The Gram-Twort variation of the standard Gram stain is the most common variant used in histopathology laboratories for the demonstration of Gram-positive and Gram-negative bacteria in formalin-fixed sections.
The standard Gram stain was developed and reported in 1884 by Hans Christian Gram (therefore Gram stain should always be spelt with a capital). He developed the technique to distinguish a certain group of bacteria in lung tissue, but noted that Typhus Bacillus was not visualised. The standard Gram stain works on the premise that the peptidoglycan rich cell wall of Gram-positive bacteria is stained by the crystal violet and the Gram-negative bacteria take up the counterstain.
Below is the author’s method of choice.
– Crystal Violet – 0.5% crystal violet in 25% alcohol
– Gram’s iodine – 1g iodine + 2g potassium iodide. Dissolve in a few mls of distilled water. Make up to 300ml with distilled water.
– Stock neutral red-fast green – 90ml of 0.2% neutral red in ethanol + 10 ml of 0.2% fast green in ethanol.
– 2% acetic acid in alcohol.
1. Take sections to water.
2. Stain with filtered crystal violet for 2 minutes.
3. Wash well in tap water.
4. Treat sections with Gram’s iodine for 2 minutes.
5. Wash well in tap water.
6. Rinse sections with acetone until colour stops leeching from them (approx 5 seconds).
7. Counterstain with filtered neutral red-fast green (dilute stock 1 in 4 with distilled water) for 5 minutes.
8. Wash well in tap water.
9. Differentiate in 2% acetic acid in alcohol until red ceases to run from section (approx 5-10 seconds).
10. Rinse in alcohol.
11. Clear and mount in DPX-type mountant.
– Always run a positive control.
– If the Gram’s iodine step is missed it takes longer to differentiate and background staining is increased.
– If having problems with differentiation try completely drying section before differentiating. Blot dry and leave for 10 minutes to ensure complete dryness.
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