Phosphates have been approved for use in meat products primarily to protect flavor and increase yields. It also is known that phosphates have antimicrobial properties. The objective of this study was to compare the effects of different phosphates in a model system. Minimum inhibitory concentrations (MICs) of selected food-grade phosphates added to early-exponential-phase cells of Staphylococcus aureus ISP40 8325 in a synthetic medium were determined to be 0.1% for sodium ultraphosphate and sodium polyphosphate glassy and 0.5% for sodium acid pyrophosphate, sodium tripolyphosphate and tetrasodium pyrophosphate. Thus, the MIC values for the very long chain-length phosphates were lower than the MIC values for shorter chain-length phosphates. Leakage of intracellular nucleotides was observed both spectrophotometrically (release of A 260 -absorbing material) and microscopically (appearance of gelatinous cellular aggregates). Treatment of the gelatinous cellular aggregates with DNase, RNase and proteinase indicated that the aggregates contained DNA, RNA and protein, thus indicating cellular lysis in the presence of phosphates.

Previous studies showed that levels as low as 0.1% sodium ultraphosphate (UP), 0.1% sodium polyphosphate glassy (SPG) and 0.5% tetrasodium pyrophosphate (TSPP) were bactericidal and bacteriolytic to early-exponential phase cells of Staphylococcus aureus ISP40 8325. In the present study, Ca 2+ (0.01 M) or Mg 2+ (0.01 M) reversed the bacteriolytic effects of UP (0.1%) and SPG (0.1%) to S. aureus . In addition, Ca 2+ (0.01 M) or Mg 2+ (0.01 M), when added to the culture medium before inoculation, protected cells from growth inhibition by UP and SPG. Moreover, the bactericidal effects of UP or SPG were reversed by Ca 2+ or Mg 2+ in metal-rescue experiments in which the metals were added to polyphosphate-containing medium after 1 h of incubation. No additive effect existed between Mg 2+ and Ca 2+ . Growth inhibition of TSPP was not reversed by Mg 2+ or Ca 2+ , but it was reversed by Fe 3+ when Fe 3+ was added to protect cells 1 h before the addition of TSPP. These studies show that the antibacterial effects of phosphates can be altered substantially by the metal-ion content of the environment.

The results of previous studies indicated that the antibacterial effects of long-chain polyphosphates (sodium polyphosphate glassy [SPG] and sodium ultraphosphate [UP]) to Staphylococcus aureus ISP40 8325 could be attributed to damage to the cell envelope (cell wall or cell membrane). Also, Ca 2+ (0.01 M) or Mg 2+ (0.01 M) reversed the bactericidal and bacteriolytic effects of polyphosphates in S. aureus . In the present study, 0.4 M sodium chloride (NaCl) protected the cells from leakage caused by SPG and 0.6 M NaCl protected the cells from leakage by UP. Polymyxin, a peptide antibiotic that causes cell membrane damage, induced leakage even in the presence of 0.6 M NaCl. In the presence of 0.4 M NaCl, bacterial leakage was significantly reduced by disodium ethylenediamine tetraacetate (EDTA), a metal chelator that causes cell wall damage. Bacterial leakage by polyphosphates was significantly greater at pH 8 than at pH 6, which suggested that metal-ion chelation was involved in the antibacterial mechanism. A dialysis membrane (MWCO 100) was used to separate free metal and polyphosphate-bound metal. Levels of free Ca 2+ and Mg 2+ in polyphosphate-treated cells were significantly lower than those of the cells without polyphosphate. This free-metal dialysis study provided Chemical evidence to show that long-chain polyphosphates interacted with S. aureus cell walls by a metal-ion chelation mechanism. In addition, long-chain polyphosphates were shown to bind to the cell wall, chelate metals, and remain bound without releasing metal ions from the cell wall into the suspending medium. A hypothesis is proposed in which the antibacterial mechanism of long-chain polyphosphates is caused by binding of long-chain polyphosphates to the cell wall of early-exponential phase cells of S. aureus ISP40 8325. The polyphosphates chelate structurally essential metals (Ca 2+ and Mg 2+ ) of the cell wall, resulting in bactericidal and bacteriolytic effects. The structurally essential metals probably form cross bridges between the teichoic acid chains in the cell walls of gram-positive bacteria.

