Evaluation of Different Methods to Detect Microbial Hygiene Indicators Relevant in the Dairy Industry

Introduction: Bacterial groups including coliforms, Enterobacteriaceae (EB), and Gram-negatives represent indicators of poor sanitation or post-pasteurization contamination in dairy products worldwide. While Petrifilms and traditional selective media are commonly used for the testing of these indicator organism groups throughout the dairy industry, new rapid methods are also being developed.

Purpose: This project was designed to evaluate the ability of different methods to detect coliforms, Enterobacteriaceae and other Gram-negatives isolated from various dairy products and dairy processing environments.

Methods: Using the Food Microbe Tracker© database, a collection of 211 coliform, Enterobacteriaceae, and Gram-negative isolates from 25 dairy-related genera were assembled for the purpose of this study. The selected isolates were tested in pure culture (at levels of 15 - 300 bacteria/test) to evaluate their detection on 3M^TM Coliform Petrifilm, 3M^TM Enterobacteriaceae Petrifilm, Violet Red Bile Glucose Agar (VRBGA), Crystal Violet Tetrazolium Agar (CVTA), Brain-Heart Infusion Agar (BHI), and a novel flow cytometry based method.

Results: Of the 211 Gram-negative isolates, 82.5% (174/211) had characteristic growth on CVTA media. Within this set of Gram-negative organisms, 175 isolates representing 19 genera in the Enterobacteriaceae group and were screened using the Enterobacteriaceae selective/differential detection methods.  Positive results were observed for 96.0% (168/175), 90.3% (158/175), and 86.3% (151/175) of isolates when tested on Enterobacteriaceae Petrifilm, VRBGA, and the flow cytometry instrument, respectively; optimization of the cut-off thresholds for the flow cytometry method may further improve the sensitivity and specificity of this method, but will require additional data.  Additionally, 73.7% (129/175) of the Enterobacteriaceae isolates tested positive as coliforms. 

Significance: The data obtained from this study indicate that detection of microbial hygiene indicator groups differs by testing methods and suggests that testing for Enterobacteriaceae or total Gram-negatives may represent more comprehensive hygiene indicators than traditionally used coliforms tests.