Protocol for Gram Staining

Protocol for Gram  Staining:

1. Primary Stain:  Crystal Violet Staining Reagent.

Solution A:

      Crystal violet (certified 90% dye content), 2g 
      Ethanol, 95% (vol/vol), 20 ml 

Solution B:

      Ammonium oxalate, 0.8 g
      Distilled water, 80 ml 

Mix A and B to obtain crystal violet staining reagent. Store for 24 h and filter through paper prior to use.

2. Mordant: Gram's Iodine

      Iodine, 1.0 g 
      Potassium iodide, 2.0 g 
      Distilled water, 300 ml

Grind the iodine and potassium iodide in a mortar and add water slowly with continuous grinding until the iodine is dissolved. Store in amber bottles. 

3. Decolorizing Agent

      Ethanol, 95% (vol/vol)

4. Counterstain: Safranin 

Stock solution:

      2.5g Safranin O 
      100 ml 95% Ethanol

Working Solution:

      10 ml Stock Solution 
      90 ml Distilled water

PROTOCOL:

1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results. 
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds. 
3. Flood slide with the mordant: Gram's iodine. Wait 1 minute. 
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Flood slide with decolorizing agent. Wait 15 seconds or add drop by drop to slide until decolorizing agent running from the slide runs clear (see Comments and Tips section).
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper.
8. Observe the results of the staining procedure under oil immersion using a Brightfield microscope. At the completion of the Gram Stain, gram-negative bacteria will stain pink/red and gram-positive bacteria will stain blue/purple.

KOH string test:

This experiment can be used as a confirmatory test for the Gram Stain. The formation of a string (DNA) in 3% KOH indicates that the isolate is a gram-negative organism. 
Procedure:
Place a drop of 3% KOH onto a glass slide.
Emulsify in KOH a loopful of the culture from a BA incubated for 18-24 hours.
Continue to mix the suspension for 60 sec and by slowly lifting the loop, observe for the formation of a string. 
Interpretation: 
Gram-negative cells form a string within 60 seconds.
Gram-positive cells are not affected.