GRAM STAIN

INTRODUCTION

· Gram stain is the most commonly used stain in microbiology.

· Discovered by Hans Christian Gram in 1884.

· Gram staining is a colouring technique to identify the unique feature of bacteria.

· This technique is help in identification & classification of bacteria.

PRINCIPLE OF GRAM STAIN

· Gram stain principle is based on two theory –

1. Ph theory:

• Gram positive cell is more acidic protoplasm which accounts for retaining basic dye more strongly than gram negative bacteria.

• Iodine make the protoplasm more acidic & serves as modrant, iodine combine with dye to form a dye-iodine complex & penetrate primary stain in positive bacteria.

2. Cell wall theory:

• Cell wall of Gram positive bacteria is thicker than gram negative due to presence of cytoplasm.

• The gram positive bacteria cell wall is less permeable, while gram negative bacteria cell wall is more permeable to acetone or alcohol during decolourisation.

REQUIREMENT FOR GRAM STAIN

# Material

A. Bacterial Culture:

• Obtain a pure culture of the bacteria to be stained.

B. Slides:

• Clean and sterile slides for preparing bacterial smears.

C. Bunsen Burner:

• For heat-fixing bacterial smears to the slides.

D. Gram stain kit

• It include:

a) Crystal Violet

b) Gram's Iodine

c) 95% Ethanol or Acetone

d) Safranin

E. Water:

• Distilled water for rinsing during the staining process.

F. Microscope:

# Method

· Take clean glass slide

· Now place one drop saline/water into the slide

· Transfer small amount of bacterial culture onto the slide

· Spread bacteria sample in thin film

· Allow it to air dry

· Heat fix the prepared smear.

PROCEDURE

· Step1- Heat fix smear is stain with Crystal violet (Primary stain) for one minute.

· Step2- Wash with rinsing water

· Step3- Now apply gram’s iodine (Modrant) for one minute.

· Step4- Wash the smear with distilled water for removed excessive iodine.

· Step5- Decolourise with acetone for 10-30 seconds. Alcohol can be substituted for acetone.

· Step6- Wash with water.

· Step7- Counterstain with safranin for 30 seconds. Dilute carbol fuchsin or neutral red may also be used as counterstain.

· Step8- Wash the smear with water for removal extra stain.

· Step9- Air dry the smear for microscopy.

INTERPRETATION OF RESULT

· Gram positive bacteria: It appears purple or violet under microscope.

· Gram negative bacteria: It appears pink or red under microscope.

MODIFICATION IN GRAM STAIN

· Jensen’s modification: Absolute alcohol is used as decolouriser & neutral red as counterstain.

· Kopeloff & Beerman’s modification: Methyl violet is used as primary stain whereas basic fuchsin as counterstain.

· Preston & morrell’s modification: Iodine-acetone is used as decoluriser.

CONCLUSION

· Gram stain is a basic but crucial method in microbiology.

· Introduce by Hans Christian Gram for differentiate bacteria based on cell wall properties.

· Bacteria classified as Gram-positive (purple/violet) & Gram-negative (red/pink) bacteria.

· Useful technique for diagnosis & treatment of bacterial infections.

· Commonly used in laboratories worldwide due to its simplicity and effectiveness.

The Principle remain same but the staining method are automated