Differential stain/ Gram's Stain
This technique was developed by Christan Gram.
This is useful to distinguish between gram positive and gram negative bacteria.
1. Gram positive bacteria
Appear dark purple colour.
2. Gram negative bacteria
Appear pink or red colour
Gram stain is colored purple.
When the stain combines with bacteria in a sample, the bacteria will either stay purple or turn pink or red.
If bacteria stays purple, they are gram positive.
If bacteria turns pink or red, they are gram negative.
Procedure of Gram's Stain
1. Make smear of given specimen and fixed it.
2. Stain with solution of crystal violet/ methylene blue for half to 1 minute.
3. Wash under water gently.
4. Flood with gram iodine solution for 1-2 minutes.
5. Wash under water
6. Add 95% of alcohol for 20 second.
7. View slide under microscope.
8. Gram positive will appear dar purple and gram negative will be colourless.
9. Wash under water
10. Flood smear with safaranin for half to one minute (Counterstaining with safaranin)
11. Wash under water
12. Observe and examine under microscope.
13. Gram negative will appear pink or red colour.
Interpretation
Gram positive; blue/ purple colour
Gram negative; pink/red colour
Result
Reporting is based on following headings;
1. Shape
2. Size
3. Gram reaction
Gram +ve bacteria; purple/blue in colour
Gram -ve bacteria; pink/red in colour
4. Arrangement
5. Gram-ve bacilli (rod shaped), Gram +ve cocci (Spherical).
6. Gram -ve cocci (spherical), Gram +ve bacilli (rod shaped).
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