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 ß-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.

Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method.

AIMS: Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. METHODS AND RESULTS: Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. CONCLUSIONS: The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time-to-results from 2-5 times shorter than the traditional testing method. SIGNIFICANCE AND IMPACT OF THE STUDY: The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.

Photophysics of the single tryptophan residue in Fusarium solani Cutinase: evidence for the occurrence of conformational substates with unusual fluorescence behaviour.

The single tryptophan residue, at position 69 in the amino acid sequence, was used as an intrinsic probe to obtain structural and dynamical information on the lipolytic enzyme Fusarium solani cutinase. In the enzyme's native state the tryptophan fluorescence is highly quenched. Time-resolved experiments reveal that the majority of the excited state species is characterized by an unusually fast decay time of approximately 40 ps, indicating the occurrence of a very efficient nonradiative relaxation process, possibly via the adjacent disulphide bond or via the peptide bonds of a nearby loop. A minority of the excited state species relaxes on a nanosecond time scale. Irradiation of the enzyme in the tryptophan absorption band causes an increase by an order of magnitude of the fluorescence quantum yield. This increase is ascribed to a photo-induced, subtle structural change of a minor subset of species whose fluorescence is not highly quenched. The structural change is accompanied by a tightening of the local environment of the tryptophan moiety, as indicated by results from time-resolved fluorescence anisotropy which reveal a complete disappearance of the segmental flexibility of the tryptophan moiety.