Evaluation of physiological staining, cryoembedding and autofluorescence quenching techniques on fouling biofilms

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Abstract
Physiological staining, cryoembedding, cryosectioning and autoftuorescence quenching techniques were evaluated for their applicability to undefined mixed population biofilms collected from environmental or engineered systems. Four different biofilms from two cooling towers, a paper 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‐phenylindole (DAPI) to distinguish respiring and nonrespiring cells. Positive CTC staining, as evidenced by the development of pink or red color, was successful in all samples examined except for paper mill biofilm. The structural integrity of frozen sections deteriorated when biofilms contained rigid or fibrous material. Autofluorescence generally impaired the ability to distinguish specific staining from natural background fluorescence. Two physical and three chemical methods were tested to quench autofluorescence. Quenching with crystal violet reduced most of the autofluorescent interference and still maintained physiological staining intensity, but contrast between CTC staining and residual autofluorescence was poor. Autofluorescence and the difficulty of sectioning thick biofilms containing abiotic materials limit the applicability of cryoembedding/staining techniques to fouling biofilms.