Development of a simple in vitro test system for the disinfection of bacterial biofilms

This paper evaluates the use of poloxamer-hydrogel biofilm-constructs for the routine evaluation of biocide formulations at use strength. Aqueous solutions of Poloxamer F127 show thermo-reversible gelation, being liquid at temperatures 15°C. Chilled poloxamer solutions (30%w/v) were made up in tryptone soya broth and inoculated with c10 4-5 cfu/ml of stationary phase cultures of eleven foodborne spoilage and pathogen test-strains, including Listeria and Salmonella spp. Drops (200μl) were placed, in triplicate, onto pre-warmed, sterile stainless steel discs (8mm diam.) held in sealed-Petri dishes. These were incubated for 5h at 30°C during which time all strains grew well giving between 10 6-7 cfu/ml. Previous work had demonstrated that cells grown within poloxamer hydrogels in this manner adopted biofilm-phenotypes which were distinct from those of planktonically grown cells and which were characteristically resistant to a range of biocides. Incubated poloxamer gels, together with their supports were transferred to solutions (10ml) of four commercial biocide formulations: (i) a buffered (pH 10) tertiary alkyl amine formulation in an amphoteric surfactant (TAAS), (ii) a hydrogen peroxide, peracetic acid, acetic acid (pH 1) formulation (HPPA), (iii) sodium hypochlorite and sodium hydroxide formulation (pH 13) (HYPO), and (iv) isopropyl alcohol in lactic acid (pH 2.3) (IPA). After 5min the test pieces were removed and transferred to neutralizer at 15°C. The gels dispersed rapidly releasing the cells and enabling viable counts to be made. All formulations effected a >5-log kill of planktonic challenges within 5min whilst the levels of killing effected within the biofilm-constructs varied between 0.5 and 3-log reductions. The results were highly reproducible with patterns of susceptibility varying both as a function of organism, biocide-type and concentration. The experiments strongly support the view that poloxamer constructs might find application in trials and testing of new disinfectant formulations.