Samples were transported to the laboratory and stored at 4°C, and analysis was started within 24 h. Using sterile scissors and forceps, 25 g of each sample was dissected at various sites of the product and placed into a sterile Stomacher strainer bag (Seward Stomacher 400 Classic Strainer bag, Worthing, United Kingdom) with 225 ml of sterile buffered peptone water (Oxoid, Basingstoke, Hampshire, United Kingdom) and homogenized for 60 s (Stomacher 400 laboratory blender, Seward, United Kingdom). Samples were incubated at 37°C for 18–22 h. After incubation, a loopful (10 μl) of each enrichment was streaked onto two parallel MacConkey agar plates (Lab M, Lancashire, United Kingdom; Scharlau Chemie s.a, Sentmenat, Spain) with 1 mg/l cefotaxime. To improve the isolation of Enterobacteriaceae listed as the highest priority by the WHO (2017b) , one of the plates was incubated at 44°C and the other at 37°C for 18–22 h. One colony from each morphologically different bacterial growth from each plate was re-streaked onto individual MacConkey agar plates with 1 mg/l cefotaxime and incubated overnight at 37°C. Bacterial colonies were re-streaked onto individual MacConkey agar plates with cefotaxime supplement until a pure culture was achieved.
Laboratory blender stomacher 400
Manufactured by Seward Medical
Sourced in United Kingdom
The Laboratory Blender Stomacher 400 is a versatile lab equipment designed for the efficient homogenization and blending of various samples. It features a robust and durable construction, allowing for consistent and reliable performance in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy powerfood microbial kit, by Qiagen (2 mentions)
Sterile buffered peptone water, by Thermo Fisher Scientific (1 mentions)
Stomacher bag, by Seward Medical (1 mentions)
Sodium chloride (nacl), by Avantor (1 mentions)
Bacteriological peptone, by Thermo Fisher Scientific (1 mentions)
Deman rogosa sharpe mrs, by Thermo Fisher Scientific (1 mentions)
Palcam agar, by Thermo Fisher Scientific (1 mentions)
Fraiser broth, by Thermo Fisher Scientific (1 mentions)
9 protocols using laboratory blender stomacher 400
1
Isolating Antibiotic-Resistant Enterobacteriaceae
2
Antimicrobial Activity of ATCE on Cooked Pork
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Fresh lean pork was purchased from the local market and cut into cube blocks (approximately 2 × 2 × 2 cm3). Then the cube blocks were sterilization for 20 min at 121 °C to obtain the sterile lean cooked pork samples. The sterile samples were treated with 0, 1, and 2 MIC of ATCE for 30 min, respectively. Approximately 103 CFU/mL of S. aureus ATCC 13565 cells was inoculated into the samples treated with ATCE. The bacterial suspension was mixed with each sample for 15 s using a sterile applicator stick. Then, the inoculated samples were sealed and stored using aseptic bags at 4 °C for 6 days. On days 0, 3, and 6, the samples (10 g) were taken out in turn and mashed (Stomacher 400 Laboratory Blender, Seward, Worthington, UK) on medium power mode for 30 s in 0.1% sterile NS (90 mL). The suspension liquid samples were appropriately diluted by NS. Finally, the number of S. aureus ATCC 13565 on lean cooked pork was enumerated on Baird-Parker (BP, Difco Labs, Detroit, Michigan, USA) plates by spreading 100 μL of the sample dilution.
3
Enumeration of E. coli O157:H7 on Leaves
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At 24 or 48 hours post-inoculation (hpi), the four inoculated leaves per plant were excised and individually transferred to sterile sampling bags with mesh (Whirl-Pak, Nasco, Modesto, CA, USA) with 5 mL of sterile 0.1% PW. E. coli O157:H7 cells were recovered via either stomaching in a Stomacher 400 laboratory blender (Seward, Westbury, NY, USA) at 250 rpm for 5 minutes or by manual homogenizing to a watery paste with a pestle passed over the bag for 1 minute. The filtered wash or homogenate was serially diluted in 0.1% PW, drop-plated onto TSAR and incubated overnight at 37°C before counting. Microbial plate counts from sample homogenates were log transformed (log CFU/mL) and the microbial load per leaf (log CFU/leaf) calculated. Results from individual leaves on the same plant were averaged to generate a single biological replicate. The limit of detection in sample homogenates was 1.7 log CFU/mL, which equaled to 2.4 log CFU/leaf. All the results in this study were above the limit of detection.
4
Isolation of Lactobacillus from Cheese
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To obtain abundant NSLAB from these cheeses, 2 portions (about 10 g) per sample were collected using a sterile knife, specifically from the rind (sampled from the first 5 mm on each side of the cheese, including the edges) and the core (sampled from the center of cheese). The samples were then mixed for continuous microbiological analysis. Cheese samples were homogenized following the procedure described by Tsafrakidou et al. (2016) . Briefly, cheese samples were homogenized with 90 mL of sterile 20 g/L sodium citrate solution at 45°C in a Stomacher 400 laboratory blender (Seward, London, UK) for 4 min at maximum speed. Dilutions (1/10) of the homogenates were prepared with a sterile solution of 0.85% (wt/vol) sodium chloride and then plated on de Man, Rogosa, and Sharpe (MRS; pH 5.7) agar plates that were incubated under anaerobic conditions at 30°C for 5 d before presumptive lactobacilli examination. Fifteen colonies per sample were randomly picked from the MRS agar plates, totaling to 120 colonies. Twenty-five colonies, which were gram-positive, catalase-negative, and able to grow at 15 and 45°C, were selected for the next test. Finally, pure cultures were frozen (-80°C) in MRS broth containing 20% (vol/vol) glycerol for storage. Isolates were activated by successive transfer in their respective medium and incubation at 37°C for production.
