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.     

Lecithinase reaction of Staphylococcus aureus strains of different origin on Baird-Parker medium

J.E.S. MATOS, R.J. HARMON AND B.E. LANGLOIS. 1995. Staphylococcus aureus produces one or more enzymes with lipolytic activity, but differences between strains have been reported (Owens and John 1975; O'Toole 1987; Rollof et al. 1987). The biological and biochemical properties of these enzymes have been investigated and results were recently reviewed (Kötting et al. 1984).
Baird-Parker medium (Baird-Parker 1962) is a selective medium commonly used for the isolation of Staph. aureus. The presence of egg yolk in this medium permits the detection of two reactions due to lipolytic activity of staphylococci: (1) Lecithinase reaction, a zone of precipitate in the medium surrounding the colonies; and (2) Lipase reaction or 'pearly layer', an iridescent film in and immediately surrounding colonies, visible by reflected light (iridescent sheen or 'oil in water').
In this study, human and bovine strains, previously biotyped according to the scheme of Devriese et al. (1984), were compared for production of a zone of precipitation, lecithinase reaction, on Baird-Parker medium.
Bovine and human strains of Staph. aureus were compared for production of the egg yolk reaction (lecithinase reaction) on Baird-Parker medium and the results were related to their biotypes and site of origin of the sample. Human strains and strains biotyped as human biotypes had higher percentage of positive results than bovine isolates and/or biotypes. However, all strains isolated from body sites of heifers produced a positive reaction regardless of the biotype.     

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.     

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.     

Selective medium for carbohydrate-utilizing transductants of Staphylococcus aureus

Murphey, W. H. (The University of Texas, Dallas), and E. D. Rosenblum. Selective medium for carbohydrate-utilizing transductants of Staphylococcus aureus. J. Bacteriol. 87:1198-1201. 1964.-The composition and properties of modified eosin-methylene blue agar are described. It is an efficient, selective medium for isolation of carbohydrate-utilizing staphylococci from large, nonutilizing populations. The medium has been used to measure the rate of transduction of mannitol fermentation and to demonstrate genetic recombination in mannitol-negative mutants of Staphylococcus aureus.     

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.     

An improved medium for growing Staphylococcus aureus biofilm

A medium (Brain Heart Infusion plus 10% human plasma) was developed, tested, and validated for growing Staphylococcus aureus biofilm in vitro. With this medium, S. aureus forms reproducible and robust biofilms in flow chambers under controlled shear flow and with increased viability recovery in static well plates.     

Methodology for recovery of chemically treated Staphylococcus aureus with neutralizing medium.

Recovery results of Staphylococcus aureus ATCC 6538 treated with phenolics and quaternary ammonium compounds on Dey and Engley (D/E) neutralizing medium at various time intervals were compared by the use of two commonly used media. Two recovery processes were utilized. In one, the chemically treated organisms were plated directly onto an agar medium. In the other, the aliquot was first put in broth and then was plated with agar. By either process, the numbers and the time period for recovery of organism were greater on D/E medium.     

An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aureus

An amperometric immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above 10(3) cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.     

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.     

The enumeration of sublethally injured populations of Staphylococcus aureus in foods

Substantiating earlier investigations, pure cultures of Staphylococcus aureus were found to be equally well recovered on Baird-Parker agar at 37°C as at 42°C, whereas Micrococcus spp. are suppressed at the latter temperature to an extent exceeding 5 log₁₀ cycles. It was also established that egg yolk dissimilation by Staph. aureus is intensified at 42°C. Heat treated (60°C) populations of Staph aureus were quantitatively recovered on Baird-Parker agar at 42°C, though acid-injured populations were not. Acid-injury (2% lactic acid at 37°C) could be completely restored by solid medium repaiar during at least 6 h at 23°C on tryptone soya peptone yeast extract egg yolk pyruvate agar. Pure culture studies were confirmed in surveys on trade samples of foods.     

Liquid modification of Baird-Parker's medium for the selective enrichment of Staphylococcus aureus

A liquid modification of Baird-Parker's medium is suggested for the detection of low (<20/g) numbers of Staphylococcus aureus. Model experiments showed that the medium had an acceptable level of selectivity and that it was non-inhibitory to injured cells. Practical evaluation demonstrated the advantage of selective enrichment procedures over both non-selective enrichment technique and direct plating methods.     

Baird-Parker Medium Supplemented with Acriflavine, Polymyxins and Sulphonamide for the Selective Isolation of Staphylococcus aureus from Heavily Contaminated Materials

Acriflavine was found to be less inhibitory to Staphylococcus aureus than to other staphylococci. Advantage was taken of this selective effect and of the synergistic action of polymyxins and sulphonamides on Proteus strains to improve the selective capacity of Baird-Parker's Staph. aureus isolation medium. A supplement mixture containing acriflavine, sodium sulphamezathine and low levels of polymyxin in addition to the usual egg yolk and potassium tellurite is proposed for use with this medium.     

Methodology for recovery of chemically treated Staphylococcus aureus with neutralizing medium

Recovery results of Staphylococcus aureus ATCC 6538 treated with phenolics and quaternary ammonium compounds on Dey and Engley (D/E) neutralizing medium at various time intervals were compared by the use of two commonly used media. Two recovery processes were utilized. In one, the chemically treated organisms were plated directly onto an agar medium. In the other, the aliquot was first put in broth and then was plated with agar. By either process, the numbers and the time period for recovery of organism were greater on D/E medium.     

Effect of free-radical scavengers on enumeration of thermally stressed cells of Staphylococcus aureus MF-31.

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

Death of Staphylococcus aureus in Liquid Whole Egg Near pH 8

Incubating and shaking Staphylococcus aureus in liquid whole egg causes a decline in viability. During the period of agitation, the natural pH of the egg rises from about 7.2 to between 8.0 and 8.2 as a result of a loss of carbon dioxide. However, if the pH of the egg is prevented from rising, either by not shaking or by addition of a buffer, S. aureus will grow. The cause of death is traced to the presence of lysozyme of egg white. Interestingly, the action of lysozyme is not attributable to its bacterial lytic property but, instead, to the basicity of the lysozyme molecule. This conclusion is supported by the fact that the lytic property of lysozyme is known to have its optimal activity near neutrality and by the finding that protamine sulfate, a nonenzymatic basic polypeptide, also caused death of S. aureus at pH 8.0 but not at 7.0. It was postulated that the rise in pH renders the bacterial cells more negatively charged, so that in the presence of positively charged molecules like lysozyme or protamine sulfate a complex is formed, agglutinating the cells.