Improved Grocott staining method with less difference among performers: Investigation of staining duration for each fungus in chromic acid ammoniacal-silver nitrate method

Abstract

It is important to determine the optimum conditions for the argentation reaction to obtain the best results of Grocott staining. Since methenamine-silver nitrate solution often causes unstable staining, chromic acid ammoniacal-silver nitrate solution is appropriate for fungal staining. However, the optimum temperature and duration of argentation reaction have not been sufficiently investigated. In this study, we examined various staining durations for both argentation reaction and gold chloride solution using a dissolver or water bath in the method using chromic acid ammoniacal-silver nitrate solution. The fungi stained here were Aspergillus, Cryptococcus, and Pneumocystis jirovecii. It was confirmed that the duration of the argentation reaction should be 5–10 min to obtain good results when a dissolver is used. Fungal staining color could be well regulated by modulating the duration of the gold chloride solution reaction, and appropriate staining was achieved when the staining duration was 1–5 min for all types of fungi. The chromic acid ammoniacal-silver nitrate method using a dissolver is considered to provide stable staining results with less difference among performers when the staining duration with the gold chloride solution is adjusted for each fungus.