Biotimer assay for counting bacterial biofilm

A growing body of evidences shows that bacterial biofilm lifestyle is comparatively more common than the planktonic one and that biofilm plays a crucial role in human health. The enumeration of the actual number of bacteria in biofilm is still a great challenge for microbiologist. The standardized method of colony forming unit (CFU) count is not reliable to quantify bacteria in biofilm. In the absence of a validated method to count bacteria in biofilm, BioTimer Assay (BTA) is presented here. BTA allows to count bacterial biofilm adherent on surfaces and employs an appropriate reagent containing an indicator able to switch as a consequence of bacterial metabolism in biofilm. The time required for indicator switch, induced by microbial metabolism, is correlated to the initial number of bacteria (N0) through a genus-specific correlation line described by the following equation t = log(1+a=N0)=k where k is growth rate and a is a function of the metabolic product responsible for the reagent switching. Moreover, BTA does not require any manipulation of samples and has been applied to count bacteria in biofilm adherent to several polymers, to verify microbiological quality of foods and to detect the antibiotic susceptibility of biofilm. BTA, providing a reliable, sensitive, rapid and easy-to perform method, could be considered a useful tool in counting bacteria in biofilm also adherent on nano-structured particles to be in vivo administered