Significance of pre‐incubation temperature and inoculum concentration on subsequent growth of Listeria monocytogenes at 14°C

The influence of the bacterial concentration of an inoculum (10 1 or 10 3 cfu ml -1 ) of two strains of Listeria monocytogenes (Scott A : serotype 4b and V7 : serotype 1) and one strain of L. innocua (Lin 11), and the time and temperature at which the inoculum was stored (cold storage : 4°C for 4 weeks, or without cold storage : - 20°C before immediate transfer), and the temperature at which cells were pre-incubated (30°C and 14°C) on subsequent growth in Richard's broth at 14°C was investigated. Richard's broth at a pH 5.9 was used to simulate potential growth in soft cheese (camembert type) and an incubation temperature of 14°C was used to simulate storage-temperature ripening of cheese. Enumeration of the number of viable cells was by plate count method, except where viable cell numbers were less than 10 3 cfu ml -1 , when the MPN (Most Probable Number) technique was used. With cold storage and an inoculum of 10 3 cfu ml -1 (high bacterial concentration) the pre-incubation temperatures (30°C and 14°C) did not significantly influence the subsequent growth curve : there was no significant lag (less than 21 h) and cell numbers peaked in about 8.5 d. However, with cold storage and an inoculum of 10 1 cfu ml -1 (low bacterial concentration) and a pre-incubation temperature of 30°C a significant shift in the growth curve was observed over that pre-incubated at 14°C, with the appearance of a lag of about 7.7 d. At a pre-incubation temperature of 14°C with the low inoculum concentration, there was a measurable lag of about I d. Without cold storage and a pre-incubation temperature of 30°C, there was a lag time of 2.3 d. Storage conditions, pre-incubation temperature and inoculum concentration therefore appear to influence the subsequent growth curve. Importantly, however, the growth curves for cultures from inocula, pre-incubated at either 30°C or 14°C, appeared to involve two distinct values of the exponential growth rate (k) : the initial portion of the growth curve described by a low value of k and the subsequent portion by a consistently and significantly greater value. The appearance of two distinct growth phases was reproduced in further data determined for all the studied strains of the microorganism. Further study to explain these unexpected and reproducible findings is being conducted.