Mechanism of Gram staining - Dr. Mobeen Syed
Put bacteria on plate (if from another plate then dilute agar first);
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Warm the plate to fix bacteria;
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Add gram stain; 20-30 seconds; wash it;
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Add iodine (mordant)
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Add drops of alcohol, to wash out the gram negative bacteria’s color - 20 - 40 seconds;
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Put safranin (counter stain) 20-40 seconds
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What Happens?
- * Gram positive (+ive) bacteria turns purple
- * Gram negative(-ive) bacteria turns red
Go to TC 2:57 in the video for illustration of how it happens:
- * Covering the Gram +ive plasma membrane (phospholipid bi-layer), there are up to 60 peptidoglycan (PG) layers.
- * The gram -ive plasma layer is covered by up to three PG layers and an outer membrane containing lipopolysaccharides.
- * Beta-Lactamase is the most important factor in the periplasmic space, enzymes that can break down penicillins.
What happens to the gram stain?
- *The small molecules of dye get trapped in the PG layer of gram +ive bacteria and in both the outer membrane and the PG layers of the gram -ive bacteria.
- * At this stage both gram +ive and gram -ive look purple under the microscope.
- * When iodine (+) is added, it attaches to the molecules of dye (-) and creates a bigger molecule, which gets trapped in the PG layers. Color does not change.
- *When alcohol is added it dissolves the lipid portion and dehydrates the cells.
- * In the gram +ive cells the water is pulled out by the alcohol and the 60 layers are compressed, trapping the molecules of dye, keeping the purple color
- * In the gram -ive cells, the alcohol dissolves the more abundant lipids, and the three PG layers are too weak to trap the dye molecules so they escape, making the cells colorless.
NB: Using too much alcohol on the gram +ive cells will eventually break down the PG layers allowing the dye to escape so you have to be careful.
- *The final step is to add safranin, a counterstain, that will turn the colorless gram -ive cells red, but will not be absorbed by the compressed gram +ive cells which stay purple.
And that is how gram staining works!
Learning objectives of this video are the following:
1. Review of the procedure of gram staining.
2. Results of gram staining in case of gram positive and gram negative bacteria.
3. How gram staining works?
Presented by Dr. Mobeen Syed
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2 Comments
naveedkashif7@*.com
Jan 13 2023, 8:16 pm
Very nice and informative
attaulhadiii@*.com
Feb 14 2020, 1:22 am
Which method is used for staining mycobacteria?