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

Poloxamer F127 is a non-toxic, di-block copolymer of polyoxyethylene and po lyoxypropylene. Aqueous solutions (30% w/v) show thermoreversible gelation, being liquid at temperatures <15 degrees C and robust gels at temperatures >15 degrees C. Chilled poloxamer (30% in tryptone soya broth) was mixed wi th an inoculum of Pseudomonas aeruginosa (10(4) cfu ml(-1)) and placed as 1 00 mu l drops onto separate glass cover-slips. These were placed into seale d Petri dishes containing moistened cotton wool and incubated at 35 degrees C. Viable counts could be performed on the poloxamer gels bg transfer of t he coverslips to diluents at < 15 degrees C. Growth curves in the gels and in liquid batch cultures were indistinguishable from one another with stati onary phase cell densities, being approximately 5 x 10(10) cfu ml(-1) in ea ch at 16 h. SDS-PAGE of cell envelope preparations showed the poloxamer-gro wn cells to exhibit a biofilm rather than planktonic phenotype. Susceptibil ity towards various concentrations of chlorhexidine, iodine and hydrogen pe roxide was assessed for 10 min at 35 OC for suspensions of broth-grown cell s and for incubated poloxamer-gels (1 and 16 h). The gels were immersed in biocide, on their glass supports, before transfer to neutralizer at 10C whe re dissolution was complete within 5 min. Further serial dilutions and plat e counts were made. While modest decreases in susceptibility towards all bi ocides 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 mi mic the localized high cell densities observed in biofilms and will also be subject to the same nutrient and chemical gradients as found within natura l 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 suitabl e for screening programmes.