5
Cheese Microbiome DNA Extraction
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A 25 g aliquot was sampled from each cheese core once and homogenized in 225 mL of buffered peptone water (LAB M, Bury, Lancashire, UK), using a Stomacher (Laboratory Blender Stomacher 400; Seward, London, UK) for 2 min at 260 rpm. Ten milliliters of the filtered homogenized sample were collected in a 15-mL conical centrifuge tube and high-quality total DNA was extracted using DNeasy PowerFood Microbial kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. DNA concentrations were measured using a fluorescence spectrometer (Qubit, Life Technologies, Carlsbad, CA, USA). The samples were stored at −20 °C until analysis.
6
Cheese Microbiome Sampling Protocol
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Ten-gram samples of cheese were collected from within a block of cheese at a depth of approximately 5 mm. The samples were aseptically weighed on one side of sterile filter stomacher bags (BioMérieux (UK) Ltd., Basingstoke, UK). Ninety milliliters of buffered peptone water were added, and the samples were homogenized in a stomacher (Laboratory Blender Stomacher 400; Seward, London, UK) for 2 min at 260 rpm. Ten milliliters of filtered homogenized sample were collected to a 15 mL conical centrifuge tube and high-quality metagenomic DNA was extracted using DNeasy PowerFood Microbial kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. DNA concentrations were measured using a fluorescence spectrometer (Qubit, Life Technologies, Carlsbad, CA, USA). The samples were stored at −20 °C.
7
Bacterial Enumeration in Fermented Meats
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For bacterial enumeration, 12 g of each fermented meat product were aseptically transferred into a stomacher bag (Seward, Worthing, West Sussex, United Kingdom) and mixed with 108 ml of recovery diluents [sterile solution of 0.85% (m/v) NaCl (VWR International, Darmstadt, Germany) and 0.1% (m/v) bacteriological peptone (Oxoid, Basingstoke, Hampshire, United Kingdom)]. The mixture was made at maximum speed for 2.5 min in a Laboratory Blender Stomacher 400 (Seward). Appropriate serial decimal dilutions in saline were prepared. Subsequently, these dilutions were spread on mannitol-salt-phenol-red agar (MSA; VWR International) and de Man-Rogosa-Sharpe (MRS) agar (Oxoid) for enumeration of presumptive CNS and LAB, respectively, after which the agar media were incubated at 30°C for 72 h. Thereafter, agar media containing 30–300 colonies were used for determining the bacterial counts and 5–30% of the colonies present were randomly selected and picked up to follow the bacterial community dynamics. The colonies picked up from the MSA and MRS agar media were transferred into brain heart infusion (BHI; Oxoid) medium and incubated overnight at 30°C to acquire grown cultures to be used for DNA extraction as well as storage at −80°C in cryovials containing 25% (v/v) of glycerol.
8
Listeria spp. Detection Protocol
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The ISO (International Organization for Standardization) 11290-1/A1-2004 method was used for Listeria spp. detection following the stated protocol ISO (2004) . Samples were weighed as 25 gr each with a precision balance (Cubis; Sartorius, Bohemia, NY, USA) under sterile conditions and the samples were transferred to sterile stomacher bags with an addition of 225 ml of Enrichment Broth (M863+SR142; Oxoid Ltd, Basingstoke, UK). Samples were homogenized in a stomacher (Laboratory Blender Stomacher 400; Seward, London, UK) for 2 minutes and incubated in a 30°C aerobic environment for 24 hours. Afterwards, 0.1 mL of the homogenates were added to 10 ml of a Fraiser Broth (CM895+SR156; Oxoid Ltd, Basingstoke, UK). Following incubation at 30°C for 24 hours, 0.1 mL of homogenate was taken from the Fraiser Broth and were cultivated into PALCAM Agar (CM 877+SR150; Oxoid Ltd, Basingstoke, UK) and Oxford Agar'a (CM 856+SR140; Oxoid Ltd, Basingstoke, UK). Mediums were incubated at 30°C under anaerobic conditions for 48 hours. Five colonies from any petri with a suspicion of Listeria spp. growth were transferred to Tryptic Soy Agar-Yeast Extract (TSA-YE, 0370; Difco, Toronto, ON) and incubated at 30°C for 24 hours for purification.
9
DNA Extraction from Cheese Samples
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The 25 gr each cheese samples were transferred to sterile stomacher bags with 0.5 mL DNase & RNase free water. Samples were homogenized in a stomacher (Laboratory Blender Stomacher 400; Seward, London, UK) for 2 minutes and 200 µL of aqueous phase were transferred to a new 1.5 mL sterile micro centrifuge tube. After that, 400 mL of lysis buffer (0.5% N-lauryl sarcosine, 50 Mm Tris-Cl, 25 mM EDTA, pH 8.0) was added to the mixture, vortexed well and centrifuged 15.000 rpm for 5 min. The pellet was resuspended with 200 µL lysis buffer and 4 µL proteinase K, vortexed well and then incubated for 1 h at 37 o C, 300 µL of NaI solution (6 M NaI in 50 mM Tris-Cl, 25 mM EDTA, pH 8.0) and 500 µL isopropanol were added to the suspension and then centrifuged at 15.000 rpm for 5 min. The pellet was washed with 35% isopropanol, dried and then suspended in 50 µL DNase & RNase free sterile water. Isolated DNA samples were stored at -20°C until the real time PCR application (Sheela and Shrinithivihahshini, 2017; Moravkova, 2017) .
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