AAT Bioquest

What are the steps of gram staining?

Posted August 23, 2022


Gram staining helps scientists determine whether bacteria in a sample are gram-positive or gram-negative based on the properties of their cell walls. These are the steps involved in gram staining: 

  • Step 1 Place a small drop of bacterial sample on a clean, grease-free slide. 
  • Step 2  Pass the slide with bacterial sample through a Bunsen burner flame 3 times (no more, no less). Three times is just right to heat fix the sample to the slide. Too much heat can melt and distort bacterial cell walls and too little heat could cause the bacteria to slide off during staining. Both scenarios could lead to inaccurate results. 
  • Step 3 Using a dropper, add crystal violet to the sample and let it stand for 30 seconds to 1 minute. Crystal violet is the primary stain for gram staining.
  • Step 4 After 1 minute, rinse the slide with a gentle stream of water five seconds (no more, no less) to get rid of the unbound crystal violet. Rinsing too long can remove too much color. Not rinsing long enough may allow excessive stain to remain on gram-negative cells. Both scenarios could lead to inaccurate results.
  • Step 5 Using a dropper, apply Gram’s iodine to the slide and let it sit for 1 minute. Gram’s iodine is a mordant, it helps to fix the crystal violet to the bacterial cell wall.
  • Step 6 After one minute, wash the slide with 95% alcohol or acetone for about 10 - 20 seconds. Gently rinse off with water. The alcohol will decolorize the gram-negative cells while the gram-positive cells will remain blue or violet. If the alcohol is kept too long on the sample, it may also decolorize gram positive cells, leading to wrong results.
  • Step 7 Apply the secondary stain, safranin, to the slide and allow it to sit for 1 minute. After 1 minute, wash it off under a gentle stream of water for no longer than 5 seconds.
  • Step 8 Blot the slide gently, allow to air dry, and observe under a microscope. The gram-negative cells will acquire a red or pink stain while the gram-negative cells will continue to appear blue or purple.  
Additional resources

Gram Staining

MycoLight™ Rapid Fluorescence Bacterial Gram Stain Kit

Gram Stain Procedure in Microbiology