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Lab Chemical-60

Product code RXSOL-60-6604-652

GRAMS IODINE FOR MICROSCOPICAL STAINING

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Add Gram's iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall. Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet.

Application

The Gram staining is one of the most crucial staining techniques in microbiology. ... The first step in gram staining is the use of crystal violet dye for the slide's initial staining. The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye.

 

  • Purpose: Acts as a mordant in the Gram staining procedure, fixing the primary stain (crystal violet) to bacterial cell walls.

Dosage

 
  1. Add 20mg safranin powder to a 100ml beaker.
  2. Pour 20ml distilled water in the beaker and make 0.1% safranin staining solution by constant stirring.
  3. Transfer 20mg of fast green dye in another 100ml beaker. ...
  4. Filter both the staining solutions to avoid particles.

How to use Gram's Iodine Solution

1. Prepare a thin smear on clear, dry glass slide.

2. Allow it to air dry and fix by gentle heat.

3. Flood with Gram's Crystal Violet (S012) for 1 minute. (If over staining results in improper decolourization of known gram-negative organisms, use less crystal violet).

4. Wash with tap water.

5. Flood the smear with Gram’s Iodine (S013). Allow it to remain for 1 minute.

6. Decolourize with Gram's Decolourizer (S032) until the blue dye no longer flows from the smear. (Acetone may be used as a decolourizing agent with caution, since this solvent very rapidly decolourized the smear).

7. Wash with tap water.

8. Counter stain with 0.5% w/v Safranin (S027) for 20 seconds and rinses off with water.

9. Wash with tap water.

10. Allow the slide to air dry or blot dry between sheets of clean bibulous paper and examine under oil immersion objective

RBC diluting fluid is isotonic with blood, hence hemolysis does not take place. Normal Saline also can be used. But it causes slight creation of red blood cells and allows rouleaux formation.