|Siragusa, Gregory -|
Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 1, 2006
Publication Date: March 1, 2006
Citation: Line, J.E., Siragusa, G.R. 2006. Biphasic microtiter method for campylobacter recovery and enumeration. Journal of Rapid Methods and Automation in Microbiology. 14:182-188. Interpretive Summary: Campylobacter is an important foodborne pathogen causing more cases of human gastrointestinal illness annually in the United States than any other bacterium. There is a need for a sensitive method for detecting and enumerating all viable campylobacters (healthy as well as stressed cells) resulting from food processing or other environmentally stressful conditions. Biphasic techniques utilizing a liquid broth over a solid agar phase for recovery of campylobacters have been documented to provide more abundant growth of the organism than other methods relying upon selective broth or solid agar recovery alone. The nature of the enrichment process, however, allows most biphasic systems to be used only for detection, not enumeration of the pathogen. We developed a method for enhanced recovery and enumeration of viable campylobacters associated with processed, raw broiler chicken carcasses by combining the enumerative capabilities of a microtiter dilution well assay with biphasic growth conditions. When tested with naturally contaminated poultry carcass rinse samples, significantly more Campylobacter were recovered by the new biphasic technique than traditional plating methods. This new technique will be helpful to scientists and technicians in industry, academia and government who are concerned with determining how many Campylobacter may be present in food products or other stressed environmental samples. This research will be useful in diagnostic work as well as determining the effectiveness of pathogen interventions and processing steps necessary to reduce human exposure to this agent of foodborne illness.
Technical Abstract: We developed a biphasic method for recovery and enumeration of campylobacters. The biphasic system was composed of 96-well microtiter plates containing Campy-Line Agar (CLA) prepared 2X for all ingredients except for 1X agar (0.1 ml per well). Samples of pure Campylobacter spp. isolates, post-chill chicken carcass rinse, or spiked carcass rinse were prepared in either twofold or fivefold dilution series across separate 96-well plates and directly transferred (0.1 ml) to the agar-containing microtiter plates to create the biphasic system. Biphasic plates were incubated for up to 48 h at 42oC either aerobically or microaerobically in a gas mixture of 5% O2, 10% CO2 and 85% N2. Wells in plates incubated aerobically were covered with 0.1 ml of sterile mineral oil prior to incubation. Results from the biphasic system were compared to standard direct surface plating methods on CLA in Petri dishes incubated similarly under microaerobic conditions. Enumeration was easily accomplished by observing the color change in the biphasic wells due to the ability of campylobacters to reduce the colorless triphenyltetrazolium chloride (TTC) in the CLA to red-colored formazan compounds. Estimation of campylobacter populations were accomplished by noting the most dilute wells demonstrating growth or by inoculating replicate wells and deriving an MPN estimation. Campylobacter was recovered in the aerobic biphasic plates; however, significantly greater Campylobacter recovery was obtained in the biphasic plates incubated microaerobically (p<0.05). Significantly higher numbers of injured campylobacters from chicken carcass rinse samples were recovered by the biphasic method compared to conventional plating techniques (p<0.05).