The use of poloxamer hydrogels for the assessment of biofilm susceptibility towards biocide treatments

P. GILBERT, M.V. JONES, D.G. ALLISON, S. HEYS, T. MAlRA AND P. WOOD. 1998. POlOXamer F127 iS a non-toxic, di-block copolymer of polyoxpethylene and polyoxypropylene. Aqueous solutions (30% w/v) show thermoreversible gelation, being liquid at temperatures 15 "C. Chilled poloxamer (30% in tryptone soya broth) was mixed with an inoculum of Pseudomonas aeruginosu ( lo4 cfu ml-I) and placed as 100 pl drops onto separate glass cover-slips. These were placed into sealed Petri dishes containing moistened cotton wool and incubated at 35 "C. Viable counts could be performed on the poloxamer gels by transfer of the coverslips to diluents at < 15 "C. Growth curves in the gels and in liquid batch cultures were indistinguishable from one another with stationary phase cell densities, being approximately 5 x 10'" cfu ml-' in each at 16 h. SDS-PAGE of cell envelope preparations showed the poloxamer-grown cells to exhibit a biofilm rather than planktonic phenotype. Susceptibility towards various concentrations of chlorhexidine, iodine and hydrogen peroxide was assessed for 10 min at 35 "C for suspensions of broth-grown cells and for incubated poloxamer-gels (1 and 16 h). The gels were immersed in biocide, on their glass supports, before transfer to neutralizer at 10 "C where dissolution was complete within 5 min. Further serial dilutions and plate counts were made. While modest decreases in susceptibility towards all biocides were associated with incorporation of the inoculum with the gel (1 h incubation), substantial changes were noted after prolonged incubation and adaptation to a biofilm phenotype (16 h incubation). The gel populations mimic the localized high cell densities observed in biofilms and will also be subject to the same nutrient and chemical gradients as found within natural biofilms. Thermoreversible gelation enables complete recovery of the test inoculum without further trauma. They therefore provide an effective model for assessing biofilm susceptibility towards biocides and would be suitable for screening programmes.