Freezing of Listeria monocytogenes and Other Microorganisms: A Review.

When the temperature of microbes is lowered rapidly, some are injured through thermal shock. Frozen cells can be injured mechanically by intra- and extracellular ice crystals. During freezing, as water is removed, there is a concentration of cell solutes which can lead to dissociation of cellular lipoprotein. Warming of frozen cells can be accompanied by growth of ice crystals which then can physically affect cells. Freeze-thaw injury of microbes is manifested by an increase in fastidiousness and by changes in cellular morphology, release of materials from the micro- and macrostructure of cells, and denaturation of macromolecules. Given the proper environmental conditions, cells can repair such injury. Cryoprotectants minimize damage to cells during freezing and frozen storage. Death and injury of Listeria monocytogenes were greater when cells were frozen and stored at -18°C rather than -198°C. Tryptose broth was more protective of cells than a phosphate buffer solution when freezing and storage were at -18°C; the reverse was true at -198°C. Repeated freezing (-18°C) and thawing (35°C) were more detrimental to cells of L. monocytogenes than were repeated freezing at -198°C and thawing at 35°C. Freezing cells at -198°C and storing them at -18°C caused more injury and death than did freezing and storage at -198°C. Glycerol was an effective cryoprotectant for L. monocytogenes . Less effective were milk fat, lactose, and casein. The extent of injury and death varied among strains of L. monocytogenes given the same treatment. Freezing and thawing increased susceptibility of L. monocytogenes to effects of lipase and lysozyme.

Transmission Electron Microscopy of Unfrozen and Frozen/Thawed Cells of Listeria monocytogenes Treated With Lipase and Lysozyme.

Unfrozen cells of Listeria monocytogenes typically contained no preplasma space exterior to the plasma membrane (PM) when viewed by transmission electron microscopy. Cells of L. monocytogenes strains Scott A, V7, and California (CA), after freezing and frozen storage, exhibited one or more of the following when viewed with transmission electron microscopy: (a) retraction of cytoplasm and infolding of the PM to form mesosomes, (b) extra-and intracellular rupture of the cell wall (CW), (c) formation of intracellular "bubbles," and (d) damage to the CW and PM that could have resulted from autolysin activity. Type and degree of effect depended on frozen storage time and strain of L. monocytogenes . Lysozyme treatment of unfrozen or frozen/stored (19 d)/thawed cells of strains Scott A, V7, and CA resulted in protoplast formation and damage to the CW. Three stages of protoplast formation were observed when cells of strain CA were frozen, stored 2 weeks, thawed, and treated with lysozyme. More damage to the CW and PM occurred when frozen storage time was extended for up to 6 weeks before treatment with lysozyme. Lipase and lysozyme treatment of unfrozen or frozen/stored (19 d)/thawed cells of strain Scott A resulted in protoplast formation with some damage to the PM and irregularity in shape of cells. Damage to the PM increased with increasing frozen storage time for up to 6 weeks. Some cells of strain CA resisted freezing, frozen storage for 6 weeks, thawing, and treatment with lipase and lysozyme.

Lysozyme and Lipase Alter Unfrozen and Frozen/Thawed Cells of Listeria monocytogenes.

Unfrozen and frozen/thawed cells of Listeria monocytogenes strains Scott A, V7, and California were treated with lipase and/or lysozyme. Cells of strain Scott A were more susceptible to the lytic action of lysozyme than were cells of strains V7 and California. Treatment of unfrozen cells with lipase before exposure to lysozyme enhanced cellular lysis. This also was true for cells held frozen for up to 6 weeks before they were thawed and treated with enzymes. Some variation existed among strains of L. monocytogenes in their susceptibility to effects of lysozyme. Frozen storage of cells of all three strains increased their susceptibility to lysis by lipase, and this was related inversely with the percentage of cells that survived freezing and frozen storage. Transmission electron microscopy showed some enzyme-treated cells formed protoplasts.

Inactivation of Listeria Monocytogenes by Chlorine.

Cells of Listeria monocytogenes strain Scott-A were harvested from cultures, washed, and then treated with a solution of sodium hypochlorite at 25°C and pH 7. The cells were more resistant to chlorine when they were (a) harvested from a 24-rather than 48-h-old culture, (b) grown in tryptose broth rather than on a slant of tryptose agar, and (c) washed and suspended using a 20 rather than 0.312 mM phosphate buffer solution. Cells of L. monocytogenes were exposed for 30 s-4 h to sodium hypochlorite solutions that contained 0.5-10 ppm available chlorine. Generally, the number of survivors decreased rapidly during the first 30 s followed by a slower decrease during the rest of the exposure time. The initial count of L. monocytogenes in the suspension (1 × 108-3.2 × 108/ml) decreased 0.49 to 6.4 orders of magnitude during the first 30 s of exposure to the chlorine solutions. The effect of the presence of organic substances on the strength of hypochlorite solutions was studied. Presence of 0.05 or 0.1% peptone caused a large and rapid loss of available chlorine. Glucose or lactose (up to 1%) had almost no effect on the concentration of available chlorine.

Temperature, pH, and Strain of Pathogen as Factors Affecting Inactivation of Listeria monocytogenes by Chlorine.

Listeria monocytogenes strain Scott-A was treated with 1 ppm available chlorine at different temperatures and pH values. Different strains of L. monocytogenes (California, Scott-A and V7) were also exposed to 1 ppm available chlorine at pH 7 and 25°C. The initial population of L. monocytogenes was 1 × 108 to 3.2 × 108 CFU/ml of sodium hypochlorite solution. Survival of L. monocytogenes was measured by surface-plating (on tryptose agar) samples taken at intervals of 30 s to 1 h of exposure to hypochlorite solution. Larger numbers of L. monocytogenes strain Scott-A survived at 25 than at 35°C. The smallest number was observed when cells were exposed to the hypochlorite solution at 5°C. The higher the pH values, in the range of 5 to 9, the greater were the numbers of survivors of L. monocytogenes strain Scott-A. Of the strains studied, California was the most resistant, while V7 was the least resistant to the hypochlorite solution.