An efficient biofilm removal method for bacterial cells exposed to drinking water

Reliable quantification of microbial growth in a drinking water environment has typically been difficult, primarily due to the development of thin, pa tchy biofilms. Therefore, initial sampling and resuspension procedures beco me critical to the subsequent biomass determination. Biofilm cells attached to polycarbonate coupons of an annular reactor (AR) have typically been re moved by aseptically scraping the coupon surface with a sterile utility kni fe. The advantage of this method is its simplicity, however, scraping often compromises sterility and is highly subject to individual variation. The p urpose of this research was to develop a method that could remove and resus pend biofilm cells efficiently, consistently and with a good recovery rate. This paper presents a comparison of removal and resuspension methods. Thre e methods used to remove cells from the polycarbonate coupons include: scra ping with a utility knife, swabbing and stomaching. In addition, four metho ds were selected for cell resuspension and involved the use of a tissue ble nder, vortex, stomacher and a sonicator. Of the removal methods examined, s tomaching consistently yielded the highest number of culturable and total b acterial cells, ranging from two to four times more cells than scraping pro cedures. For one experimental set, the number of colonies enumerated by het erotrophic plate count (HPC! from the stomacher ranged from 1.0-1.7 x 10(6) CFU/cm(2), whereas numbers obtained using the scraping method were 4.6-4.9 x 10(5) CFU/cm(2). It was found that the number of HPCs recovered with sto maching was significantly greater at the 5% level than that obtained using the scraping method. Similarly, for cell resuspension, stomaching provided the highest enumeration. Once removal was achieved, sonication also provide d good resuspension. An analysis of variance showed that, compared to the r esuspension step, the removal step is more significant at the 5% level to t he recovery of biofilm cells. The stomacher has the unique advantage of com bining cellular removal and resuspension into a single step. This method wa s, therefore, selected as an ideal method for recovery of biofilm cells. Su bsequent optimization measures using the stomacher showed that sterile deio nized water was a suitable diluent for recovering cells from a drinking wat er environment. At normal speed (i.e., 230 rpm+/-5%), the optimal stomacher run length for maximum cell removal was 2 min. (C) 1999 Elsevier Science B .V. All rights reserved.