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.     

Application of current methods for isolation and identification of staphylococci in raw bovine milk

Samples of raw milk were examined for counts of somatic cells, total viable bacteria, staphylococci (Schleifer & Kramer's medium) and Staphylococcus aureus (Baird-Parker medium, Baird-Parker medium with pig plasma and Baird-Parker medium with additional antibiotics). For the isolation of staphylococci from raw milk, Schleifer & Kramer's medium was found to be very selective and in general performed satisfactorily. From the results obtained with the three remaining media the continued use of Baird-Parker medium for isolation of Staph. aureus from raw milk is recommended with the proviso that colonies selected for identification should include those that clear and do not clear the egg yolk and are not limited to colonies with diameters greater than 1 mm. Staphylococci isolated from raw milk were identified by key tests using a multipoint inoculation procedure. A selected number were also examined by the API STAPH system in conjunction with the API LAB computer programme for identification of staphylococci. Of the staphylococci examined, 90.0% were identified using the multipoint procedure. For strains identified as Staph. aureus, Staph. hyicus subsp. hyicus, Staph. epidermidis, Staph. simulans, Staph. xylosus or members of the Staph. hominis/Staph. warneri/Staph haemolyticus group, the API system provided confirmatory evidence. With strains identified by the multipoint procedure as Staph. hyicus subsp. chromogenes, Staph. sciuri subsp. sciuri and Staph. sciuri subsp. lentus the API system did not always provide concurring results. Several strains which could not be identified by the multipoint procedure could be identified by the API system. Staph. aureus, Staph. hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains isolated from milk were examined for production of enterotoxin A-E. Only 3.9% of Staph. aureus strains examined produced detectable enterotoxin (type C). None of the Staph. hyicus subsp. hyicus or Staph. hyicus subsp. chromogenes strains produced any of the known enterotoxins.     

Isolation and characterization of staphylococci from goats milk produced in Northern Ireland

Goats milk was examined for total viable bacteria and staphylococci (Baird-Parker medium, Schleifer & Kramer's (SK) medium, SK medium with a reduced sodium azide content (SKR) and SK and SKR with 5% added sheep blood). Staphylococcus aureus, Staph. epidermidis and Staph. simulans were the predominant species isolated overall. SK medium proved inhibitory with respect to the isolation of Staph. caprae and Staph. chromogenes. This was reduced by plating on the modified SK media. Representative strains of each species isolated were examined for production of enterotoxins A-E. Enterotoxin C alone was produced by 35% of the Staph. aureus strains tested. None of the other staphylococcal species examined produced any of the known enterotoxins.     

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.     

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.     

Enumeration of Vital and Thermally Stressed Staphylococcus aureus in Foods Using Baird-Parker Pig Plasma Agar (BPP)

Baird-Parker (BP), modified BP medium (egg yolk replaced with pig plasma, BPP) and modified Vogel and Johnson agar (phosphatidyl choline, deoxyribonucleic acid, with catalase added, PCVJ) were equally efficient for enumerating both non-stressed and thermally-stressed populations of Staphylococcus aureus from pure cultures and naturally contaminated foods. Vogel and Johnson agar was inferior. All colonies exhibiting coagulase activity on BPP were subsequently confirmed as Staph. aureus ; this was not the case with presumptive colonies from BP and PCVJ. Based on selectivity, definitive diagnostic characterization of colonies and increased sensitivity (more sample plated), BPP should be considered as the reference method for the routine enumeration of Staph. aureus in foods.     

Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.     

THE PERFORMANCE OF AN EGG YOLK-TELLURITE MEDIUM IN PRACTICAL USE

SUMMARY: Observations by seven laboratories that have used egg-tellurite-glycine-pyruvate agar (ETGPA) (Baird-Parker, 1962) for isolating Staph. aureus from foods are reported and discussed. The results indicate that ETGPA is superior to media currently used for detecting this organism. The main disadvantages of the medium are the similar appearance of Staph. aureus shown by some strains of Proteus vulgaris and the occasional finding of egg yolk nonclearing strains of Staph. aureus.     

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.     

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.     

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.     

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph. aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50 degrees C there was no marked variation in coagulase production by the surviving strains but at 55 and 62.5 degrees C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62.5 degrees C resulted in loss of enterotoxin production by all the survivors except S6 and 234, the surviving cells of which still produced enterotoxin B after heat treatment at 55 degrees C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.     

