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What is gram staining procedure in Microbiology?

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more than 4 weeks ago
Gram staining is a common technique used in microbiology to differentiate between two major groups of bacteria based on their cell wall composition. The procedure was developed by Danish bacteriologist Hans Christian Gram in 1884.

The Gram staining procedure involves the following steps:

1. A bacterial smear is prepared on a glass slide from a bacterial culture.
2. The slide is heat-fixed to adhere the bacteria to the slide.
3. The slide is flooded with crystal violet stain, which stains all bacteria purple.
4. The slide is then flooded with iodine solution, which acts as a mordant to fix the crystal violet stain to the bacterial cell wall.
5. The slide is washed with alcohol or acetone, which acts as a decolorizer. This step is crucial as it differentiates between Gram-positive and Gram-negative bacteria.
6. The slide is then counterstained with safranin, which stains Gram-negative bacteria pink or red.
7. The slide is washed and allowed to dry before being examined under a microscope.

Gram-positive bacteria retain the crystal violet stain and appear purple, while Gram-negative bacteria lose the crystal violet stain and take up the safranin counterstain, appearing pink or red. This differentiation is due to differences in the cell wall structure of the two types of bacteria. Gram-positive bacteria have a thick layer of peptidoglycan in their cell wall, while Gram-negative bacteria have a thinner layer of peptidoglycan and an outer membrane.

Gram staining is a valuable tool in microbiology for quickly identifying and classifying bacteria based on their cell wall composition, which can help in determining appropriate treatment strategies for bacterial infections.
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more than 4 weeks ago
Gram Staining Procedure


To differentiate between bacteria based on their cell wall structure and Gram reaction (positive or negative).


- Bacterial culture
- Crystal violet stain
- Gram's iodine solution
- Alcohol (ethanol or isopropanol)
- Safranin counterstain


1. Preparation of Bacterial Smear:

- Clean a glass slide with alcohol.
- Suspend a loopful of bacterial culture in a drop of water on the slide.
- Heat-fix the smear over a flame.

2. Crystal Violet Staining:

- Flood the smear with crystal violet stain for 1 minute.
- Gently rinse the slide with water.

3. Gram's Iodine Treatment:

- Flood the smear with Gram's iodine solution for 1 minute.
- Gently rinse the slide with water.

4. Decolorization:

- Tilt the slide at an angle and gently add alcohol drop-wise until the runoff becomes clear. Caution: Do not over-decolorize.

5. Safranin Counterstaining:

- Flood the smear with safranin counterstain for 10 seconds.
- Gently rinse the slide with water.

6. Drying and Observation:

- Blot the slide dry using absorbent paper.
- Examine the smear under a microscope using the oil immersion lens.


- Gram-positive bacteria: Retain the crystal violet-iodine complex and appear purple.
- Gram-negative bacteria: Lose the crystal violet-iodine complex during decolorization and appear pink from the safranin counterstain.


- Gram's iodine is a mordant, which helps the crystal violet bind more strongly to the cell wall of Gram-positive bacteria.
- Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane, which make them less retentive of the crystal violet stain.

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