Antibiotic resistance among enterococci and fecal streptococci was examined by testing 149 isolates from pork, water, and clinical material, as well as 50 strains of 13 known species, for resistance to 27 different antimicrobial agents. Tests were performed by using the MicroScan Pos MIC type 6 panels. Pork isolates exhibited less resistance than either water or clinical isolates to most antibiotics, although a larger proportion of pork isolates than others was resistant to tetracycline. Comparisons of antimicrobial-resistance patterns between enterococcal species revealed that Enterococcus faecium was most resistant to β-lactam antimicrobials, especially ampicillin, whereas Enterococcus faecalis seemed to be the most resistant to the synergistic effects of antimicrobial combinations. Vancomycin resistance was observed in one Enterococcus hirae isolate from water. Enterococcal isolates from any of the sources tested did not show multiple resistance to antibiotics (such as gentamicin, ampicillin, streptomycin, and vancomycin) used to treat serious infections caused by gram-positive cocci.

The purpose of this study was to determine the numbers and species of enterococci encountered on pork carcasses during different stages in the slaughter process as well as on pork products. Three hog slaughtering plants were surveyed, each 3 times at four processing points. Each hog was swabbed at two sites on the carcass. Specimens were plated on two different enterococcal media, KF streptococcal agar and fluorescent gentamycin-thallous-carbonate agar. Retail and spoiled pork sausage products also were examined. Isolates were speciated by using the API Rapid Strep and Baxter MicroScan Pos ID panels. Contamination levels varied between plants as the carcasses progressed down the processing line; the highest counts were obtained directly before packaging in plants A and C. The highest count for plant B occurred at the first stage of sampling. More Enterococcus faecalis than Enterococcus faecium were isolated from the pork carcasses. Pork sausage results also are presented. Enterococci are useful as an indicator of pork sanitation and to detect critical control points during processing. In some instances, high levels of enterococci are associated with spoilage of pork sausage.

Six-ounce (151-g) Whirl-Pak® bags containing 3.05 g of dehydrated Presence-Absence (P-A) Broth and 5 mg of 4-methylumbelliferyl-β-D-glucuronide (MUG) were pasteurized with 10 kGy of gamma irradiation. To conduct a “bag” P-A test, 100 ml of water sample were added to a bag. The bag was closed, the medium was dissolved by massaging the bag for about 15 sec, and the bag was then placed in a rack for incubation. The bag method was compared with P-A tests conducted in 160-ml glass bottles and 200-ml polysulfone bottles, as well as with a 5-tube Laury] Tryptose Broth (LTB) most-probable-number (MPN) method. Twenty-nine surface-water samples (11 streams, 7 rivers, and 11 lakes), 9 well-water samples, and 2 sewage effluents were examined. Chi-square analyses of the results revealed that no significant difference (P≤0.1) existed among the different P-A tests. The hydrolysis of MUG provided excellent Escherichia coli detection and was easiest to determine in the bags.

Recoveries of coliforms in water and food samples were increased by adding sodium pyruvate to the Tryptic Soy Agar (TSA) base layer, and Violet Red Bile (VRBA) overlay, of the Modified VRBA procedure described in Standard Methods for the Examination of Dairy Products. Six pyruvate levels (0, 0.005, 0.01, 0.05, 0.1, and 1.0%) were tested. Counts were significantly lower (P≤0.05) on media containing 0% and 1.0% pyruvate than on the other media. Although 0.05% yielded the highest counts overall, there were no significant differences (P≤0.05) among 0.005, 0.01, 0.05, and 0.1% pyruvate. Analysis by using a general linear model procedure revealed that 0.02% pyruvate was the statistically predicted optimal level to use in the Modified VRBA procedure.

