A COMPARATIVE-STUDY OF 4 DIFFERENT STAINING METHODS FOR ESTIMATION OFLIVE YEAST FORM CELLS OF PARACOCCIDIOIDES-BRASILIENSIS

A comparative study of four different staining methods for estimation of live yeast form cells of Paracoccidioides brasiliensis was carried out. The staining methods used were fluorescent staining, vital dye ex clusion tests with erythrosin B and by Janus green and lactophenol cot ton blue staining. Colony forming units (cfu) of the yeast form of eig ht P. brasiliensis isolates on brain heart infusion agar (BHIA) supple mented with 4% horse serum plus 5% P. brasiliensis cell extract (BHIA + HS + EXT) were examined for reliability of staining in determining t he number of live fungal units in eight different isolates. Cfu on BHI A + HS + EXT plates showed an excellent plating efficiency over 96% in all isolates tested. The percentage of the live cells indicated by fl uorescent staining (FL) or vital dye exclusion test with erythrosin B (EB) or Janus green (JG-1) was lower than that of cfu. By contrast, th e percentage due to modified dye exclusion test with Janus green (JG-2 ) and that due to lactophenol cotton blue staining (LPCB) showed a clo se correration to that of cfu. Our results indicate that the modified dye exclusion test with Janus green and lactophenol cotton blue staini ng are useful for estimating cell viability of yeast form cells of P. brasiliensis.