Submitted to: Journal of Microbiological Methods
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/9/2003
Publication Date: 11/1/2003
Citation: CHEN, C., NACE JR, G.W., IRWIN, P.L. A 6X6 DROP PLATE METHOD FOR SIMULTANEOUS COLONY COUNTING AND MPN ENUMERATION OF CAMPYLOBACTER JEJUNI, LISTERIA MONOCYTOGENES, AND ESCHERICHIA COLI. JOURNAL OF MICROBIOLOGICAL METHODS. 2003. Interpretive Summary: Bacterial enumeration is important in evaluating the degree of bacterial contamination in foods. There are advantages and disadvantages in the various methods used for enumeration, but almost all are laborious and require a large amount of resources such as culture media, investment in specialized equipment, and incubator space. Campylobacter is among the most common foodborne pathogens worldwide. Due to the microaerophilic nature of this bacterium (prefers lower oxygen levels for optimal growth), expensive gas jars and packets that generate the desirable atmosphere or specialized incubators are necessary for culturing it. We sought to develop a new enumeration procedure that is fast, accurate, precise and at the same time saves material and space for laboratories that are limited in budget and space. By utilizing readily available 96-well plates for serial dilutions and multichannel pipettes for plating, we developed a 6x6 drop plate method that combines the advantages of being able to count the bacterial colonies that form and to enumerate by the most probable number (MPN) techniques. This method is very accurate and precise, and can be applied to a wide range of bacterial concentrations. The 6x6 drop plate method will reduce laboratory costs by: 1) shortening sample processing time reducing labor cost; 2) lowering expenditures on growth media and supplies; and 3) lessening the need for expensive, specialized incubator space, or gas jars/gas packets.
Technical Abstract: For organisms which require unique cultural conditions, such as Campylobacter spp., incubator space is costly and limited. To maximize space utilization, a protocol was developed using standard 96-well microtiter plates and mutlichannel pipettes for serial dilutions followed by 6x6 drop plating on agar. This method does not require expensive equipment and entails only minor modifications of existing practices. Data collected with this protocol can be used to calculate CFU/mL, in the conventional way, as well as to determine a most probable number (MPN), thus, combining the advantages of plating (colony counts with morphology) with MPN into one easy-to-perform enumeration approach. This scheme was tested by enumerating Campylobacter jejuni, Listeria monocytogenes, and E. coli whereupon detection limits were 18 ± 6, 8 ± 3, and 7 ± 6 CFU/mL, respectively. Across all tested organisms, the average observed change (m) in 6x6 drop plate-based bacterial levels with known concentrations was close to unity (r^2 = 0.98; m = 0.99), arguing for both excellent precision and accuracy. For samples of unknown concentration, the 6x6 drop plate protocol is exceptionally time- and cost-efficient, since far fewer agar plates are needed than are required by the more established drop or spread plate enumeration techniques.