Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.     

Comparative studies of selective media for recovery of Staphylococcus aureus from natural waters

Several selective media currently used for the enumeration of Staphylococcus aureus from different sources were evaluated in order to establish their quantitative recovery, specificity and degree of selectivity, using different types of water samples. The highest selectivity and reliability in the enumeration of Staph. aureus from the samples was obtained on Borrego-Florido-Romero-0 (BFR-0) and KRANEP agars. The method that produced the highest recovery of Staph. aureus was BFR-0 agar with membrane filter and incubation at 36 degrees C for 48-72 h.     

Comparative studies of selective media for recovery of Staphylococcus aureus from natural waters

Several selective media currently used for the enumeration of Staphylococcus aureus from different sources were evaluated in order to establish their quantitative recovery, specificity and degree of selectivity, using different types of water samples. The highest selectivity and reliability in the enumeration of Staph. aureus from the samples was obtained on Borrego-Florido-Romero-0 (BFR-0) and KRANEP agars. The method that produced the highest recovery of Staph. aureus was BFR-0 agar with membrane filter and incubation at 36°C for 48–72 h.     

Evaluation of culture media for the recovery of Mycobacterium avium subsp. paratuberculosis from Cheddar cheese

AIMS: The study evaluated the efficacy of four Mycobacterium avium subsp. paratuberculosis (MAP) culture media in suppressing commonly used starter cultures and typical nonstarter microflora present during the manufacture and ripening of Cheddar cheese, with a view to identify a suitable medium for the enumeration of MAP during laboratory-scale Cheddar production. METHODS AND RESULTS: Four Cheddar starter cultures and Cheddar cheese manufactured with these starters were inoculated onto Herrold's egg yolk medium (HEYM); HEYM supplemented with vancomycin, amphotericin B and nalidixic acid (HEYM/VAN); Middlebrook 7H10 agar containing polymyxin, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) antibiotic supplement; and BACTEC 12B radiometric medium with and without a preliminary decontamination step (0.75% w/v hexadecylpyridinium chloride (HPC), 5 h). The inclusion of a decontamination step inhibited all Cheddar cheese starter and nonstarter micro-organisms. The medium 7H10/PANTA and to a lesser extent HEYM/VAN were effective inhibitors of cheese microflora when no decontamination step was employed. CONCLUSIONS: Middlebrook 7H10 medium, supplemented with PANTA antibiotics, suppressed all micro-organisms associated with ripening Cheddar cheese manufactured with pasteurized milk. SIGNIFICANCE AND IMPACT OF THE STUDY: A MAP culture medium has been identified, which may be used to enumerate this bacterium during the laboratory manufacture and ripening of Cheddar cheese and hence facilitate further research into the persistence of this pathogen in the product.     

Comparison of selective media for coagulase-positive enterotoxigenic Staphylococcus aureus.

Different culture media were used to detect enterotoxigenic Staphylococcus aureus strains from peptone salt solution and inoculated minced meat. When recovery, reactions, and counting mistakes were considered, it was concluded that Baird-Parker agar was the best medium.     

Antialgal substances for iodine-disinfected swimming pools.

Forty substances were tested for antialgal activity against Chlorella pyrenoidosa (Wis. 2005) and Phormidium inundatum (Wis. 1093). C. pyrenoidosa exhibited greater resistance to adverse effects of test compounds than did P. inundatum. Although several structurally unrelated compounds were inhibitory to both alga species, even at an initial concentration of 1.0 mg/liter, methylthio-s-triazines, ametryne, prometryne, and terbutryne, at 0.1 mg/liter, restricted growth to less than 25% of control (untreated) cultures. The methylthio-s-triazines were virtually chemically unreactive with free iodine. Inhibition of Staphylococcus aureus by free iodine dosages of 0.25, 0.5, 0.75, and 1.0 mg/liter was unaffected by the presence of 2.0 mg of terbutryne per liter.     

Dry collection and culture methods for recovery of methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains from indoor home environments

Staphylococcus aureus in home environments may serve as a reservoir for human colonization, making sampling of indoor surfaces relevant to exposure assessment. Using laboratory experiments and application to homes of asthmatic children in Barbados, we characterize microbiological methods adapted for settings with transportation delays between sampling and initiation of culture.     

A simple method for counting Staphylococcus aureus in swimming pool water

Staphylococcus aureus counts from swimming pool water were determined by the membrane filtration technique. Water samples were passed through a membrane filter and then put on Baird-Parker media. After incubation, the filters were transferred to nutrient agar, and incubated at 37 degrees C, for 3 h. After removal of the filters, the plates were incubated at 60 degrees C for 2 h. An overlay of toluidine blue agar was added and the plates reincubated for 4 h at 37 degrees C. The formation of thermonuclease correlated with the formation of coagulase, and the results indicated that Staphylococcus aureus could be present in swimming pool water without the presence of either coliform or faecal coliform bacteria.     

