Staining Gram-Positive Bacteria, Gram staining, Theory of Gram staining, Preparation of stain solution.

A. Staining of gram-positive bacteria:

Staining increases the contrast between bacteria and their background. This improves colorless bacteria more clearly visible under a microscope. Staining helps

• 1. to study the morphology of bacteria,

• 2. to determine pure culture media,

• 3. To determine types of bacteria in culture media

• 4. To differentiate bacteria as per their affinity to stain.

Gram staining is used to differentiate bacteria depending upon the affinity of bacteria to Gram stain. Bacteria that accept the color of gram stain are called Gram-positive bacteria. Bacteria that do not accept the color of gram stain are called Gram-negative bacteria.

Theory of Gram staining: Crystal violet, iodine, alcohol, and safranin are used in gram staining of bacteria. Bacteria accept the pink color of crystal violet. Iodine increases the affinity of crystal violet with the internal cellular components of bacteria. This enhances the coloration of internal cellular components of bacteria. Alcohol or acetone is used as a decolorizing agent. They wash out the pink color of the internal cellular components of bacteria. Bacteria that retain the pink color of crystal violet even after their washing with alcohol or acetone are called Gram-positive bacteria. Bacteria that do not retain the pink color of crystal violet after washing with alcohol or acetone are called Gram-negative bacteria. Safranin is used as a counterstaining solution. Gram-negative bacteria accept the color of safranin.

Preparation of stain solution

1. Crystal violet solution

• Solution-A

• i. Crystal violet 1 gm

• ii. Ethyl Alcohol 10ml

• Solution-B

• i. Ammonium oxalate 0.4gm

• ii. D. Water 40ml

• it is in an amber colour-dropping bottle.

2. Iodine solution

• i. Iodine 500 mg

• ii. Potassium Iodi de 1 gm

• Store it in an amber colour-dropping bottle

3. Decolourising agent: Alcohol 95% v/v and acetone in equal amounts.

4. Safranin solution

• i. Safranin O 0.17 gm

• ii. Alcohol 95% v/v 5 ml

• iii. D. Water 45 ml.

Procedure:

1. Smear Preparation:

• i. Sterilise the inoculation loop under the flame of the burner,

• ii. Transfer specimen culture on dry grease-free slide by using a sterilise inoculation tube,

• iii. Make a smear of specimen sample on the slide,

• iv. Dry the smear in the air

• v. Fix the dry smear by passing it through the flame of the burner 3 to 4 times.

• Filter and store in an amber-colored bottle.

2. Gram Staining

• i. Cover the smear with crystal violet stain solution for 1 minute

• ii. Wash out the crystal violet solution by using tap water.

• iii. Cover the smear with iodine solution for 1 minute,

• iv. Wash the iodine solution by using tap water

• v. Cover the stain by using decolorizing agent alcohol or acetone or a mixture of alcohol-acetone for 30 seconds. or purple bubbles stop to come out from the smear.

• vi. Wash the decolorizing agent by using tap water,

• vii. Cover the smear with counterstain safranin for 10 seconds,

• viii. Drain out the counter stain and allow the smear to dry in air. Dry it carefully by using blotting paper

• ix. Examine it under a microscope under a low power objective and then under high power objective and then under oil immersion objective.

3. Result:

• i. Dark purple: Gram-positive bacteria

• ii. Pale to dark Red: Gram-negative bacteria

• B. Culture of gram-positive bacteria: It Shall be discussed under individual gram-positive bacteria

• C. Biochemical characteristics of gram-positive bacteria: It Shall be discussed under individual gram-positive bacteria

• D. Antibiotics related to gram-positive bacteria: It Shall be discussed under individual gram-positive bacteria.

Dr Pramila Singh