Selective isolation and enumeration of low numbers of Staphylococcus aureus by a procedure that relies on elevated-temperature culturing

A procedure for the selective detection of Staphylococcus aureus was elaborated, consisting of three parts: (i) enrichment in liquid Baird-Parker medium; (ii) isolation on Baird-Parker agar incubated at 43 degrees C, and (iii) confirmation by means of the test for thermostable nuclease. Elevated-temperature incubation of Baird-Parker medium did not make it inhibitory to cells stressed by heat treatment or osmotic shock.     

Selective isolation and enumeration of low numbers of Staphylococcus aureus by a procedure that relies on elevated-temperature culturing.

A procedure for the selective detection of Staphylococcus aureus was elaborated, consisting of three parts: (i) enrichment in liquid Baird-Parker medium; (ii) isolation on Baird-Parker agar incubated at 43 degrees C, and (iii) confirmation by means of the test for thermostable nuclease. Elevated-temperature incubation of Baird-Parker medium did not make it inhibitory to cells stressed by heat treatment or osmotic shock.     

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph, aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50°C there was no marked variation in coagulase production by the surviving strains but at 55 and 62–5dE C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62mD5dE C resulted in loss of enterotoxin production by all the survivors except S₆ and 234, the surviving cells of which still prodused enterotoxin B after heat treatment at 55dE C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.     

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.     

The inhibition of Staphylococcus hyicus on a selective medium for staphylococci

Growth of Staphylococcus hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains was found to be severely inhibited when broth cultures of these organisms were streaked on Schleifer and Kramer's staphylococcal (SK) medium. Of the selective agents contained in SK medium, potassium thiocyanate was found to be inhibitory towards both subspecies of Staph. hyicus and sodium azide had an additional inhibitory effect on Staph. hyicus subsp. chromogenes. Of six different media supplements examined, sheep blood and Tween 80 were found to improve the growth of both Staph. hyicus subspecies when added to SK medium. These findings were confirmed in subsequent work where the supplemented SK media were used to isolate potential enterotoxin-producing organisms from simulated raw milk (Staph. aureus, Staph. hyicus subsp. hyicus, Staph. hyicus subsp. chromogenes, Staph. intermedius ). SK medium supplemented with sheep blood proved more effective in allowing satisfactory recovery of Staph. aureus, Staph. hyicus subsp. hyicus and Staph. intermedius. However, neither supplement enabled satisfactory recovery of Staph. hyicus subsp. chromogenes to be achieved.     

The inhibition of Staphylococcus hyicus on a selective medium for staphylococci

Growth of Staphylococcus hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains was found to be severely inhibited when broth cultures of these organisms were streaked on Schleifer and Kramer's staphylococcal (SK) medium. Of the selective agents contained in SK medium, potassium thiocyanate was found to be inhibitory towards both subspecies of Staph. hyicus and sodium azide had an additional inhibitory effect on Staph. hyicus subsp. chromogenes. Of six different media supplements examined, sheep blood and Tween 80 were found to improve the growth of both Staph. hyicus subspecies when added to SK medium. These findings were confirmed in subsequent work where the supplemented SK media were used to isolate potential enterotoxin-producing organisms from simulated raw milk (Staph. aureus, Staph. hyicus subsp. hyicus, Staph. hyicus subsp. chromogenes, Staph. intermedius). SK medium supplemented with sheep blood proved more effective in allowing satisfactory recovery of Staph. aureus, Staph. hyicus subsp. hyicus and Staph. intermedius. However, neither supplement enabled satisfactory recovery of Staph. hyicus subsp. chromogenes to be achieved.     

Design and performance of a new medium for the quantitative recovery of Staphylococcus aureus from recreational waters

Several reports have suggested that staphylococci, and especially Staphylococcus aureus , are useful indicators of pollution of recreational waters. The lack of a selective, accurate and reliable recovery system for the quantification of Staph. aureus from water has been the principal obstacle to the evaluation of their use as indicators. In this study, several inhibitory substances and different nutrient sources have been evaluated for the formulation of a new selective medium. The medium designed, BFR-0 agar, recovers more than 75% of staphylococci and allows Staph. aureus to be identified.     

Design and performance of a new medium for the quantitative recovery of Staphylococcus aureus from recreational waters

Several reports have suggested that staphylococci, and especially Staphylococcus aureus, are useful indicators of pollution of recreational waters. The lack of a selective, accurate and reliable recovery system for the quantification of Staph. aureus from water has been the principal obstacle to the evaluation of their use as indicators. In this study, several inhibitory substances and different nutrient sources have been evaluated for the formulation of a new selective medium. The medium designed, BFR-0 agar, recovers more than 75% of staphylococci and allows Staph. aureus to be identified.