After the treatment, the samples of chicken skin or stainless steel and their respective controls were placed in a cold storage room (4°C). In the cold room, the samples were placed in 15 × 80 cm stomacher bags containing 20 ml of PBS. The samples were gently massaged by hand and homogenized for 1 min at level 3 (MiniMix, INTERSCIENCE, Wiesbaden, Germany). Thereafter, the samples were taken to the laboratory in an ice-bath box and diluted to a 10−3 dilution level. All dilutions and the undiluted sample were plated on XLD plates (Xylose Lysine Deoxycholate agar, Oxoid Deutschland GmbH, Wesel, Germany).
Minimix
Manufactured by Interscience
Sourced in France
The MiniMix is a compact laboratory mixer designed for gentle blending and homogenization of small sample volumes. It features a variable speed control and a compact design for convenient benchtop use.
Automatically generated - may contain errors
Lab products found in correlation
Oxalic acid, by Merck Group (3 mentions)
Stonebrink, by BD (2 mentions)
L wenstein jensen, by BD (2 mentions)
Bagpage, by Interscience (1 mentions)
Genolyse isolation kit, by Hain Lifescience (1 mentions)
Genotype mtbc assay, by Hain Lifescience (1 mentions)
Novobiocin, by Thermo Fisher Scientific (1 mentions)
Modified tryptone soy broth, by Thermo Fisher Scientific (1 mentions)
Ct smac, by Thermo Fisher Scientific (1 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (1 mentions)
8 protocols using minimix
1
Microbial Analysis of Chicken Skin and Stainless Steel
2
Lymphatic Tissue Isolation and Processing
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After thawing, the lymphatic tissue was isolated by removing surrounding tissues and the lymph node capsule by using sterile scissors. The lymphatic tissue from each individual was then divided into two parts, from which one was homogenized with physiological solution (for conventional bacteriological study) and second with 5% oxalic acid (Sigma-Aldrich, St. Luis, MO, USA) (for mycobacterial culture) in a stomacher apparatus (MiniMix, Interscience, France) for 3 min at a frequency of 12 beats/sec in bags with side filtering membrane. The resulting suspension was poured into 50 mL tubes and subjected to accordingly conventional bacteriological examination or mycobacterial culture.
3
Oyster Tissue Homogenization for OsHV-1 Detection
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The primary inoculae were freshly prepared, clarified, and filtered homogenates of mantle and gill tissue. Oysters were thawed at 4 °C, opened by removing the superior valve and the excised gill and mantle tissues were weighed and placed into a stomaching bag with 10× volume of sterile ASW. Tissues were homogenized in a stomaching machine (MiniMix, Interscience, France) at maximum speed for 1 min. The homogenate was then transferred into 50 mL sterile tubes and centrifuged at 1000× g for 10 min at 4 °C (Beckman Coulter, Brea, CA, USA). The supernatant was diluted with three volumes of ASW and filtered successively through 5 µm, 0.8 µm, 0.45 µm filters and twice through a 0.22 µm syringe filter (Sartorius, Göttingen, Germany). The clarified tissue homogenate was then stored briefly at 4 °C awaiting quantification of OsHV-1 DNA by real-time quantitative PCR (qPCR) prior to use. Tissues were processed in a class 2 biosafety cabinet with cross contamination between specimens and control groups prevented by use of autoclaved or sterile disposable equipment and surface disinfection using 1% Virkon-S for 15 min.
4
Oyster Tissue Homogenization and Bacterial DNA Isolation
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Each sample was prepared from soft tissues remaining after the gill and mantle biopsy and after removal of the digestive gland. The tissues were transferred into a pre-weighed stomacher bag (Interscience, France), weighed and disrupted by stomaching with 4× (w/v) sterile ASW, in a bag mixer (MiniMix, Interscience, France) for 1 min, at maximum (9) speed. On Day 0, individual oysters were homogenized and thereafter, tissues from 4 oysters from each treatment and the same batch were pooled and homogenized. A coarsely clarified homogenate (5 ml) was obtained by collecting the material filtered through the inner mesh (porosity <250 μm) of the stomaching bag (BagPage®, Interscience, France). These homogenates were used directly for bacterial culture and stored at -80 °C for molecular studies of bacterial content.
Preparation of homogenates for bacterial molecular studies was by bead beating as previously described, with 150 μl of homogenates, 390 μl lysis/binding solution (MagMax™ - 96 Viral RNA Isolation Kit) in a 2 ml microcentrifuge tube containing 0.4 g of silica-zirconia beads. Homogenates were centrifuged at 900 g for 10 min and 180 μl of the supernatant was used for the nucleic acid purification with the MagMax™-96 Viral Isolation Kit. Nucleic acids were stored at -20 °C until bacterial DNA quantification.
Preparation of homogenates for bacterial molecular studies was by bead beating as previously described, with 150 μl of homogenates, 390 μl lysis/binding solution (MagMax™ - 96 Viral RNA Isolation Kit) in a 2 ml microcentrifuge tube containing 0.4 g of silica-zirconia beads. Homogenates were centrifuged at 900 g for 10 min and 180 μl of the supernatant was used for the nucleic acid purification with the MagMax™-96 Viral Isolation Kit. Nucleic acids were stored at -20 °C until bacterial DNA quantification.
5
Mycobacterium Culture and Genotyping Protocol
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The culture was performed according to the standard procedure used at the National Veterinary Research Institute (NVRI) in Pulawy (Poland). Briefly, the collected material was decontaminated with a 5% solution of oxalic acid (Sigma-Aldrich, Saint Louis, MO, USA). Homogenization was performed in a stomacher (MiniMix, Interscience, France) for three minutes (1500 g, 10 min). The supernatant was removed and the pellet was washed twice with sterile physiological sodium chloride solution (Polfa, Lublin, Poland).
