Toluidine blue O staining Reagents

• Fixing solution: measure 75ml glacial acetic acid into a beaker standing in a water-bath containing ice (temperature <10°C). Slowly add 25 ml concentrated sulphuric acid while stirring the mixture constantly. When ready, the mixture is stored in a staining jar with a tight fitting lid. The shelf life is 1 week at RT.

• Toluidine blue stain: dissolve 0.45g Toluidine blue O in 90ml distilled water. Add 3ml concentrated HCl and 210ml absolute ethanol. The shelf life is 1 year at RT.

• Xylene or substitute, for example, Histoclear (National Diagnostics).

Positive Pneumocystis carinii control

A positive specimen is stored at -40°C in small amounts. To conserve material, strongly positive specimens may be diluted with treated negative BAL material. For use, a portion of control specimen is thawed and placed on slides as for clinical specimens.

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