Gram stain quiz (4pts)

What are the 4 Gram stain steps, in order?

Crystal violet → Iodine → Alcohol → Safranin

• (“Come In And Stain”)

What is the function of crystal violet?

Primary stain. It stains all cells purple.

What is the function of iodine?

Mordant. It binds crystal violet to form a large CV–I complex that is harder to wash out. Iodine is not a stain.

What is the function of alcohol?

Decolorizer. It removes the CV–I complex from Gram-negative cells only. This is the critical step.

What is the function of safranin?

Counterstain. It stains the decolorized Gram-negative cells pink/red.

What are the final colors of Gram-positive and Gram-negative cells?

Gram-positive is purple.

Gram-negative is pink/red.

Why do Gram-negative cells lose the crystal violet?

Alcohol dissolves the lipid outer membrane (LPS). The thin peptidoglycan layer can’t hold the Crystal Violet-iodine, so it washes out.

Why do Gram-positive cells keep the crystal violet?

Alcohol dehydrates the thick peptidoglycan, which shrinks its pores and traps the Crystal Violet-iodine complex inside.

What happens if you over- or under-decolorize?

Over-decolorizing makes Gram-positive cells look falsely pink.

Under-decolorizing makes Gram-negative cells look falsely purple.

How many stains does a differential stain use, and what can it tell you that a simple stain can’t?

2 stains. It distinguishes different bacteria (for example, by cell wall type), tells a pure culture from a mixed one, and helps with identification.

A simple stain uses 1 stain, so everything appears the same color.

What are examples of differential stains?

Gram stain, negative/capsule stain, endospore stain

What does the Gram stain differentiate bacteria based on?

Differences in their cell wall components

How do Gram-negative and Gram-positive cell walls differ?

Gram-negative has a thin peptidoglycan layer plus an outer membrane with LPS.

Gram-positive has a thick peptidoglycan layer and no outer membrane.

What are the 2 parts of the Gram stain procedure?

1. Smear, which means preparing a slide with the specimen.

2. Stain, which makes the colorless sample visible (purple or pink/red).

Why do dyes bind to the bacterial cell wall?

The cell wall is negatively charged and the dye chromophores are positively charged. Opposite charges attract, so the dye binds to the peptidoglycan.

If proper methods are followed, will alcohol decolorize Gram-positive cells?

No. Only the wrong timing (over-decolorizing) causes that.