The enumeration ofStaphylococcus aureus in foods

Summary Staphylococcus aureus can be enumerated in foods by the use ofChapman’s special medium (3), under application of the drop plate technique, recommended byMiles andMisra (6). Colonies ofS.aureus so obtained can easily be macroscopically differentiated from aerobic sporeformers which grow also on this medium. The colonies can be used immediately for testing their coagulase reaction.     

A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods

The coagulase reaction of Staphylococcus aureus on the PPSA (pork plasma for S. aureus) agar of Devoyod et al. was found to be fibrinogen-deficient. By including bovine fibrinogen (BFG) in the medium, the fibrin halos around S. aureus colonies became more distinct, preparations of pork plasma previously unacceptable for inclusion in the original PPSA agar were performing well, and the amount of pork plasma required in PPSA agar could be reduced by nearly 90%. In the modified medium, designated PPF (pork plasma fibrinogen) agar, the agar base (Baird-Parker agar without egg yolk) was unchanged. After surface plating, the base was covered with 8 mL of a modified overpour agar: 2.5% pork plasma, 0.38% BFG, and 0.0015% soy trypsin inhibitor in 0.7% Bacto agar. Most S. aureus strains could be enumerated after 24 h of incubation at 35 degrees C; the others required 44 h. Without soy trypsin inhibitor, a number of strains showed considerable fibrinolysis between 24 and 44 h of growth; this activity was neutralized by the inhibitor. The S. aureus counts of 27 food samples on PPF agar were essentially the same as the confirmed S. aureus counts obtained by the Baird-Parker method.     

A highly selective two-stage isolation method for the enumeration of Staphylococcus aureus in foods

A plate method for enumerating Staphylococcus aureus is described which combines a 1-h recovery period for stressed cells on a relatively non-selective Baird-Parker agar base followed by a 24-h growth phase in a highly selective, supplemented Baird-Parker medium added as an overlay. Tests with pure cultures showed satisfactory recovery of stressed Staph. aureus and other bacteria. Similar results were obtained with the conventional Baird-Parker procedure and with the two-stage isolation method for shrimps and poultry neck skins, but for raw minced meat, recoveries were higher with the combined method than with the conventional medium. All colonies visible after 24 h on the two-stage medium can be counted as Staph. aureus, whereas longer incubation times and confirmatory tests are necessary to differentiate it from other organisms on conventional Baird-Parker medium.     

A highly selective two-stage isolation method for the enumeration of Staphylococcus aureus in foods

A plate method for enumerating Staphylococcus aureus is described which combines a 1-h recovery period for stressed cells on a relatively non-selective Baird-Parker agar base followed by a 24-h growth phase in a highly selective, supplemented Baird-Parker medium added as an overlay. Tests with pure cultures showed satisfactory recovery of stressed Staph. aureus and other bacteria. Similar results were obtained with the conventional Baird-Parker procedure and with the two-stage isolation method for shrimps and poultry neck skins, but for raw minced meat, recoveries were higher with the combined method than with the conventional medium.
All colonies visible after 24 h on the two-stage medium can be counted as Staph. aureus , whereas longer incubation times and confirmatory tests are necessary to differentiate it from other organisms on conventional Baird-Parker medium.     

Catalase and enumeration of stressed Staphylococcus aureus cells.

The effects of catalase on the enumeration of stressed (heated, reduced water activity, or freeze-dried) Staphylococcus aureus cells on several selective media were examined. The addition of catalase greatly increased the enumeration of stressed cells. The beneficial effects of catalase were most pronounced on those media least efficient in enumeration of stressed staphylococci, showing increases in enumeration of up to 1,100-fold. The effects of catalase appear to be due to the reduced ability of stressed cells to repair and form colonies in the absence of an exogenous decomposer of H2O2. Thermally stressed cells were more sensitive to H2O2 than unstressed cells. During recovery, stressed cells overcame the requirement for catalase. These findings implicate H2O2 as a factor in the failure of certain selective media to adequately enumerate stressed cells and demonstrate that the addition of catalase to these media markedly increases their productivity.     

