All (fluoro)quinolone-resistant isolates (n = 99) were tested for moxifloxacin susceptibility. Briefly, isolates were recovered from −80 °C on CBA (Fannin Ltd) for 24 h at 42 °C under microaerobic conditions, and subcultured to CBA for 20 ± 2 h at 42 °C under microaerobic conditions. In microtiter plates, 100 µL serial dilutions of moxifloxacin (Sigma Aldrich) were prepared in of Mueller Hinton broth with lysed horse blood (Thermo Fisher Scientific, Waltham, MA, USA) ranging from 0.125–16 mg/L. A 0.5 McFarland inoculum was prepared in 5 mL demineralised water (Thermo Fisher Scientific) and 100 µL was transferred to 11 mL of Mueller Hinton broth with lysed horse blood (Thermo Fisher Scientific). moxifloxacin serial dilutions were inoculated with 100 µL of cell suspension and incubated for 20 ± 2 hat 42 °C under microaerobic conditions.
Lysed horse blood
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Lysed horse blood is a laboratory product that consists of red blood cells derived from horses that have been broken down or lysed. This product is intended for use in various in vitro diagnostic and research applications.
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Lab products found in correlation
Modified charcoal cefoperazone deoxycholate agar, by Thermo Fisher Scientific (1 mentions)
Nutrient broth no 2, by Thermo Fisher Scientific (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Genbox microaer, by bioMérieux (1 mentions)
Genbox jar, by bioMérieux (1 mentions)
Buffered peptone water (bpw), by Lab M (1 mentions)
Vancomycin, by Merck Group (1 mentions)
Trimethoprim, by Merck Group (1 mentions)
Nalidixic acid, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Erythromycin, by HiMedia (1 mentions)
Ciprofloxacin, by HiMedia (1 mentions)
Ampicillin, by HiMedia (1 mentions)
Gentamicin, by HiMedia (1 mentions)
Imipenem, by HiMedia (1 mentions)
Sr0155, by Thermo Fisher Scientific (1 mentions)
Brucella broth, by Thermo Fisher Scientific (1 mentions)
Mccda, by Thermo Fisher Scientific (1 mentions)
Cycloserine, by Thermo Fisher Scientific (1 mentions)
14 protocols using lysed horse blood
1
Moxifloxacin Susceptibility in Quinolone-Resistant Isolates
2
Isolation and Cultivation of H. pylori
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This study was approved by the University of Malaya Medical Centre (UMMC) Medical Ethics Committee and biopsy samples for culturing were obtained with informed and written consent from consecutive and non-repetitive patients who presented for endoscopy at UMMC. Gastric biopsy samples obtained from antrum and body of the stomach were homogenized in different tubes and plated directly onto non-selective and selective chocolate agar supplemented with 7% lysed horse blood (Oxoid, UK). Selective chocolate agar contained vancomycin (10 µg/ml), amphotericin B (5 µg/ml), trimethoprim (5 µg/ml) and nalidixic acid (20 µg/ml) [31] . The inoculated agar plates were incubated for 3–10 days in a humidified 10% CO2 incubator at 37°C. H. pylori were successfully cultured from 110 patients and were confirmed by positivity for urease test. Amongst these, 102 were naive to standard H. pylori therapy whilst the remaining eight were from patients with treatment failure. Clonal strains derived from well-isolated H. pylori colonies of the same patient were tested for homogeneity by random amplification of polymorphic DNA (RAPD) and representative clonal strains were used for this study.
3
Gastric Biopsy Culturing Protocol
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Gastric biopsy samples obtained from the antrum of the stomach were homogenized and plated onto non-selective and selective chocolate agar supplemented with 7% lysed horse blood (Oxoid, Hampshire, UK). Selective chocolate agar contained vancomycin (10 μg/ml), amphotericin B (5 μg/ml), trimethoprim (5 μg/ml) and nalidixic acid (20 μg/ml) [25 ]. The inoculated agar plates were incubated for 3–10 days in a humidified 10% CO2 incubator at 37°C.
