Development of a simple recovery-enrichment system for enhanced detection of heat-injured Listeria monocytogenes in pasteurized milk

A simple anaerobic recovery-enrichment system, semisolid Penn State Univers ity (ssPSU) broth, that enhances recovery of heat-injured Listeria monocyto genes, was rapidly achieved in 10-ml screw-capped tubes by adding Bacto-aga r (2.5 g/liter) and L-cysteine (0.5 g/liter) to Penn State University broth . Glucose was removed from the formulation far ssPSU broth to prevent the g rowth of thermoduric lactobacilli. Ferric ammonium citrate was added to ssP SU broth to detect esculin hydrolysis and to indicate the presumptive prese nce of L. monocytogenes. Replacement of phosphate buffer with 3-[N-morpholi no]propanesulfonic acid (MOPS) buffer and addition of magnesium sulfate (15 mM) enhanced recovery and detection of L. monocytogenes heat treated at 62 .8 degrees C for 20 min. D-Serine, at a concentration of 150 mM, was found to inhibit germination of Bacillus spp. spores but did not inhibit severely heat-injured L. monocytogenes, Finally, ssPSU broth was modified (to mPSU broth) to contain the following: (i) Bacto-agar, 2.5 g/liter; (ii) ferric a mmonium citrate, 0.5 g/liter; (iii) MOPS buffer, pH 7.0; (iv) D-serine, 13. 7 g/liter; (v) D-alanine, 11.6 g/liter; and (iv) magnesium sulfate, 1.81 g/ liter. Incubation temperature significantly affected the recovery and detec tion of severely heat-injured L. monocytogenes. L. monocytogenes that were heat challenged in filter-sterilized whole milk at 62.8 degrees C for 20, 2 5, and 30 min could not be detected at incubation temperatures greater than or equal to 30 degrees C but were consistently detected after incubation a t 25 degrees C for 174, 199, and 330 h, respectively. Heat-injured cells of L. monocytogenes that were added to various commercial brands of pasteuriz ed whole milk were also detected using mPSU broth. When clostridial spores (10(4) spores per mi) were added to filter-sterilized milk containing eithe r heat-injured or non-heat-injured L. monocytogenes, only the latter could be detected in mPSU broth. The mPSU broth system requires no purging with n itrogen gas to create anaerobic conditions and permits recovery, growth, an d detection of L. monocytogenes in one vessel in the presence of thermoduri c background microflora commonly found in pasteurized milk.