BIPHASIC MICROTITER METHOD FOR CAMPYLOBACTER RECOVERY AND ENUMERATION

Authors: Line, John; Siragusa, Gregory

Submitted to: Campylobacter Helicobacter and Related Organisms International Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 6/15/2003
Publication Date: 12/6/2003
Citation: Line, J.E., Siragusa, G.R. 2003. Biphasic microtiter method for Campylobacter recovery and enumeration. International Journal of Medical Microbiology. 293:10.

Interpretive Summary:

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 2 or 5-fold 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, the significantly greater Campylobacter recovery was obtained in the biphasic plates incubated microaerobically (P<0.05). Significantly higher numbers of injured campylobacters from the chicken carcass rinse samples were recovered by the biphasic method compared to conventional plating techniques (P<0.05).