A DIRECT VIABLE COUNT METHOD SUITABLE FOR USE WITH LISTERIA-MONOCYTOGENES

A method was developed to determine viable Listeria monocytogenes via direct microscopic observation. The test sample is mixed with tryptic soy broth containing 6 g/L yeast extract and 4-mu-g/L of novobiocin, and incubated for 6 h at 35-degrees-C. Cells are then collected on a membrane filter, stained with acridine orange, and elongated cells counted using an epifluorescent microscope. Viable cells enlarged from 0.8 to 1.5-mu-m to a length of 2.4 to 4.8-mu-m and did not divide during the incubation. Without novobiocin in the medium, the cell population increased 10-fold. The method, as described, is only useful for pure culture studies since no differential reaction was used. Survival of L. monocytogenes Scott A during starvation at 21-degrees-C in 0.8% saline and phosphate buffer diluent was determined using the direct viable count method. Results from using phosphate buffer indicate that the viable count method can detect greater numbers of starved cells than conventional nonselective plate counts.