Explain Precautions for Gram Staining of Bacterial Cultures?

1. Heat fix the smear, otherwise cells would be washed off.

2. Fresh 24 hrs old culture should be used. Gram variability can be seen in old culture. Since with age gram Positives lose their ability to retain primary stain, in gram variability some cells appear purple and others appear red.

3. Decolourization is a critical step. Decolourization is done till the colour of the primary stain comes out. Over-decolourization can make gram positives appear gram negatives and under decolourization can cause gram negatives to appear as gram positives.

4. Slide should be thoroughly washed after every step to remove excess reagent under running water or water applied with an eye dropper.