| Histopathology Lab, University of Nottingham http://www.nottingham.ac.uk/pathology/protocols/gram.html
SECTIONS
4µm paraffin wax sections
SOLUTIONS
1. Hucker-conn ammonium oxalate - Crystal violet
- Crystal violet 2g
- 95% Alcohol 20cm3
- Ammonium oxalate 0.8g
- Distilled water 80cm3
Dissolve the crystal violet in the alcohol, and the ammonium oxalate in the distilled water, and mix the 2 solutions. Stable for about 2 years but may need occasional filtering.
2. Weigerts Iodine
- Potassium iodide 2g
- Iodine crystals 1g
- Distilled water 100ml
Dissolve the potassium iodide in 2-3mls of the distilled water and then dissolve the iodine crystals. Dilute to 100mls in the rest of the distilled water.
3. 0.1% Nuclear fast red
- Nuclear fast red 0.1g
- Aluminum sulphate 2.5g
- Distilled water 100mls
METHOD
- Take sections to water
- Stain in filtered crystal violet for 2 minutes
- Rinse in water
- Treat with Weigerts iodine for 2 minutes
- Rinse in water briefly
- Differentiate in acetone until the section is colourless
- Rinse briefly in water
- Counterstain in nuclear fast red for 5 minutes
- Dehydrate, clear . Mount sections in DPX
RESULTS
- Gram positive organisms and some fibrin - Blue/black
- Gram negative organisms and cell nuclei - Red
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