A rapid twofold dilution method for microbial enumeration and resuscitation of uninjured and sublethally injured bacteria

AIMS: A rapid and simple method for enumerating uninjured and sublethally injured bacterial cells, the twofold dilution method (2FD), was developed and evaluated. METHODS AND RESULTS: Following twofold serial dilution of samples in a 96 well microtiter plate, double strength selective broth or nonselective broth was added to each well. For resuscitation of heat-injured (55 degrees C for 10 min) coliforms, the selective broth was added to the wells after 3 h preresuscitation time in buffered peptone water. The results of the 2FD were compared to plating methods for total and coliform plate counts from mixed cultures and beef carcass surface tissue samples. CONCLUSION: The 2FD method results were not significantly different for uninjured cells (P > 0.05) from those obtained using Petrifilm and standard plating. Correlation of the scatterplot of spread plating and 2FD indicated a high level of agreement between these two methods (R(2)=0.98 for total counts and R(2)=0.96 for coliforms from mixed cultures; R(2)=0.98 for total cell counts and R(2)=0.94 for coliforms from faeces inoculated beef carcasses). SIGNIFICANCE AND IMPACT OF THE STUDY: The twofold dilution method recovered significantly higher numbers of heat-injured coliforms compared to conventional plating methods (P < 0.05).     

Rapid enumeration of Staphylococcus aureus in foods by direct demonstration of enterotoxigenic colonies on membrane filters by enzyme immunoassay.

Based on enzyme-linked immunosorbent assay, a convenient method has been devised for the direct demonstration of enterotoxin B production by Staphylococcus aureus colonies grown for 24 h on membrane filters. The problem of false-positive reactions due to binding of immunoglobulin G to protein A was turned to advantage by conjugating horseradish peroxidase directly to protein A, which then mediated the labeling of the antitoxin. The test requires 3 h to complete and yields a purple stain at the site of enterotoxin B-producing colonies, thus allowing direct enumeration of confirmed S. aureus in foods within 27 h. The method should be applicable to other enterotoxins of S. aureus.     

Stability of ribosomes of Staphylococcus aureus S6 sublethally heated in different buffers.

Cells of Staphylococcus aureus heated at 52 degrees C in magnesium-chelating buffers [pH 7.2, 50 mM potassium phosphate or 50 mM tris(hydroxymethyl)-aminomethane containing 1 mM ethylenediaminetetraacetic acid] leaked 260-nm absorbing material, shown to be RNA, and suffered destruction of their ribosomes. These cells did not regain their salt tolerance when repair was carried out in the presence of actinomycin D (5 microgram/ml). Cells similarly heated in magnesium-conserving buffers [pH 7.2, 50 mM tris(hydroxymethyl)aminomethane containing 10 mM MgCl2 or piperazine buffer] did not leak RNA, suffered no ribosomal damage when heated for 15 min, and recovered, at least partially, in the presence of actinomycin D. Ribosomal damage, is therefore, a consequence of Mg2+ loss and is not an effect of heat per se. Cells suspended in either Mg2+-chelating or Mg2+-conserving buffers lost salt tolerance to about the same extent during heating at 52 degrees C. Therefore, sublethal heat injury can not be attributed to ribosomal damage.     

The enumeration of proteolytic bacteria in foods

Summary For the enumeration of proteolytic bacteria in foods, regularly or sometimes obtained by microbial fermentation, a surface plating technique, using Frazier's gelatin/agar, incubated for two days at 31±1°C. and thereupon developed with sublimate solution, appeared useful.     

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.     

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods.

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.     

Accelerated procedure for the enumeration and identification of food-borne Staphylococcus aureus.

A procedure was developed for accelerating to 29 h the enumeration and identification of both healthy and stressed cells of Staphylococcus aureus in foods. Baird-Parker agar medium was incubated for 24 h; S. aureus was identified within 5 additional h by using a simplified thermonuclease test.     

A semi-homogeneous amperometric immunosensor for protein A-bearing Staphylococcus aureus in foods

Summary A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at ca. 30⁴ cfu/ml, and at ca. 10⁵ cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperomatric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.Offprint requests to: R. G. Kroll     

Egg yolk-free Baird-Parker medium for the accelerated enumeration of foodborne Staphylococcus aureus

A simplified procedure is described for the accelerated enumeration of foodborne Staphylococcus aureus. This involves the replacement of egg yolk in the Baird-Parker medium with Tween 80 and MgCl2. These compounds, along with pyruvate, allow the recovery of stressed cells of S. aureus on a medium which contains potassium tellurite, LiCl, and glycine as selective agents. Black colonies are identified as S. aureus by the simplified thermonuclease test.