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.     

Repair and Enumeration of Injured Coliforms in Frozen Foods

Two strains of Escherichia coli manifested death and repairable injury after being frozen in water or sterile foods at -20 C. The injured survivors were inhibited from forming colonies on violet red bile agar (VRBA) or deoxycholate lactose agar; this inhibition was greater when enumeration was done by the pour plate method instead of the surface or surface-overlay method. Injured cells repaired rapidly in Trypticase soy broth (TSB), and the repair was about maximum after 1 h at 25 C. When the injured cells were added to different foods and incubated at 25 C, repair also occurred; however, recovery was better and more uniform when the samples were mixed with TSB and incubated 1 h at 25 C. Cell multiplication was not evident until after 90 to 120 min at 25 C. The enumeration of coliforms from commercially frozen foods was increased when the thawed samples were mixed with TSB and the cells were allowed to repair 1 h at 25 C. In some samples, the repair permitted at least a 20-fold increase in the coliform count. The associated flora in the commercially frozen foods gave no evidence of impairing the repair of coliforms, nor did they start multiplication prior to 90 min after being incubated in TSB at 25 C. Generally, the plating gave more reproducible recovery of coliforms than did the most probable number method. Also, a higher number of coliforms were obtained by the surface-overlay method of plating using VRBA.     

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.     

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.     

Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

Injury and recovery of Escherichia coli after sublethal acidification.

Over 99% of the viable cells of Escherichia coli K-12 were injured after a 60-min exposure to 0.3 M sodium acetate buffer at pH 4.2. Injured cells were those able to grow on Trypticase soy agar but unable to grow on violet red bile agar. The extent of both death and injury of acid-treated cells increased with decreasing pH or increasing concentration of acid. Injured cells were able to recover their colony-forming ability on violet red bile agar by incubation in Trypticase soy broth or potassium phosphate buffer before plating on the agar media. Direct neutralization of the injury medium did not allow recovery and, in fact, was lethal to the population. The addition of metabolic inhibitors to the Trypticase soy recovery broth was used to study the repair process. It was not affected by the presence of inhibitors of protein, cell wall, deoxyribonucleic acid, or ribonucleic acid syntheses. The addition of 2,4-dinitrophenol to the recovery medium also did not inhibit recovery. Actinomycin D and N,N'-dicyclohexylcarbodiimide were lethal to a proportion of the acidified cells but allowed another fraction of the population to recover. There were no detectable amounts of 260- or 280-nm-absorbing materials leaked during the course of acid injury.     

Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

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.     

Injury and recovery of Escherichia coli after sublethal acidification.

Over 99% of the viable cells of Escherichia coli K-12 were injured after a 60-min exposure to 0.3 M sodium acetate buffer at pH 4.2. Injured cells were those able to grow on Trypticase soy agar but unable to grow on violet red bile agar. The extent of both death and injury of acid-treated cells increased with decreasing pH or increasing concentration of acid. Injured cells were able to recover their colony-forming ability on violet red bile agar by incubation in Trypticase soy broth or potassium phosphate buffer before plating on the agar media. Direct neutralization of the injury medium did not allow recovery and, in fact, was lethal to the population. The addition of metabolic inhibitors to the Trypticase soy recovery broth was used to study the repair process. It was not affected by the presence of inhibitors of protein, cell wall, deoxyribonucleic acid, or ribonucleic acid syntheses. The addition of 2,4-dinitrophenol to the recovery medium also did not inhibit recovery. Actinomycin D and N,N'-dicyclohexylcarbodiimide were lethal to a proportion of the acidified cells but allowed another fraction of the population to recover. There were no detectable amounts of 260- or 280-nm-absorbing materials leaked during the course of acid injury.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation.

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Effect of aeration on minimal medium recovery of heated Salmonella typhimurium.

The effect of presence or absence of air on minimal medium recovery of heated Salmonella typhimurium was investigated. It was determined that the expression of minimal medium recovery is not only dependent on heat and a nutritionally complex medium but also on air. Unlike in the presence of air, in the presence of nitrogen, cells were able to recover their ability to grow on Trypticase soy agar enriched with 0.5% yeast extract (TSY) when incubated in TSY broth. It was established that in the presence of nitrogen the number of heat-TSY- induced, single-straneded breaks in deoxyribonucleic acid (DNA) were less than in the presence of air. Furthermore, the DNA breaks in nitrogen were repaired, whereas DNA breaks in air were not. The ability of cells to grow on TSY agar corresponded well with their ability to repair damage to DNA.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Plating procedure for the enumeration of coliforms from dairy products.

A "repair-detection" procedure consisting of pour plating of food samples with Trypticase soy agar, followed by 1-h repair incubation at room temperature and subsequent overlay with violet red bile agar, was found to be an effective method for the detection of injuried and uninjuried coliforms from dairy products. This method was relatively less effective for the detection of coliforms in many semipreserved foods as compared with dairy products, but more effective than the most-probable-number method.     

Repair and Enumeration of Injured Coliforms by a Plating Procedure

Surface plating of coliforms on Trypticase soy agar, followed by 1 to 2 h of incubation at 25 C and subsequent overlay with violet red bile agar, was found to be a useful method for the repair and enumeration of coliforms injured by freezing.     

Radiation resistance and injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25 degrees C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and -30 degrees C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at -20 degrees C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at -20 degrees C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at -20 degrees C, nor did storage at -20 degrees C alter the cell's resistance to irradiation at 25 degrees C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36 degrees C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36 degrees C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5 degrees C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36 degrees C for 1 day than at 5 degrees C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

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.     

Revival and Subsequent Isolation of Heat-Injured Bacteria by a Membrane Filter Technique

Cultures containing mixed flora from raw milk were heated at 62.8 C for 15, 20, 25, and 30 min. Dilutions were filtered through membrane filters, and the filters were incubated on Trypticase soy broth (TSB) and on TSB plus NaCl (TSBS). The TSB count indicated the total population which survived heating and included injured and uninjured cells. The colonies on TSBS indicated the uninjured cells and were marked by perforating the membrane near the colony. This membrane was then transferred to fresh TSB and incubated further. The injured organisms recovered and formed colonies which could be distinguished from previous colonies of uninjured organisms. Transfer counts on TSB were not substantially different from the initial TSB counts at 15, 20, 25, and 30 min of heating.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

Recovery of Spores of Bacillus stearothermophilus from Thermal Injury

Bacillus stearothermophilus grown in nutrient broth produced a product which promoted recovery from thermal injury of its spores. This phenomenon was observed with nutrient agar as the plating medium but not with a medium composed of Trypticase, Phytone, dextrose and phosphate (TPDP). Recovery of injured spores was greatest in such a medium if it contained starch or charcoal. Trypticase soy agar and dextrose tryptone agar were markedly inferior to TPDP medium.