Influence of Milk Components on the Injury, Repair of Injury, and Death of Salmonella anatum Cells Subjected to Freezing and Thawing

Fast freezing and slow thawing Salmonella anatum cells in various milk components inactivated from 20 to 98% of the cells and damaged 40 to 90% of the cells surviving the treatments. Injured cells failed to form colonies on a selective medium (xylose-lysine-peptone agar with 0.2% sodium deoxycholate) but did form colonies on a nonselective plating medium (xylose-lysine-peptone agar). The major milk components-lactose, milk salts, casein, and whey proteins-influenced the extent of injury, repair of injury, and death. The percentages of cells injured and inactivated were decreased by the presence of any milk components except whey proteins. Also, repair of injury was promoted by the presence of each milk component except whey proteins, which in contrast inhibited repair. Phosphate was the most influential milk salts component that protected the cells and promoted repair of injury. These individual milk components may have decreased the extent of freezing-induced death and cellular damage by stabilizing the S. anatum cell envelope.     

Repair of Injury in Freeze-Dried Salmonella anatum

Repair of injury induced by freeze-drying Salmonella anatum in nonfat milk solids occurred rapidly after rehydration. Injury in surviving cells was defined as the inability to form colonies on a plating medium containing deoxycholate. Death was defined as inability to form colonies in the same medium without this selective agent. The rate of repair of injury was reduced by lowering the temperature from 35 C to 10 C and was extremely low at 1 C. Repair was independent of influence of pH between 6.0 and 7.0. Repair did not require synthesis of protein, ribonucleic acid, or cell wall mucopeptide, but did require energy in the form of adenosine triphosphate (ATP) synthesized through oxidative phosphorylation. The requirement for ATP was based on dinitrophenol or cyanide interference with repair. Dinitrophenol activity was pH-dependent; no repair occurred at pH 6.0 and some repair was observed at pH 6.5 and above. Injured cells were extremely sensitive to low concentrations of ethylenedinitrilotetraacetate. This indicated that freeze-drying injury of S. anatum may involve the lipopolysaccharide portion of the cell wall and that repair of this damage requires ATP synthesis.     

Effect of Rehydration on Recovery, Repair, and Growth of Injured Freeze-Dried Salmonella anatum

From 70 to 90% of the Salmonella anatum cells that survived freeze-drying in nonfat milk solids were injured. After rehydration, these injured survivors failed to grow on a selective plating medium containing deoxycholate but could form colonies on a nonselective medium. In a suitable environment after rehydration, injury disappeared in most of these cells. The rate of this repair at 25 C was very rapid initially and, in a medium containing milk solids, was completed within 1 hr after rehydration. The repaired cells initiated growth about 1 hr later than normal cells and grew at a slower rate. In a medium containing milk solids, initial recovery, extent of repair of injury, initiation of growth, and rate of growth were not influenced by supplementation with extra nutrients in other rehydration media. Rehydration controlled by modifying the concentrations of lactose, sucrose, or milk solids in the rehydration medium influenced the recovery of cells and the time that growth was initiated. Glycerol failed to increase recovery. Higher numbers of cells were recovered by rehydrating at 15 to 25 C, but an earlier initiation of growth and more rapid growth were observed at 35 C.     

Characterization of the Repair of Injury Induced by Freezing Salmonella anatum

Fast freezing and slow thawing of Salmonella anatum cells suspended in water resulted in injury of more than 90% of the cells that survived the treatment. The injured cells failed to form colonies on the selective medium (xyloselysine-peptone-agar with 0.2% sodium deoxycholate) but did form colonies on a nonselective (xylose-lysine-peptone-agar) plating medium. In Tryptic soy plus 0.3% yeast extract broth or minimal broth, most of the injured cells repaired within 1 to 2 hr at 25 C. Tryptic soy plus yeast extract broth supported repair to a greater extent than minimal broth. Phosphate or citrate at concentrations found in minimal broth supported repair of some cells. MgSO(4), when present with inorganic phosphate or citrate or both, increased the extent of repair. The repair process in the presence of phosphate was not prevented by actinomycin D, chloramphenicol, and D-cycloserine, but was prevented by cyanide and 2,4-dinitrophenol (only at pH 6). This suggested that the repair process might involve energy metabolism in the form of adenosine triphosphate. The freeze-injured cells were highly sensitive to lysozyme, whereas unfrozen fresh cells were not. In the presence of phosphate or minimal broth this sensitivity was greatly reduced. This suggested that, at least in some of the cells, the injury involved the lipopolysaccharide of the cell wall and adenosine triphosphate synthesis was required for repair.     

