Topic 7: Preparation of Smears and Simple Staining

Ex

methylene blue chloride ⇌ methylene blue + (chromophore) + Cl -

Direct stain

simple stain that stains the bacteria

Negative stain

simple stain that stains the background, not the bacteria

Heat-fix

dry smear is passed through a bunsen burner flame several times, may not kill all the bacteria

Chemically fix

cover the smear with 95% methyl alcohol for 1 min

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

benzene

What is a chromophore?

charged colored ions in dyes

If chromophore is a positive ion, the stain is considered a ___ stain.

basic

If chromophore is a negative ion, the stain is considered a ___ stain.

acidic

What are most bacteria stained with?

basic stain

How does the basic stain "stain" the bacterium?

permeates the cell wall and adheres by weak ionic bonds to the negative charges of the bacterial cell

What are stains that only use one stain called?

simple stains

What is the simple stain that stains the bacteria called?

direct stain

What is the simple stain that stain the background and NOT the bacteria called?

negative stain

What must be done to a slide before bacteria can be stained?

smear must be prepared and placed on slide

How is a smear made?

spreading bacterial suspension on a clean slide and allowing it to air-dry

What must occur after placing the smear and before the staining process?

smear needs to be fixed

Is bacteria usually alive or dead after fixing?

dead

What causes the bacteria to stick to the slide?

coagulated proteins from the cells

How does the process of heat-fix occur?

dry smear is passed through a bunsen burner flame several times

Does heat-fixing kill bacteria every time?

no - not all bacteria may die

How does the process of chemical fixation occur?

cover smear with 95% methyl alcohol for 1 min

What is autolysis?

denaturing of bacterial enzymes preventing them from digesting cell parts, which causes the cell to break

Fixing uses what process that denatures bacterial enzymes?

autolysis

True or False: Fixing enhances the adherene of bacterial cells to the microscope slide.

true