4
Campylobacter coli Cultivation Protocol
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On each experimental occasion, C. coli ATCC® 43478™ (LGC Standards GmbH, Wesel, Germany), previously stored at −80°C in nutrient broth No. 2 (Oxoid, Basingstoke, England) supplemented with 5% lysed horse blood (Oxoid) and 20% glycerol (BDH VWR, Lutterworth, England), was recovered on Columbia blood agar (CBA; bioMérieux, Marcy-l’Étoile, France), then isolated on modified charcoal cefoperazone deoxycholate agar (CCDA; Oxoid) and subcultured once more on CBA. Plate incubation was always performed at 41.5 ± 1°C for 24 h under microaerobic conditions (GENbox microaer and GENbox jar, bioMérieux). For the generation of pure C. coli suspensions, one pure colony from CBA was inoculated in 100 ml of cation-adjusted Mueller-Hinton broth (CAMHB; BBL™ BD, Franklin Lakes, NJ) following incubation at 37 ± 1°C for 20 ± 2 h under microaerobic conditions. The growth state of the bacterial culture was approximately estimated at the end of the incubation by measuring the optical density (OD) at 600 nm using a spectrophotometer (BioPhotometer®; Eppendorf, Hamburg, Germany). The estimated count was confirmed by subjecting three aliquots of 100 ml to qPCR, as described below, and by performing 10-fold serial dilutions in buffered peptone water (BPW, LabM, United Kingdom) followed by spread plating on double CBA plates.
5
Gastric Biopsy for H. pylori Detection
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Tissue biopsies from both gastric antrum and corpus were extracted during routine gastroscopy for rapid urease test (RUT), culture for H. pylori and histopathological investigations. Gastric biopsies for H. pylori culture were homogenized in different tubes and plated directly on non-selective and selective chocolate agar supplemented with 7% lysed horse blood (Oxoid, UK), and contained vancomycin (10μg/ml), amphotericin B (5μg/ml), trimethoprim (5μg/mL) and nalidixic acid (20μg/mL) (Sigma, USA). The inoculated plates were incubated for 3–10 days in a humidified 10% CO2 incubator at 37°C.
6
Antibiotic Resistance Evaluation in Campylobacter
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The disc diffusion method was used for evaluating the bacterial resistance to antibiotics. An overnight bacterial culture of the isolates in Mueller-Hinton (MH) broth supplemented with sodium pyruvate, sodium metabisulphite, and ferrous sulphate (Oxoid, UK) was used. The culture density was estimated at 0.5 McFarland (absorbance at 600 nm is 0.063), then a swab was streaked onto MH agar supplemented with 5% lysed horse blood (Oxoid, UK). Plates were allowed to dry before distributing the discs containing the different antibiotics on the agar′s surface [34 ]. The antibiotics were gentamicin, imipenem, ampicillin, at 10 μg, aztreonam and tetracycline at 30 μg, erythromycin (15 μg), and ciprofloxacin (5 μg) (Himedia, India). Plates were incubated at 42 °C for 48 h, then the diameter of the inhibition zone was measured in millimeters (mm) using a digital caliper. Proposed zone diameters for defining the breakpoints cut-off for Campylobacter were determined according to the breakpoints proposed by the European Committee for Antimicrobial Susceptibility Testing [35 ].
7
Campylobacter Isolation from Greek Pigs
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A total of 200 Campylobacter isolates, originating from 16 commercial pig farms from six different Greek regions were examined. The median size of the farms was 550 breeding sows (230–1600). Among these isolates, 100 have been previously identified as C. jejuni and 100 as C. coli. Speciation of Campylobacter isolates has been made according to a multiplex PCR protocol [20 ]. All samples were cryopreserved at −70 °C, in Brucella broth (OXOID, UK) supplemented with 5% lysed horse blood (OXOID, UK) and 15% glycerol.
The cryopreserved Campylobacter samples were defrosted, revived, and inoculated on to modified charcoal cefoperazone deoxycholate agar (mCCDA-OXOID, UK) with the selective supplement SR0155 (OXOID, UK). The inoculated plates were incubated under microaerobic conditions (85% N2, 10% CO2, and 5% O2) at 41.5 °C for 48 h.
