The study was conducted from February to August 2019. A total of 100 milk samples were collected from different sources (47 samples from dairy farms, 27 samples from retail markets, and 26 samples from farmers’ houses) in Qena, Egypt. These samples were collected under aseptic conditions in a clean, sterile 15 mL falcon tube and transferred instantly in an icebox to the bacteriological laboratory in the Department of Microbiology, Faculty of Veterinary Medicine, South Valley University. One milliliter of milk samples were fed in 9 mL buffer peptone water (Oxoid) and incubated at 37°C for 18-24 h. Subsequently, a sterile loop was used to transfer bacteria from the inoculated buffer peptone water and was inoculated on a MacConkey agar plate (Oxoid). Plates were incubated at 37°C for 24 h. The suspected colonies were inoculated in eosin methylene blue (Oxoid). After the procedure, green metallic sheen colonies were selected for biochemical identification using the IMViC reaction and triple sugar iron test [12 ].
Buffer peptone water
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Buffer peptone water is a nutrient-rich medium used for the cultivation and enumeration of a wide range of microorganisms, including bacteria and fungi. It serves as a general-purpose diluent and enrichment broth for microbiological analyses.
Automatically generated - may contain errors
Lab products found in correlation
Macconkey agar plate, by Thermo Fisher Scientific (2 mentions)
Mannitol salt agar, by Thermo Fisher Scientific (1 mentions)
Mannitol salt agar plate, by Thermo Fisher Scientific (1 mentions)
Baird parker agar, by Thermo Fisher Scientific (1 mentions)
Macconkey agar medium, by Thermo Fisher Scientific (1 mentions)
Automated system, by BD (1 mentions)
Eosin methylene blue, by Thermo Fisher Scientific (1 mentions)
Triple sugar iron, by Thermo Fisher Scientific (1 mentions)
Urea agar, by Thermo Fisher Scientific (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Xylose lysine deoxycholate agar, by Thermo Fisher Scientific (1 mentions)
7 protocols using buffer peptone water
1
Isolation and Identification of E. coli in Milk
2
Isolation and Identification of Coagulase-Negative Staphylococci
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CoNS were isolated according to El-Nagar et al. (2017 ) and Shokry et al. (2018 ) by inoculating 10 g of samples into 90 ml of buffer peptone water (Oxoid Limited, Thermo Fisher Scientific Inc., UK) and incubating at 37 °C for 24 h. A loopful of enriched samples was then streaked on Baird Parker agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) and Mannitol Salt agar plates (Oxoid Limited, Thermo Fisher Scientific Inc., UK), and incubated at 37 °C for 48 h. The plates were examined for the presence of black colonies without clear or opaque zones on Barid Parker and yellow or red colonies on Mannitol Salt agar. The suspected CoNS colonies were identified according to Holt et al. (1994 ) using different biochemical tests such as coagulase, catalase, sugars fermentation (mannose, sucrose, mannitol, trehalose, maltose, lactose, and raffinose), urea, Voges-Proskauer, ONPG, and novobiocin sensitivity (Table S1 ).
3
Isolation of E. coli from Samples
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For isolation of E. coli from a collected sample, the sample was at first inoculated into a test tube containing buffer peptone water (Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37°C overnight for primary enrichment. The overnight culture was streaked on MacConkey agar medium (Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37°C for 24 h. Bright pink-colored large colonies yielded on to MacConkey agar plate were suspected as the growth of E. coli. Such colonies were streaked onto EMB agar plate (Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37°C for 24 h. Based on “green metallic sheen,” colony yielded on this medium was taken as the growth of E. coli, which was later confirmed by applying standard biochemical tests recommended for the identification of E. coli.