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.     

Comparison of methods for recovery of Escherichia coli and Staphylococcus aureus from seeded laundry fabrics.

To assess the effect of laundry procedures on fabric-associated bacteria, a standard method of enumeration is needed. We evaluated six methods for enumeration of Escherichia coli and Staphylococcus aureus seeded (10(2) and 10(5) CFU/100 cm2 of fabric area) onto sterilized hospital sheets and terry . Two methods involved maceration of seeded swatches in broth followed by passage of the broth through a 0.45-micron-pore-size, 47-mm-diameter filter membrane. Three methods involved agitation of seeded swatches in broth with a paint shaker and membrane filtration of the broth to recover eluted bacterial cells, and the final method involved direct enumeration of cells on fabrics by overlaying seeded swatches with agar containing triphenyltetrazolium chloride as an indicator. The most convenient recovery method employed a 90-s agitation followed by serial dilution of broths and membrane filtration. This method provided 44/57% (low seed/high seed) recovery of E. coli from sheets and 133/31% from terry and 34/74% recovery of S. aureus from sheets and 58/57% from terry . Although maceration provided similar recovery of E. coli and S. aureus, it is a less-practical method. The direct enumeration method was ineffective for enumerating gram-positive bacteria. We conclude that either the agitation or maceration method used enumerated the seeded bacteria to within 1 log10 of their expected number and can be used to assess the bactericidal effectiveness of various steps in the laundering process.     

Ribosome assembly during recovery of heat-injured Staphylococcus aureus cells.

The process of ribosome formation during repair of sublethal heat injury was examined in Staphylococcus aureus. Sublethal heating of this organism results in the degradation of the 30S ribosomal subunit and alteration of the 50S subunit. Cells recovering from sublethal injury were examined for changes with time in the sedimentation and electrophoretic properties of ribonucleoprotein particles and ribonucleic acid, respectively. When cells were allowed to recover in [3H]uridine, the label could be followed into ribonucleic acid species that coelectrophoresed with 23S and 16S ribonucleic acid. Three ribonucleoprotein particles (49S, 36S, and 30S) were isolated from repairing cells by sedimentation through sucrose gradients. Polyacrylamide gel electrophoresis showed that the 49S particle contained 23S ribonucleic acid, the 36S particle contained both 23S ribonucleic acid and 16S precursor and mature ribonucleic acid, and the 30S particle contained 16S and precursor 16S ribonucleic acid. Particles with similar sedimentation properties were found in unheated cells.     

Antialgal substances for iodine-disinfected swimming pools

Forty substances were tested for antialgal activity against Chlorella pyrenoidosa (Wis. 2005) and Phormidium inundatum (Wis. 1093). C. pyrenoidosa exhibited greater resistance to adverse effects of test compounds than did P. inundatum. Although several structurally unrelated compounds were inhibitory to both alga species, even at an initial concentration of 1.0 mg/liter, methylthio-s-triazines, ametryne, prometryne, and terbutryne, at 0.1 mg/liter, restricted growth to less than 25% of control (untreated) cultures. The methylthio-s-triazines were virtually chemically unreactive with free iodine. Inhibition of Staphylococcus aureus by free iodine dosages of 0.25, 0.5, 0.75, and 1.0 mg/liter was unaffected by the presence of 2.0 mg of terbutryne per liter.     

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.     

Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner’s 2nd Agar or Broth (R2A or R2AB), Brain–Heart Infusion Broth (BHIB), Mueller–Hinton Broth (MHB), and Ashdown’s (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.     

Evaluation of several selective media for recovery of Aeromonas hydrophila from polluted waters.

Eleven media were studied for their suitability in the selective isolation of Aeromonas hydrophila. Preliminary results showed that five of them (inositol-brilliant green-bile salts agar, bile salts-brilliant green agar, Rimler-Shotts agar, xylose-sodium deoxycholate-citrate agar, and dextrin-fuchsin-sulfite agar) allowed the growth of several microorganisms that are usually present in the same samples in which A. hydrophila is found. Six media (mA agar, modified Rimler-Shotts agar, DNase-toluidine blue-ampicillin agar, Pril-xylose-ampicillin agar, MacConkey-trehalose agar, and starch-bile salts agar) were selected for evaluation as recovery selective media on the basis of their efficiency in the isolation of A. hydrophila from natural water samples. mA agar showed the best recovery rate and also an acceptable specificity, but its selectivity was low. Another medium that can be considered is DNase-toluidine blue-ampicillin agar, which showed good accuracy, but its specificity was low.