suspension on a clean slide and allowing it to air-dry.

Smear must be fixed to kill the bacteria; coagulated proteins from the cells will cause cells to stick to the slide.

• Heat-fix: dry smear is passed through a bunsen burner flame several times, may not kill all the bacteria

• Dry smear can be placed on a 60 °C slide warmer for 10 minutes or until chemically fixed

• Chemically fix: cover the smear with 95% methyl alcohol for 1 min.

Fixing denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell to break → process called $autolysis$$$autolysis$$

Fixing also enhances the adherence of bacterial cells to the microscope slide