In situ visualisation of fungi in soil thin sections: Problems with crystallisation of the fluorochrome FB 28 (Calcofluor M2R) and improved staining by SCRI Renaissance 2200

Fluorescent stains offer an effective means of visualising bacteria and fungi in soil or litter samples. Fluorescent brightener (FB) 28 (also known as Calcofluor White M2R) is commonly used to stain such microorganisms. However, during production of soil thin-sections we observed erratic crystallisation of this stain, particularly in soils colonised by Rhizoctonia solani. We report on the evaluation of alternative stains to FB 28 for their propensity to crystallize in interaction with fungi, staining efficiency, and suitability for application in soil thin-section production. All of the additional candidate stains namely Fluorescent Brightener Agent (FBA) 15/25, FBA 71 and SCRI Renaissance 2200 (SR 2200) were highly effective in staining agar-cultured hyphae, but differed in the degree to which they stained hyphae cultured in soil. All stains tested, except SR 2200, stained hyphae of R. solani insufficiently when growing on or through soil. These stains also showed extensive crystallisation in solutions that had been in contact with R. solani colonised soil. However, SR 2200 stained hyphae of R solani growing over soil as effectively as hyphae growing on agar and showed no evidence of crystallisation; the intensity of staining exceeded that of the 'benchmark' FB 28 for hyphae grown in two soil types. These excellent fluorescent properties of FBA 220 persisted in soil thin-sections, resulting in bright hyphae that could be readily visualised in situ in undisturbed soils.