The microbiological control in industrial contest , is still today majority based on growth capability on standard medium. This approach is time consuming allowing only retrospective actions . In addition it is now recognized that some micro-organisms may not be cultured while maintaining a viability . Over the last 20 or so years no alternatives have been developed to fully substitute the official plate count technique.
The objectives of the proposed research and development project are to develop and validate for industrial routine applications , new ultra rapid techniques for microbiology testing. The analytical procedures developed will have to meet the needs of the pharmaceutical and the water industries and their performance will be tested in actual plant environment.
A sterility test based on esterase activity and membrane integrity has been developed ; this test allow the detection of viable micro-organisms down to one cell per volume filtered within 3 hours for a TVC (total viable count). It has been validated on a large number of micro-organisms and showed a good correlation with the traditional plate count method. The initial validation on pharmaceutical products have revealed the presence of false positives which have been later on eliminated by addition of a counter stain in the protocol ; the improved protocol is now proposed to several pharmaceutical plants for internal acceptance. Specific detection of E. coli has been approached by using the in situ hybridation technique ; application to naturally water samples has shown the requirement of a signal amplification due to a low level of ribosomal RNA which can be achieved by additional enzyme amplification. The total reaction time, however, exceed the two hours objective. An improved hybridation approach using peptide nucleic acid probe is currently tested to decrease the response time. No monoclonal antibodies tested so far have been found suitable for specific E. coli or coliforms detection. However, for enterobacteriacae where a monoclonal antibody exist (Chemunex antibody) a combination of this antibody and the viability marker used for sterility testing show that this approach allow the specific detection of viable micro-organisms.
To meet these objectives, three types of tests will be developed -an ultra rapid sterility test on liquid samples , based on a direct fluorescent labeling of cells using a viability marker ; different process waters as well as chlorinated water samples will be used to validate this approach. - specific labeling techniques for rapid detection and counting of specific type of bacteria : E.coli , coliforms , Enterobacteriacoea .The test will use either a nucleic acid probe or monoclonal antibodies . The total response time should not exceed two hours . For these two approaches , no cell multiplication will be applied in order to satisfy both sensitivity and rapidity and overcome the problem of viable but non culturable micro-organisms -a combination of viability and specificity for rapid micro-organisms detection; in many industrial situations, it may be of interest to count only the live micro-organism of a particular strain.
Funding SchemeCSC - Cost-sharing contracts
CM19 5AD Harlow
RG2 0JN Reading