PHYSIOLOGICAL METHODS TO STUDY BIOFILM DISINFECTION

This report reviews the development of a rapid in situ approach to stu dy the physiological responses of bacteria within biofilms to disinfec tants. One method utilized direct viable counts (DVC) to assess the di sinfection efficacy when thin biofilms were exposed to chlorine or mon ochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after dis infection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-mu m) sections of biofilm communi ties and examination by fluorescence microscopy. The fluorogenic stain s used in this approach included 5-cyano-2,3-ditolyl tetrazolium chlor ide (CTC), which indicates cellular electron transport activity and Rh odamine 123, which responds specifically to proton motive force. The u se of these stains allowed the microscopic discrimination of physiolog ically active bacteria as well as heterogeneities of active cells with in thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel c oupons indicated biocidal activity of chlorine-based disinfectants occ urred initially at the bulk-fluid interface of the communities and pro gressed toward the substratum. This approach provided a unique opportu nity to describe the spatial response of bacteria within biofilms to a ntimicrobial agents and address mechanisms explaining their comparativ e resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative appro ach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate bi ocide efficacy. Hence the alternative approach described here more acc urately indicates the ability of bacteria surviving disinfection to re cover and grow as well as demonstrate spatial heterogeneities in cellu lar physiological activities within biofilms.