The cryopreserved Campylobacter samples were defrosted, revived, and inoculated on to modified charcoal cefoperazone deoxycholate agar (mCCDA-OXOID, UK) with the selective supplement SR0155 (OXOID, UK). The inoculated plates were incubated under microaerobic conditions (85% N2, 10% CO2, and 5% O2) at 41.5 °C for 48 h.
8
Disinfection of Hospital Surfaces
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All sites used chlorine-based cleaning products for the disinfection of hard surfaces and floors, and time (hours) since cleaning was recorded when samples were collected. Rooms at all sites were cleaned at least once daily. Sponge-sticks (3M, Saint Paul, Minnesota), moistened with sterile water, were used to sample a 5 × 20-cm area of flat surface, or the entire surface of the call bells, or a 13.3-cm length of bed rails (representing the same surface area). After transport to Leeds, swabs were placed in 50 mL neutralizing solution (0.1% [wt/vol] sodium thiosulfate, 3% [wt/vol] Tween80, 0.3% [wt/vol] lecithin in phosphate-buffered saline) and homogenized for 10 minutes before the solution was pulled through Microfil V 0.22-µM filters with 100-mL funnel onto the integral membrane using a gantry and pump assembly. Membranes were placed onto Brazier’s agar (Oxoid, UK) supplemented with 250 mg/L cycloserine and 8 mg/L cefoxitin (Oxoid) and 2% lysed horse blood (Oxoid) (CCEY agar). All plates were incubated anaerobically (A95 workstation; Don Whitley, Bradford, UK) for 48 hours before enumeration of typical colonies (gray-brown, irregular edge, typical odor). Atypical colonies were identified using matrix-assisted lazer desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF; Bruker, Billerica. MA).
9
Campylobacter Surveillance and Susceptibility Testing
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As part of routine Campylobacter surveillance in Spain, isolates were sampled and cultured on blood agar plates (bioMérieux) and incubated for 48 h at 37°C under microaerophilic conditions using Campygen atmosphere generation system packs (Oxoid, Basingstoke, U.K.). Subcultured colonies were harvested and suspended in sterile water to a standardized cell density (0.5 McFarland turbidity). Fifty microliters of this suspension was added to 11 ml of Mueller‐Hinton broth (TREK Diagnostics Systems, Waltham, MA) supplemented with 5.5% lysed horse blood (Oxoid). The solution was poured onto EUCAMP2 microdilution plates (TREK Diagnostics Systems) which were incubated under microaerophilic conditions for 48 h at 37°C as previously described (Florez‐Cuadrado et al., 2017 ). The interpretation of the quantitative data was performed according to the European Committee of Antimicrobial Susceptibility Testing, EUCAST (http://www.eucast.org/ ; last accessed: 06/2017).
10
Biofilm Formation Assay for Bacterial Isolates
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All cultured bacteria were isolated and tested for their ability to form biofilms. The biofilms biomass metrics procedure was held at the Department of Biopathology and Clinical Microbiology of the “Aeginiteion” Hospital, Athens Medical School. Silicone squares of the same size (5 mm) were UV sterilized for 10 min and placed in Falcon tubes under aseptic conditions in a biological safety cabinet class IIB [42 (link)]. All bacterial strains isolated from the clinical samples by standard culture techniques were prepared in suspensions; S. pneumoniae, Streptococcus spp., S. aureus, and M. catarrhalis, were inoculated in 3 mL of Mueller-Hinton broth (MHB) (Scharlau™, Barcelona, Spain), while H. influenzae was inoculated in 3 mL of Brain-Heart infusion (BHI) broth (Scharlau™, Barcelona, Spain) supplemented with 5% lysed horse blood (Oxoid Ltd., Basingstoke, UK). Bacterial suspensions were incubated at 37 °C for 48 h at 150 rpm agitation to promote biofilm formation. After the incubation, the broth was removed with the aid of a vacuum pump device. The silicone discs were rinsed thrice with distilled water so that the non-adherent cells could be removed and left overnight to dry in a laminar flow hood.
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