Counts from samples that contained low numbers of bacteria were determined by mixing the samples with double-strength agar media in 42-oz (1.2-L) Whirl-Pak bags. The bag-plate method was compared with other direct-plating methods and the most-probable-number and membrane-filtration procedures. Results obtained by using the bag method were as reliable as methods commonly used for analysis of samples that contain low numbers of viable bacteria.

A modified Bacillus subtilis disc-plate assay could detect 0.015 I.U. of penicillin G per ml of milk, whereas the lower detection limit of the standard assay was 0.050 I.U. per ml. Likewise, a modified Bacillus stearothermophilus disc-plate assay could detect 0.003 I.U. of penicillin G per ml of milk, whereas the lower detection limit of the standard assay was 0.005 I.U. per ml. Increased sensitivities were accomplished by preloading assay discs with “critical” concentrations so that minute quantities of antibiotic above the “critical” concentrations would produce zones of inhibition. Only a few alterations in routine laboratory procedure were required to perform the assays. Use of these assays should assure a milk supply that would not cause allergic reactions in humans or significantly inhibit dairy starter cultures. The general principal of “critical” concentration might have applications other than those that we have described, to increase the sensitivity of radial-diffusion analyses for biologically active compounds.

Salmonella isolation and identification follow an inseparable continuum of steps from beginning to end. This paper, therefore, encompasses both aspects of the problem. The importance of recovering injured cells is mentioned, and problems in automating the isolation process are described. Rapid serological tests for identification of salmonellae directly in enrichment media are discussed. Suggestions are made for improvements in antiserum preparation and for automating certain serological tests. Various kits, procedures and automated equipment on sale or under development for the rapid and convenient physiological characterization of salmonellae and other Enterobacteriaceae are described. Examples are given of comparative efficiencies and potential problems that might be encountered. Finally, a prediction is made about the possible nature of future generations of highly efficient and inexpensive automated systems for Salmonella identification.

Major events that led to acceptance of 30 to 300 as the most suitable number of colonies on plates for counting were reviewed. Three new sets of data were collected, involving triplicate plates of fifteen 1: 1.4 serial dilutions of 65 samples of raw milk. Statistical methods were developed to analyze bias (variability introduced primarily by crowding and analyst counting errors) and variance (sampling and dilution errors). Bias and variance were combined as mean-squared error, which was expresed as a function of the number of colonies per plate, The counting range that minimized the mean squared error could then be determined for selected dilution series. For two-fold, five-fold and ten-fold dilution series, respectively, the most suitable limits on plates for counting were 70 to 140, 40 to 200 and 25 to 250 colonies/plate. A range of 25 to 250 colonies/plate was suggested for the analysis of dairy products. Limitations in application of the data to other systems are discussed.

This is a review that concentrates primarily on recent developments in the modification of conventional methods for recovery of injured coliforms and salmonellae; 43 of the 58 references were published in 1975 or later. The review encompasses four areas of activity: ( a ) most probable numbers procedures, ( b ) membrane filter procedures, ( c ) direct plating methods, and ( d ) the influence of other variables.

The literature directly pertinent to coliform analyses at 30 C was reviewed. Original data were presented on effects of time of incubation of violet red bile (VRB) agar plates at 30 C and the effects of autoclave-sterilization and storage of VRB agar. When boiled VRB agar was used, an incubation period of at least 24 h was required for dairy products; an incubation period of 48 h was required for water, frozen vegetables, and other materials that contained coliforms that were slow in growth initiation. Additional observations should be made after incubation of plates for another 24 h to discover if substantial increases in colony count occurred. If autoclave-sterilized VRB agar was used, the recommended incubation periods should be extended for an additional 12–24 h. The pH of prepared VRB agar should be 7.2. A pH of 6.9 or below was indicative of extensive flaws in the method of preparation and/or storage. although some deterioration occurred in the absence of marked pH changes.

It has been suggested recently that the maximum holding time of dilutions of raw milk to be used for the standard plate count be reduced to 5 min. The data upon which this suggestion was based are reassessed by normalizing each sample to equal weight before summary computations are made. Results indicate that the average increases of counts did not exceed 10% during holding times of up to 20 min.