Laboratory Protocols

• Cryotomy
• Histological Staining Techniques
  • Index
• Histological fixatives
• Peroxidase-anti-peroxidase Technique
• Preparation of tissue section adhesives
• Processing tissues for light microscopy (basics) (PDF 1.1mb)
• Processing tissues for light microscopy (detailed)
• Sample collection guidelines
• Setting up the light microscope
• Specimen preparation for electron microscopy

Histological Staining Techniques

Gram for bacteria
Gram, 1884.
Use a positive control section.

• Dewax sections, rinse in alcohol, rinse in water.
• Stain with Lillie's crystal violet - 1 minute. (Filter stain onto slide.)
• Wash off with tap water.
• Treat with Gram's iodine - 2 minutes.
• Wash in tap water.
• Differentiate in acetone 1-2 seconds.
• Wash in tap water and check the staining using a microscope. The background should not be purple.
• Rinse in distilled water.
• Counterstain with 1% neutral red for 3-5 minutes. (Filter the stain onto the slide.)
• Wash in distilled water.
• Dehydrate, clear and mount. Prolong the alcohol step until the red stops coming out.

Results:

Gram +ve organisms - purple.
Gram -ve organisms - red.
Nuclei - red

Lillie's crystal violet
Dissolve 10g crystal violet in 100mls 95% alcohol
Dissolve 4g ammonium oxalate in 400mls distilled water
Add the two solutions together and stir the mixture for 3 hours. Filter.

Gram's iodine
Iodine 1g.
Potassium iodide - 2g
Distilled water - 300mls
Place the iodine and potassium iodide in a small quantity of the water and allow them to dissolve fully before adding the remainder of the water.

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