Baird parker rpf agar

Microbiological analyses were carried out in duplicate as previously described by Alemán et al. [27 (link)]. Briefly, 10 g of the sample were transferred into sterile bags (Sterilin, Stone, Staffordshire, UK) and mixed with 90 mL of buffered 0.1% peptone water (Oxoid, Basingstoke, UK) in a vertical laminar-flow cabinet (mod. AV 30/70 Telstar, Madrid, Spain). After 1 min shaking in a Stomacher blender (model Colworth 400, Seward, London, UK), appropriate dilutions were prepared for the following determinations: total bacterial counts (TBC) on pour plates of Plate Count Agar (PCA) incubated at 30 °C/72 h; Enterobacteriaceae on double-layered plates of Violet Red Bile Glucose agar (VRBG, Oxoid) incubated at 30 °C/48 h; lactic acid bacteria on double-layered plates of De Man, Rogosa and Sharpe (MRS) agar (Oxoid) incubated at 30 °C/72 h; Staphylococcus coagulase positive (S. aureus and other species) on Baird Parker-RPF agar supplemented with rabbit plasma and fibrinogen (BioMérieux S.A., Marcy l’Etoile, France). Microbiological counts were expressed as the log of the colony-forming units per gram (Log cfu/g) of sample.

One hundred microliter of the recovered suspension was serially diluted for quantification of the total aerobic bacteria using TSA agar plates (Tryptone Soya Agar, Oxoid), Enterobacteriaceae as an indicator of faecal bacteria using Violet Red Bile Glucose (VRBG) plates (Bacto VRBGA; Oxoid) and S. aureus using Baird Parker Agar (Baird Parker-RPF agar, Biomerieux). The remaining sample volume of ca 100 μl was inoculated onto Clostridium difficile Selective Agar (BBL C. difficile Selective Agar, Becton Dickinson) and incubated anaerobically for the detection of C. difficile. Because of some differences in the actual sample surface sizes all CFU counts were normalized to 10 cm² before data analysis. To be able to assess the cleaning efficiency, the acceptance level criteria was set to ≤ 5 CFU/cm² for total aerobic microorganisms and ≤ 1 CFU/cm² for S. aureus and C. difficile [22 ].

The detection of E. coli, coliforms, Staphylococcus aureus, and enterococcus faecalis was done using the surface plate technique. Chromocult coliform agar was used for both E.coli and coliforms (Chromocult; Merck, Darmstadt, Germany) (Byamukama et al., 2000 (link)). A step by step procedure as explained in Mawioo et al. (2016b) was applied to prepare and incubate the samples for the E. coli and coliforms analysis. Dark blue to violet colonies were classified as E. coli while red to pink colonies were identified as coliforms (Byamukama et al., 2000 (link), Sangadkit et al., 2012 ). Staphylococcus aureus and enterococcus faecalis were grown using Baird-Parker RPF agar, and Slanetz and Bartley agar media, respectively (bioMérieux SA, Marcy l'Etoile, France). The preparation involved spreading 0.1 mL of the sample or its dilutions on the surface of the agar plate. The inoculated plates for enterococcus faecalis were incubated at 37 °C for 48 h and the colonies were identified as red, brown or pink. For the Staphylococcus aureus, the inoculated plates were incubated at 37 °C for 24 h but prolonged for an additional 24 h if no characteristic colonies were formed. Their colonies were identified as grey-black. The average number of colonies were used to calculate the viable-cell concentrations in the samples and expressed in either CFU/g TS or CFU/L of the test sample.