Histological Staining Techniques
Gram for bacteria
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
Wash in distilled water.
Dehydrate, clear and mount. Prolong the alcohol step until the
red stops coming out.
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.
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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