Gram Staining

Gram staining (or Gram's method) is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative).

Gram-Positive

Before: Pick a colony and dilute in 10µl water OR Take 10µl glycerol stock culture from -80ºC freezer Protocol:
  1. Add 5µl culture to a clean slide
  2. Gently pass over flame 3 times
  3. Add crystal violet solution (primary stain)  to cover fixed sample.
  4. Incubate for 10 seconds (or up to 1 minute).
  5. Gently rise with water.
  6. Add gram's iodine solution (mordant) to cover stain.
  7. Wait for 10 seconds (or up to 1 minute).
  8. Quickly rinse slide with Acetone/Ethanol for 3 sec (too long may decolourise Gram positive cells).
  9. Wash immediately with water.
  10. Add safarin (secondary stain).
  11. Wait for 10 seconds (or up to 1 minute).
  12. Rinse gently with water
  13. Dry slide with filter paper and examine under oil immersion 
Outcome: Gram-Positive stains (e.g. E.coli, Magnetospirillum) will retain crystal violet and show violet colour under the microscope. Gram-Negative strain (e.g. Bacillus, Staphylococcus Aureus) will lose primary stain and show secondary stain and show a red colour under the microscope.
Gram-positive anthrax bacteria (purple rods) in cerebrospinal fluid sample. If present, a Gram-negative bacterial specie
Gram-positive anthrax bacteria (purple rods) in cerebrospinal fluid sample. If present, a Gram-negative bacterial species would appear pink. (The other cells are white blood cells).

Example Gram-Positive:

Example Gram-Positive: Asource)
Gram-stained micrograph reveals chains, and solitary Gram-negative Legionella pneumophila bacteria found within a sample
Gram-stained micrograph reveals chains, and solitary Gram-negative Legionella pneumophila bacteria found within a sample taken from a victim of the 1976 Legionnaires’ disease outbreak in Philadelphia.
Example Gram-Negative:source)