Development of a nondestructive fluorescence‐based enzymatic staining of microcolonies for enumerating bacterial contamination in filterable products

Aims:  Develop a nondestructive fluorescence-based staining procedure to rapidly detect and enumerate bacteria in filterable samples. Methods and Results:  The study consists in the development of a staining solution and a protocol to fluorescently detect microcolonies on cellulose membranes. After detection, membranes can be re-incubated on media to yield colonies. Carboxyfluorescein diacetate was selected among other carboxyfluorescein derivatives for its staining efficiency and the absence of background. Several permeabilizers were evaluated for their ability to promote dye uptake into cells without affecting viability. We demonstrated that a combination of n-Octyl β-d-glucopyranoside, sodium hexametaphosphate, lithium chloride and rubidium chloride significantly increased the staining efficiency of bacteria without affecting their viability. The method developed allowed the detection in <9 h of all tested aerobic bacteria and in 48 h of the anaerobic slow grower Propionibacterium acnes. Conclusions:  This method allows the rapid detection of bacteria in filterable samples in at least three to five times faster than traditional microbiological method. Significance and Impact of the Study:  The advantage of this nondestructive procedure is to allow contaminants identification after membrane re-incubation. This method could be easily applied in routine in pharmaceutical, clinical and food and beverage industries to monitor contaminations.