Gram Stain Lab

Differential stain

A stain that distinguishes organisms on the basis of reactions to the staining procedure

What does Gram stain differentiate?

gram positive and gram negative bacteria

Gram positive bacteria features

Thick peptidoglycan cell wall

1 plasma membrane

Stains purple

Gram negative bacteria features

Thin peptidoglycan cell wall

Outer and inner plasma membranes

Periplasm inbetween inner and outer membranes

Naturally resistant to many antibiotics

Has endotoxin and can cause septic shock

Stains pink

Crystal violet and iodine CV-I

Large complexes that are insoluble in H2O so they don’t easily leave the cell wall

Which type of bacteria will old bacteria look like when stained?

Gram negative

What happens if you don’t decolorize long enough?

Gram negative will look like Gram positive

What happens if you decolorize too long?

Gram positive will look Gram negative

What is the key differentiating step?

Alcohol or ethanol

What is the primary stain?

Crystal violet

What is the mordant?

Gram’s iodine

What is the decolorizing agent?

Alcohol or ethanol

What is the counter stain?

Safranin

Why do the Gram negative bacteria cells loose the primary stain?

The decolorizing agent, alcohol dissolves the outer membrane and the CV-I complex wash out through the thin peptidoglycan layer

4 main steps of Gram staining procedure

1. Cover a heat fixed smear with crystal violet for 30 seconds and gently rinse with DI H2O

2. Cover smear with Gram’s iodine for 1 minute and gently rinse with DI H2O

3. Rinse smear with ethanol for 5 seconds followed by a DI H2O rinse

4. Cover smear with safranin for 30 seconds and gently rinse with DI H2O

Why would a hospital perform a Gram stain on a sample from a patient?

A quick way to get results.

Helps identify the bacteria cell wall which allows the medical team to determine the type of antibiotic needed