Stomacher bag

To evaluate the efficacy of our MALDI-TOF database, 197 specimens isolated from Brucella-infected animals and processed by the IZSAM were investigated. Briefly, the preferred tissues from the slaughtered animals were removed aseptically and cleaned of foreign material. Small pieces were then homogenized using Stomacher bags (VWR International, Radnor, PA), seeded onto selective Farrel and Theyer-Martin-modified solid medium, and then incubated at 37°C ± 2°C in air supplemented with 5–10% (v/v) CO₂ for up to 6 weeks. Colonies with distinctive morphology and positive urease-oxidase test were isolated and subjected to the specific PCR assays described above. After the addition of 10% formic acid for bacterial inactivation and protein extraction, as described in our protocol, extracts were stored at 4°C and then sent to UCSC within 48–72 h as blind-coded samples for MALDI-TOF investigation.

For the first objective, treated wheats were aseptically sampled (25 g) at the following time intervals: 0.5, 2, 6, 12, 18, and 24 h upon application of the tempering treatments; 25 g of wheat was mixed with 225 mL buffered peptone water (BPW) in stomacher bags (VWR, Radnor, PA, USA) and homogenized (2 min) using a stomacher (Seward, Islandia, NY, USA). Serial decimal dilutions in 0.1% peptone water and appropriate dilutions were spread plated on TSA (tryptic soy agar). Plates were incubated (37 °C, 24 h), and STEC counts were enumerated. This resulted in a detection limit of 2.0 log CFU/g.

A quantity of 5 g skin of each breast or drumstick was weighed into stomacher bags (VWR) and filled up to 50 g with sterile saline solution added with peptone buffered water (0.85% NaCl, 0.1% peptone) (VWR). The 1:10 dilution was stomachered for 2 min at 230 rpm (Stomacher 400 Circulator, Seward Ldt., Worthing, United Kingdom) and further serial tenfold dilutions up to 10⁻⁶ were prepared. The samples from the breast fillets were used for quantitative analysis of TVC (according to ISO 4833–1:2013). In brief, a volume of 1 ml of the dilution was pipetted into a petri dish and 12–15 ml plate count agar (CM0325, Oxoid) was added followed by incubation for 72 h at 30°C. The inoculated and treated drumsticks were examined quantitatively for Campylobacter spp. (according to ISO 10272–1), therefore 0.1 ml of the dilution was pipetted onto CCD-Agar plates (Oxoid) and spread evenly. The plates were incubated for 48 h at 41.5°C in a microaerobic atmosphere. For both Campylobacter spp. and TVC numbers plates between 5 and 300 colonies were counted and results were expressed in log₁₀ cfu/g skin. The limits of detection were 10 cfu/g for TVC and 100 cfu/g for Campylobacter spp.