Repair of Injury Induced by Freezing Escherichia coli as Influenced by Recovery Medium

Freezing an aqueous suspension of Escherichia coli NCSM at -78 C for 10 min, followed by thawing in water at 8 C for 30 min, resulted in the death of approximately 50% of the cells, as determined by their inability to form colonies on Trypticase soy agar containing 0.3% yeast extract (TSYA). Among the survivors, more than 90% of the cells were injured, as they failed to form colonies on TSYA containing 0.1% deoxycholate. Microscope counts and optical density determinations at 600 nm suggested that death from freezing was not due to lysis of the cells. Death and the injury were accompanied by the loss of 260- and 280-nm absorbing materials from the intracellular pool. Injury was reversible as the injured cells repaired in many suitable media. The rate of repair was rapid and maximum in a complex nutrient medium such as Trypticase soy broth supplemented with yeast extract. However, inorganic phosphate, with or without MgSO₄, was able to facilitate repair. Repair in phosphate was dependent on the pH, the temperature, and the concentration of phosphate.     

Injury and Death of Streptococcus lactis due to Freezing and Frozen Storage

Cells of Streptococcus lactis were harvested in the early stationary phase, washed, and resuspended in either skim milk (10% nonfat milk) or buffered distilled water (0.0003 m dipotassium phosphate, pH 7.2). Samples of each suspension were frozen and stored at -20 C for intervals up to 28 days. Colony counts of the frozen culture were made using lactic agar and a “restricted” lactic agar medium (Tryptone reduced to 0.5%) to determine injury and death. Death was determined by the difference in plate counts on lactic agar before and after freezing. Injured cells were determined by the difference in plate counts on the two plating media. Greatest injury of the cells occurred during early stages of frozen storage and decreased with time, and death continuously increased. Injury and death were more pronounced when cells were frozen in water than when frozen in 10% nonfat milk solids. Certain cultures survived better when frozen rapidly, whereas with others survival was greater when freezing was slow. Successive freezing, thawing, and propagation of the culture gradually eliminated cells which showed injury by freezing.     

Minimal Medium Recovery of Thermally Injured Salmonella senftenberg 4969

Exposure of Salmonella senftenberg 4969 to sublethal heating in phosphate buffer, pH 7·0, at 52· produced thermally injured cells characterized by their relative inability to form colonies on trypticase soy yeast extract agar compared to minimal medium (M9) agar. During subsequent incubation at 37· in liquid media, more injured cells were capable of repair in M9 than in nutrient media used for pre-enrichment purposes. M9 was superior to lactose broth as a liquid holding medium to restore the ability of injured cells to grow on both rich and selective agar media. The addition of food products produced a more favourable environment for the repair of thermally injured cells in M9 rather than lactose broth. Pre-enrichment in M9 was 100 times more effective than using lactose broth as the preliminary step in the detection of S. senftenberg in laboratory pasteurized liquid egg albumen.     

