Lab Exercise 3 - Staining Methods: Gram Stain

differential stain

a stain that detects differences between organisms through the use of many dyes and reagents

Gram stain

a differential stain that differentiates Gram-positive from Gram-negative bacteria, based on the amount of peptidoglycan in the cell wall

origin/development of the Gram stain

developed by Hans Christian Gram, Danish physician

mid-1880s

Gram wanted to differentiate bacteria causing pneumonia from deceased patients’ lung tissue

crystal violet was used to stain cells; some cells would not retain purple when cleaned up with ethanol

stain was originally considered a failure

procedure of Gram staining

on a heat-fixed stain:

Apply crystal violet stain, let it sit for 1 minute. Quickly and gently wash off with water.

Apply Gram’s iodine, let it sit for 1 minute. Quickly and gently wash off with water.

Apply 6-8 drops of 95% ethanol, let it sit up to 8-10 seconds (NO LONGER THAN 12 SECONDS). Quickly and gently wash off with water.

Apply safranin, let it sit for 1 minute. Quickly and gently wash off with water.

Gently blot with bibulous paper until dry. Observe stain.

purpose of crystal violet in Gram staining

primary dye

colors Gram-positive bacteria for identification

purpose of Gram’s iodine in Gram staining

mordant

helps crystal violet stick to the cells

purpose of 95% ethanol in Gram staining

decolorizer

washes off crystal violet from Gram-negative cells to be counterstained

purpose of safranin in Gram staining

secondary dye, counterstain

colors Gram-negative cells for identification (because the purple didn’t stick)

results of Gram staining

Gram-positive cells will be purple

Gram-negative cells will be pink