GRAM STAIN

SECTIONS

4µm paraffin wax sections

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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
  

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METHOD

1. Take sections to water
   
2. Stain in filtered crystal violet for 2 minutes
   
3. Rinse in water
   
4. Treat with Weigerts iodine for 2 minutes
   
5. Rinse in water briefly
   
6. Differentiate in acetone until the section is colourless
   
7. Rinse briefly in water
   
8. Counterstain in nuclear fast red for 5 minutes
   
9. Dehydrate, clear . Mount sections in DPX
   

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RESULTS

• Gram positive organisms and some fibrin - Blue/black
  
• Gram negative organisms and cell nuclei - Red
  

NOTE: No responsibility is assumed by The University of Nottingham or the Queens Medical Centre NHS Trust for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. It is the users responsibilty to ensure that all procedures are carried out according to appropriate Health and Safety requirements.

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© Copyright 1997 University of Nottingham Medical School Division of Histopathology. This page was last built on Mon, May 5, 1997 with Frontier. Thanks for looking in. Comments to James.Lowe@nottingham.ac.uk