Preparation of gram

staining reagents

Presented by
Samina Akram

1. Primary Stain: Crystal Violet

Staining Reagent.
Solution A for crystal violet staining reagent

Crystal violet.
20g

Ethanol, 95% 95
ml

FOR USE

Solution B for crystal violet staining reagent

Ammonium oxalate.. 9 g
Distilled water
1L

Mix A and B to obtain crystal violet staining

reagent

Filter through paper prior to use

Shelf life:
Caution:
Store in amber
bottle at room
temperature for 5-6
months

Ammonium Oxalate is very toxic

Handle it with care
Ethanol or Methanol are highly
flammable

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.

Storage and Shelf Life:

Store below 30C in tightly closed container and away
from bright light.

3. Decolorizing
Agent
Ethanol, 95%
OR
Acetone, 50 ml
Ethanol (95%),
50 ml

Acetone and ethanol are highly flammable.Keep

away from heat ,flame or sparks

4.Counter stain:Gram safranin

Stock solution:

2.5g Safranin O
100 ml 95% Ethanol

Working Solution:

10 ml Stock Solution
90 ml Distilled water

THANK YOU!