4
Microbiological assessment of meat and related samples
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Three hundred samples from various slaughterhouses and meat stores in different areas of Rawalpindi, Islamabad, Pakistan were collected during 2018–2019. The sample size for this study was calculated using prevalence formula in a software N-Query Advisory (STATCON Gmbh, Germany, Version 7.0) (Pourhoseingholi et al., 2013 (link)). A total of 300 samples were collected: 40 from each of working area, tools (knives, hooks), butcher hands and beef, 30 from each of chicken and mutton, 20 from each of nasal and rectal swabs. Sterile cotton swabs were first dipped in buffer peptone water (Oxoid, United Kingdom) rubbed horizontally and then vertically on the selected materials (Adugna et al., 2013 (link)). Then swabs were placed with proper labeling in airtight zip bags and stored at −20°C. Collected samples were processed through standard operating procedures. The raw meat samples (1 g) were first added to 10 ml tryptone water (10 g/l tryptone and 5 g/l NaCl) and properly mixed. This solution was used as inoculum for making serial dilutions. This makes the first dilution i.e., 10–1. The dilutions 10–3, 10–4, and 10–5 were used for inoculation. The swab samples were placed in peptone water-containing falcon tubes and then centrifuged at 5,000 rpm until all material on swab is dissolved fully in peptone water.
5
Enterococcal Isolation from Pig Farms
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The large-scale swine farm (approximately 1,000 breeding pigs and 10,000 fattening pigs) is located in Sichuan, one of China’s major pig raising provinces. In October and November 2021, Fresh fecal swabs were taken from 150 breeding pigs in the breeding pig section of the farm and from 450 fattening pigs aged between approximately 2, 4, and 6 months (150 fattening pigs each stage) in the fattening pig section of the farm. The swab samples were inoculated into 3 ml of Buffer Peptone Water (Oxoid, Basingstoke, United Kingdom) containing 8 mg/l florfenicol. The culture was incubated overnight at 37°C and 180 RPM, and following incubation, 0.1 ml aliquots of culture were streaked onto Pfizer Enterococcus Selective Agar (Qingdao Hope Bio-Technology, China) supplemented with florfenicol (8 mg/l), and the selective plate was incubated overnight at 37°C. If the plate had colonies, we randomly selected one enterococcal colony from the plate. Then, we used an automated system (BD Diagnostic Systems, Sparks, MD, United States) to identify the enterococcal isolates.
6
Isolation and Identification of Bacteria from Brain Tissue
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Bacteria were isolated and identified, as previously described [25 ]. Briefly, pre-enrichment of brain tissue used Buffer Peptone Water (Oxoid®, UK) incubated at 37°C for 16-18 h. One-tenth mL of pre-enrichment medium was transferred to Modified Semisolid Rappaport-Vassiliadis medium (LabM®, UK) and incubated at 41.5°C for 24 h. Furthermore, 1 mL was transferred to MKTTn broth (LabM®, UK) and incubated aerobically for 24 h. The samples were then streaked onto XLD (LabM®, UK) and SS (Oxoid®, UK) agar plates and incubated at 37°C for 24 h, aerobically. Typical colonies were identified by biochemical tests (Urea agar, Triple sugar iron, and Lysin iron) (Oxoid®, UK).
7
Isolation and Identification of Salmonella
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Each cloacal swab was inoculated into 10 mL buffer peptone water (Oxoid) and incubated at 37C for 18 hrs. Then, subcultured into tubes containing 10 mL Rappaport-Vassiliadis soy broth (RVS) (Oxoid) and incubated at 41.5 C for 24 hrs. A loopful from inoculated RVS broth was streaked onto MacConkey agar (Oxoid); Xylose lysine-deoxycholate (XLD) agar (Oxoid) and incubated at 37 ⸰ C for 24 h under aerobic condition. A pure culture of suspected salmonella colonies was then obtained through three successive subcultures. Biochemical and morphological characterizations were applied to identify salmonella isolates according to procedures described by Macfadian [17] and Brenner [18] . Sub-culture of the pure isolates onto semi-solid nutrient agar slant for preservation and further identification were carried out according to Wilson and Miles [19] .
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