Gram Staining

Goal

Assess whether the biological sample contains Gram positive or Gram negative bacterial strains.

Materials

Gas burner
Crystal Violet
95% alcohol
Iodine
Safranin
3x Bucket or beaker glass (or wash in between stainings)
Tap water
Blotting paper (alternatively: Tork roll or toilet paper)
Clear nail polish
Acetone
Cotton swab

Composition of the stains

Crystal violet: 1 g of dry crystal violet in 10 mL 95% ethanol
Iodine solution: 1 g of iodine in 100 mL water
Safranin: 0.25 g of safrin in 10 mL 95% ethanol and 90 mL water

Method

Prepare a drop of sample on your microscope slide according to the Method
Leave the sample to air dry for 5 to 10 minutes.
Fixate by moving the slide back and forth through a flame for a few seconds.
Stain the sample by a droplet of crystal violet and let it stain for max 60 seconds.
Remove excess stain by bathing / dipping your glass slide into a beaker glass / bucket with tap water.
Add the iodine solution for 60 seconds.
Remove excess stain as described before.
Decolourise using 95 % ethanol and immediately rinse with tap water. Don’t leave the ethanol on the slide for too long.
Counter stain with safranin for about 60 seconds.
Remove excess stain as described before.
Remove excess water using blotting paper.
Let slide air dry to get rid of the remaining water.
Put 2 drops of clear nail polish onto the sample.
Cover the sample with a cover slide.
Try to get rid of air bubbles by putting light pressure on the cover slide.
Remove excess nail polish with a cotton swab with acetone.
Take a look with the microscope with full light intensity (open diaphragma)

Interpreting the results

Gram positive cells appear violet
Gram negative cells appear red

Back to BHA5 - Class 4

Microscopic image of a Gram stain of mixed Gram-positive cocci (Staphylococcus aureus ATCC 25923, purple) and Gram-negative bacilli (Escherichia coli ATCC 11775, red). Magnification: 1.000x - Picture by Y tambe CC BY SA 3.0 license