EVALUATION OF PHYSIOLOGICAL STAINING, CRYOEMBEDDING AND AUTOFLUORESCENCE QUENCHING TECHNIQUES ON FOULING BIOFILMS

Physiological staining, cryoembedding, cryosectioning and autofluoresc ence quenching techniques were evaluated for their applicability to un defined mixed population biofilms collected from environmental or engi neered systems. Four different biofilms from two cooling towers, a pap er mill machine and the effluent ditch of a wastewater treatment plant were tested. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride ( CTC) was used in combination with the DNA stain 4',6-diamino-2-phenyli ndole (DAPI) to distinguish respiring and nonrespiring cells. Positive CTC staining, as evidenced by the development of pink or red color, w as successful in all samples examined except for paper mill biofilm. T he structural integrity of frozen sections deteriorated when biofilms contained rigid or fibrous material. Autofluorescence generally impair ed the ability to distinguish specific staining from natural backgroun d fluorescence. Two physical and three chemical methods were tested to quench autofluorescence. Quenching with crystal violet reduced most o f the autofluorescent interference and still maintained physiological staining intensity, but contrast between CTC staining and residual aut ofluorescence was poor. Autofluorescence and the difficulty of section ing thick biofilms containing abiotic materials limit the applicabilit y of cryoembedding/staining techniques to fouling biofilms.