Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.
Other fixatives are likely to be satisfactory.
Method
Bring sections to water via xylene and ethanol.
Place in eosin solution for 5 minutes.
Rinse with tap water.
Place in crystal violet 1 minute.
Rinse with tap water.
Flood with Gram's iodine for 1 minute.
Rinse with tap water.
Gently blot the section, being careful not to damage it.
Decolorise the section with aniline-xylene.
Rinse with several changes of xylene to remove all aniline.
Mount with a resinous medium.
Expected results
Gram positive bacteria – blue
Fibrin – blue
Background – pink
Notes
Control the differentiation with aniline-xylene microscopically.
To examine, place the section in xylene to stop dye removal.
Return to aniline-xylene if more differentiation is needed.
Stop differentiation when the target element has good contrast.
Increasing the aniline content of the aniline-xylene will increase the speed of dye removal.
Decreasing it will slow dye removal.
If the background is not pink enough, increase the time in eosin, stain in eosin at elevated temperature, or increase the eosin concentration.
The eosin counterstain may be omitted entirely if wished.
