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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS Title: Comparison of methods for detecting live, stressed, and dead cells of Campylobacter jejuni

Authors
item Chen, Chin-Yi
item He, Yiping

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: January 20, 2007
Publication Date: July 8, 2007
Citation: Chen, C., He, Y. 2007. Comparison of methods for detecting live, stressed, and dead cells of Campylobacter jejuni. Abstract. International Association for Food Protection. P3-50.

Technical Abstract: The microaerophilic gram-negative bacterium Campylobacter jejuni is the leading cause of food-borne illnesses. It can be found in high prevalence in poultry products and in many environments, yet it is difficult to culture in the laboratory. When stressed, it can assume a "viable but non-culturable" (VBNC) state with a concomitant change in morphology from a spiral to a coccoid-shape. The purpose of this study was to compare several methods for detecting and/or quantifying live/viable and dead bacteria to evaluate cell viability under different growth and stress conditions. Ethidium monoazide in combination with real-time PCR (EMA-PCR), BacLight viability straining with confocal microscopy, and plate counting on Mueller-Hinton (MH) agar were used to quantify cells in defferent growth stages and under stress conditions such as exposure to oxygen (in air/ 1 week), heat (boiling/ 10 min), and isopropanol (30 min). Campylobacter jejuni strain 81-176 was cultured in MH broth or on MH agar under microaerobic conditions (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 16 h prior to stress treatments or before dilution for growth studies. Plate counting, microscopy and EMA-PCR showed varying degrees of detection sensitivity for live, dead, and stressed (coccoid) cells. The plate count failed to detect culturable cells under oxygen-stressed conditions even when both EMA-PCR and BacLight straining showed that the majority of cell populations (>96%) was viable. The EMA-PCR method, though sensitive, could not differentiate cell populations containing more than 99.9% dead cells thus may overestimate the number of viable cells. Microscopy with viable-straining is labor-intensive and not amiable for routine laboratory testing. The ability to detect viable bacteria is critical in evaluating the efficacy of intervention strategies and for risk assessment. Conventional culturing methods could underestimate the bacterial counts especially for cells in the VBNC stage, resulting in "fail-danger status" in intervention processes.

Last Modified: 12/21/2014