Modified toluidine blue O stain for Pneumocystis carinii: further evaluation of some technical factors.

A modified toluidine blue O (TBO) stain for Pneumocystis carinii cysts was evaluated with regard to the influence of (i) the age and extent of use of the sulfation reagent, (ii) the source of TBO, (iii) the TBO content of the staining solution, and (iv) the amount of TBO present in the alcohol wash solutions. All TBOs evaluated, except for a new TBO obtained from Roboz Surgical Instrument Co., Inc., Washington, D.C., produced satisfactory results. Each lot of TBO should be quality controlled before use to ensure that the P. carinii cysts stain lavender against a blue background. We have ourselves decided to use only certified TBO with a high dye content. As extensively used sulfation reagent provided less satisfactory results than did either freshly prepared or 1-week-old unused sulfation reagent, we have decided to prepare fresh sulfation reagent at least weekly and to discard used sulfation reagent after 10 slides have been processed.