Discrepancies in the Enumeration of Escherichia coli

Stationary-phase cells of Escherichia coli were enumerated by the pour plate method on Trypticase soy agar containing 0.3% yeast extract (TSYA), violet red-bile agar, and desoxycholate-lactose agar, and by the most-probable-number method in Brilliant Green-bile broth and lauryl sulfate broth. Maximum counts were assumed to be those on TSYA. In general, numbers detected were lower with the selective solid media and higher with the selective liquid media. Inhibitory effects, especially on selective solid media varied with the strains of E. coli. The lower detection on selective solid media was partly due to the stress induced in some cells by the temperature of the melted media used in the pour plate method. These cells apparently failed to repair and form colonies in the selective media. Improved detection on the selective solid media was achieved by using 1% nonfat milk solids, 1% peptone, or 1% MgSO(4).7H(2)O in the dilution blanks. Higher detection on selective agar media was effected by surface plating or by surface-overlay plating of the cells. The surface-overlay method appeared to be superior for the direct enumeration of E. coli in foods.     

Role of Suspending and Recovery Media in the Survival of Frozen Shigella sonnei

Shigella sonnei was frozen at -20 C in saline, nutrient broth, and milk, and plated, after thawing, upon synthetic medium, nutrient agar, and blood heart infusion agar. There was a difference in the numbers of cells recovered when the frozen and thawed cells were grown on different media. The synthetic medium was unable to recover cells injured by freezing or did so only poorly compared to the complex media. The addition of meat extract, peptone, or Casamino acids to the synthetic medium improved its ability to recover injured cells as measured by bacterial colony counts. This is suggestive of metabolic injury caused by the freezing processes since the cells which survived freezing required an enriched medium for growth. In this paper the term metabolic injury is used to express a change in the nutritional requirements of the organisms which resulted in an increase in growth factor requirements. Freezing the cells in saline resulted in greater injury compared to cells frozen in nutrient broth or milk; this suggested that these suspending agents possessed some protective quality. The metabolic injury increased with an increase in the length of time the cells were held in the frozen state.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

Heat-induced injury inListeria monocytogenes

Summary Heating ofListeria monocytogenes (Scott A strain) in potassium phosphate buffer (0.1 M, pH 7.2) at 52°C for 1 h led to injury, with the heat-injured cells failing to produce colonies on agar medium containing 5% NaCl. The detection of injury was based on the use of differential media: plating on tryptose phosphate broth+2% agar and 1% sodium pyruvate (TPBA+P) and on tryptose phosphate broth+2% agar and 5% NaCl (TPBA+S). Only non-injuredListeria formed colonies on TPBA+S whereas both heat-injured and non-injured cells formed colonies on TPBA+P. The bacterial count on TPBA+P minus that on TPBA+S represents the extent of heat injury. A large number of selective agars were tested and compared to TPBA+P for their ability to support repair and colony formation of heat-injuredL. monocytogenes. Media containing 0.025% phenylethanol, 0.0012–0.0025% acriflavin, 0.1–0.2% potassium tellurite, 0.001% polymyxin B sulfate, 5% NaCl or a combination of these ingredients were detrimental to the recovery of heat-injuredL. monocytogenes. Media currently in use forL. monocytogenes are not satisfactory for the recovery of injured cells.     

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).     

Injury recovery of foodborne pathogens in high hydrostatic pressure treated milk during storage

Bacteria are expected to be injured or killed by high hydrostatic pressure (HHP). This depends on pressure levels, species and strain of the microorganism and subsequent storage. Injured bacteria may be repaired which could affect the microbiological quality of foodstuffs with an important safety consideration especially in low acid food products. In this study two Gram-positive (Listeria monocytogenes CA and Staphylococcus aureus 485) and two Gram-negative (Escherichia coli O157:H7 933 and Salmonella enteritidis FDA) relatively pressure resistant strains of foodborne pathogens were pressurized at 350, 450 and 550 MPa in milk (pH 6.65) and stored at 4, 22 and 30 degrees C. The results of shelf life studies indicated two types of injury, I1 and I2, for all the pathogens studied. It is obvious that I2 type injury is a major injury and after its repair (I2 to I1), the cells can form colonies on non-selective but not on selective agar. The formation of colonies on both selective and non-selective agar occurs only after full recovery of injury (I1 to AC). The results presented in this study show that even if injured cells are not detected immediately after HHP treatment, I2 type injury could be potentially present in the food system. Therefore, it is imperative that shelf life studies must be conducted over a period of time for potential repair of I2 type injury either to detectable injury (I1) or to active cells (AC) to ascertain microbiological safety of low acid food products.     

