Development and application of a bioluminescence ATP assay method for rapid detection of coliform bacteria

Abstract

The conventional bacterial quantitative and qualitative methods are complex and require a lot of skills and time. Therefore a rapid microorganism detection system (RMDS) method was developed. With this method, yeast cells can be detected without cultivation, while some other microorganisms can be often detected after only 4–5 h of cultivation. The food sample is placed on a special membrane filter and cultivated on a plate until a sufficient amount of ATP is obtained. The membrane filter containing the microorganisms is then sprayed with the extractive and luminescent reagents and luciferin-luciferase reacts with the living cells. By this method, only the bioluminescent living cells are detected as spots. Thus, the RMDS method can detect the number but not the type of bacteria. An attempt to develop a method for the identification of coliforms, which is an index bacteria in food hygiene inspection, was made. In order to inhibit the growth of microorganisms which are closely related to the Pseudomonas genus and which form red colonies similar to coliforms as well as the germination of fungi spores, they were cultivated under anaerobic conditions in a new selective medium. Typical microorganisms which are often detected in foods, including yeasts, fungi, Gram-positive bacteria, Gram-negative bacteria (containing coliforms) and bacterial spores, were used. The sample was cultivated in an anaerobic L-shaped test-tube using the new coliform selective medium and then detected by the RMDS method. The results show that under this condition, the growth of both Pseudomonas sp. and fungi are suppressed, and coliforms could be detected within 6–7 h. This method is more rapid than the conventional method and has great potential for practical hygiene testing of both food beverages and water.