A DIRECT VIABLE COUNT METHOD FOR THE ENUMERATION OF ATTACHED BACTERIAAND ASSESSMENT OF BIOFILM DISINFECTION

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obt ained with this technique were compared to two other enumeration metho ds, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The i n situ DVC method was applied to directly assess the viability of bact eria in biofilms without disturbing the integrity of the interfacial c ommunity. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation an d total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilitie s of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor sh owed no significant change in susceptibility to disinfectants during t he period of biofilm formation. In addition, the attached cells did no t reveal any more resistance to disinfection than planktonic cells. Th e disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have compa rable disinfection efficiencies at 25-degrees-C. Although being a weak er disinfectant, monochloramine was more effective in removing attache d bacteria from the substratum than free chlorine. The in situ DVC met hod always showed at least one log higher viable cell densities than t he PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated th at the in situ DVC method can provide more accurate information regard ing the cell numbers and viability of bacteria within biofilms followi ng disinfection.