Protein-bound SH groups in frozen-thawed egg cells of the sea urchin

Unfertilized egg cells of the sea urchin St. intermedius could survive slow freezing to ?15 °C for a short period of time, but at the same freezing temperature extracellular freezing became fatal within a few hours. Such freezing injury resulted in “black” or “white” cytolysis in frozen-thawed cells. “Black” cytolysis took place in the process of both freezing and thawing, while “white” cytolysis occurred only on thawing. Rapid rewarming consistently produced “white” cytolysis in extracellularly frozen cells. The observed behavior of the injured cells during freeze-thawing appeared favorable for the explanation of freezing injury by the SH-SS hypothesis. Protein-bound SH groups were quantitatively determined in both whole cell and cortex with plasma membrane before and after freeze-thawing. However, no significant change in the SH value was observed between freeze-thaw cytolysed materials and unfrozen ones.     

Diluent sensitivity in thermally stressed cells of pseudomonas fluorescens.

Thermally injured cells of Pseudomonas fluorescens were unable to produce colonies on Trypticase soy agar (TSA) after dilution with 0.1% peptone. Nutritional exigency could not be used as the criterion for this injury, since varying the composition of the plating medium had little effect on the number of colonies that developed. The injured cells had no requirement for compounds known to leak out during the heat treatment in order to recover. The cells did not exhibit injury if dilution preceded heat treatment on the plating medium, demonstrating that the heat treatment sensitized the cells to the trauma of dilution. Substitution of 0.1% peptone with growth medium as the diluent largely offset the previously observed drop in TSA count. Little difference in survival was observed when monosodium glutamate or the balance of the defined medium was used as the diluent. The diluent effect was ionic rather than osmotic. The presence of cations was important in maintaining the integrity of the injured cell, and divalent cations enhanced this protective effect. The role of these cations at the level of the cell envelope is discussed.     

Thermal injury of Yersinia enterocolitica

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

Thermal injury of Yersinia enterocolitica.

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

Diluent sensitivity in thermally stressed cells of pseudomonas fluorescens.

Thermally injured cells of Pseudomonas fluorescens were unable to produce colonies on Trypticase soy agar (TSA) after dilution with 0.1% peptone. Nutritional exigency could not be used as the criterion for this injury, since varying the composition of the plating medium had little effect on the number of colonies that developed. The injured cells had no requirement for compounds known to leak out during the heat treatment in order to recover. The cells did not exhibit injury if dilution preceded heat treatment on the plating medium, demonstrating that the heat treatment sensitized the cells to the trauma of dilution. Substitution of 0.1% peptone with growth medium as the diluent largely offset the previously observed drop in TSA count. Little difference in survival was observed when monosodium glutamate or the balance of the defined medium was used as the diluent. The diluent effect was ionic rather than osmotic. The presence of cations was important in maintaining the integrity of the injured cell, and divalent cations enhanced this protective effect. The role of these cations at the level of the cell envelope is discussed.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Pulsed electric fields cause sublethal injury in Escherichia coli

AIMS: The objective was to investigate the occurrence of sublethal injury in Escherichia coli by pulsed electric fields (PEF) at different pH values. METHODS AND RESULTS: The occurrence of sublethal injury in PEF-treated E. coli cells depended on the pH of the treatment medium. Whereas a slight sublethal injury was detected at pH 7, 99.95% of survivors were injured when cells were treated at pH 4 for 400 micros at 19 kV. The PEF-injured cells were progressively inactivated by a subsequent holding at pH 4. CONCLUSIONS: PEF cause sublethal injury in E. coli. The measurement of sublethal injury using a selective medium plating technique allowed prediction of the number of cells that would be inactivated by subsequent storage in acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could be useful for improving food preservation by PEF technology and contributes to the knowledge of the mechanism of microbial inactivation by PEF.