Survival and recovery of viable but nonculturable Listeria monocytogenes cells in a nutritionally depleted medium

Survival of a desiccated five-strain Listeria monocytogenes mixture during storage in sand at 4 degrees C for 2 months was determined using the acridine orange direct count method with novobiocin and plate counts. Samples of inoculated sand were taken every 2 weeks, incubated at 37 degrees C for 6 h, stained with acridine orange, and then examined with a fluorescence microscope. Elongated viable but nonculturable cells were most frequently observed during weeks 2 and 4. At weeks 6 and 8, most of the cells either remained viable or were dead. In each microscopic field, only one or two viable but nonculturable cells were observed among hundreds of other viable culturable cells, indicating that L. monocytogenes does not generally become viable but nonculturable. Therefore, viable but nonculturable cells are not a concern when plating environmental samples or desiccated L. monocytogenes cells on nonselective media. Tryptic soy agar with 0.6% (wt/vol) yeast extract (TSAYE) and Columbia agar were used as nonselective plate count media. Modified Oxford agar and TSAYE + 5% (wt/vol) sodium chloride were used as the selective plate count media. The effects of aerobic or anaerobic incubation and media supplementation with 0.1% or 1% (wt/vol) sodium pyruvate were tested to optimize recovery of desiccated cells. Nonselective media showed better recovery when TSAYE and Columbia agar contained 0.1% (wt/vol) pyruvate and were incubated aerobically. These two culture methods were equally effective (P > 0.05) for recovering desiccated L. monocytogenes cells.