Assessing the Survival of Listeria monocytogenes in a Domestic Freezer by Analyzing Subsequent Growth at 30°C Using a Novel Reference Method

Abstract

Listeria monocytogenes is a serious pathogen capable of extensive survival under frozen storage. Using optical density and multiple initial inocula in multiple identically prepared microtiter plates, the effect of storage time at −22°C on the subsequent growth at 30°C of the organism when defrosted was studied using a technique that compared the growth (through time to detection) of a test plate (previously frozen) with that of an identically prepared control plate, analyzed at the start of the experiment. Experiments were carried out using tryptic soy broth (TSB) or TSB supplemented with 3% salt. Plates were stored and frozen for up to 6 months (10 days, 20 days, 2 months, and 6 months). As storage time increased, there was only a small relative increase in the lag and the variance in the time to detection observed. When compared with storage in 3% salt TSB, which reduced the specific growth rate relative to growth in standard TSB, there were only marginally greater increases in lag and data variance. After 6 months storage in 3% salt TSB, there were some indications of inactivation (observed as small reductions of the initial optical density (equal to 1 × 109 CFU/ml) equivalent to a 50% inactivation. The method and the analyses suggest that this technique could allow easy examination of the effect of frozen storage on given cultures, with respect to the effects of pH, water activity, and also the effect of preservatives commonly used as extra hurdles in foods.