The obtained pellet was seeded on two solid media: Stonebrink (Becton Dickinson, Franklin Lakes, NJ, USA) and Löwenstein–Jensen (Becton Dickinson, Franklin Lakes, NJ, USA). The media were incubated at 37 °C. Growth was assessed every seven days for 12 weeks.
The DNA was isolated from the cultures and the genotype determined as described previously [1 (link)]. Briefly, DNA isolation was performed using a Genolyse isolation kit (Hain Lifescience, Nehren, Germany). Strains were determined using the GenoType®MTBCassay (Hain Lifescience, Nehren, Germany).
The obtained pellet was seeded on two solid media: Stonebrink (Becton Dickinson, Franklin Lakes, NJ, USA) and Löwenstein–Jensen (Becton Dickinson, Franklin Lakes, NJ, USA). The media were incubated at 37 °C. Growth was assessed every seven days for 12 weeks.
The DNA was isolated from the cultures and the genotype determined as described previously [1 (link)]. Briefly, DNA isolation was performed using a Genolyse isolation kit (Hain Lifescience, Nehren, Germany). Strains were determined using the GenoType®MTBCassay (Hain Lifescience, Nehren, Germany).
6
MTBC Diagnostic Culture Protocol
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MTBC diagnostic culture was carried out as described previously (Didkowska et al. 2020) . Briefly, the clinical material was decontaminated with a 5% solution/.n of oxalic acid (Sigma-Aldrich, St. Louis, MO, USA) and then homogenized in a stomacher (MiniMix, Interscience, France). After centrifugation (1500 g, 10 minutes), the supernatant was removed and the pellet washed twice with sterile physiological sodium chloride solution (Polfa Lublin, Poland). The obtained pellet was placed on two solid media: Stonebrink (Becton Dickinson, Holdrege, NE, USA) and Löwenstein--Jensen (Becton Dickinson, Holdrege, NE, USA). Both media were incubated at 37°C for 12 weeks and assessed every seven days.
7
Detecting E. coli O157 in Food Samples
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Upon processing, 25 ml of maximum recovery diluent (MRD, Oxoid, UK) was added to each RAMS and homogenized for 1 min with a stomacher blender (Minimix, Interscience).
For E. coli O157 detection, 10 ml thereof was added to an equal volume of doubleconcentrated modified tryptone soy broth (Oxoid, UK), containing 20 mg/ml of novobiocin (Oxoid, UK) (mTSBn), and subsequently incubated for 6 h at 37°C (Cobbaut et al., 2008) .
Immuno-magnetic separation (IMS) was performed on 1 ml of enrichment broth, using 20 l of anti E. coli O157 Dynabeads (Invitrogen, US), according to the manufacturer's recommendations.
The washed beads were then spread-plated onto sorbitol-MacConkey agar supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC) (Oxoid, UK), and incubated for 24 h at 37C. Following incubation, up to 4 non-sorbitol fermenting (NSF) colonies per plate
For E. coli O157 detection, 10 ml thereof was added to an equal volume of doubleconcentrated modified tryptone soy broth (Oxoid, UK), containing 20 mg/ml of novobiocin (Oxoid, UK) (mTSBn), and subsequently incubated for 6 h at 37°C (Cobbaut et al., 2008) .
Immuno-magnetic separation (IMS) was performed on 1 ml of enrichment broth, using 20 l of anti E. coli O157 Dynabeads (Invitrogen, US), according to the manufacturer's recommendations.
The washed beads were then spread-plated onto sorbitol-MacConkey agar supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC) (Oxoid, UK), and incubated for 24 h at 37C. Following incubation, up to 4 non-sorbitol fermenting (NSF) colonies per plate
8
Pathogen Recovery from Pear Wedges
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Recovery of pathogen populations were performed at day 0 and after 3 and 8 days of storage under the two conditions. Before opening the trays, the headspace gas composition was determined using a handheld gas analyser (CheckPoint O2/CO2, PBI Dansensor, Denmark).
To recover the pathogens from the wedges, a plug (1.2 cm in diameter and 1 cm deep, approximately 1 g plug -1 ) containing the entire well was removed using a sterile cork borer. One plug per repetition was placed in a sterile filter bag (80 mL, BagPage®, Interscience BagSystem, Saint Nom, France) and diluted with 9 mL of buffered peptone water (BPW, Oxoid). The mixture was homogenized in paddle blender for 2 min at high speed (MiniMix, Interscience, France), and aliquots of the mixture were then serially diluted in SP and plated on XLD for enumerating S. enterica or on Palcam agar for L. monocytogenes. The agar plates were incubated at 37 ± 1 °C for 24 h (S. enterica) or 48 h (L. monocytogenes). The data were transformed to CFU g -1 pear. Three determinations per pathogen were made at each sampling point in duplicate.
To recover the pathogens from the wedges, a plug (1.2 cm in diameter and 1 cm deep, approximately 1 g plug -1 ) containing the entire well was removed using a sterile cork borer. One plug per repetition was placed in a sterile filter bag (80 mL, BagPage®, Interscience BagSystem, Saint Nom, France) and diluted with 9 mL of buffered peptone water (BPW, Oxoid). The mixture was homogenized in paddle blender for 2 min at high speed (MiniMix, Interscience, France), and aliquots of the mixture were then serially diluted in SP and plated on XLD for enumerating S. enterica or on Palcam agar for L. monocytogenes. The agar plates were incubated at 37 ± 1 °C for 24 h (S. enterica) or 48 h (L. monocytogenes). The data were transformed to CFU g -1 pear. Three determinations per pathogen were made at each sampling point in duplicate.
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