For broth inoculation, the above procedure for the preparation of inocula for caeca was followed with just a dilution difference; the broths were diluted five times to 10−5 in 9 ml MRD and then transferred to 99 ml of eitherBolton broth (CM0983, Oxoid, Cambridge, UK) or brain heart infusion broth (CM1135B, Oxoid, Cambridge, UK) to provide an inoculum with approximately 1 log10 CFU/ml. Plate counts were performed to confirm this concentration.
Cm1135b
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The CM1135B is a laboratory centrifuge designed for general purpose applications. It has a maximum speed of 6,000 rpm and can accommodate a variety of sample tubes and bottles. The centrifuge is equipped with a digital display and controls for adjusting speed and time settings.
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Lab products found in correlation
5 protocols using cm1135b
1
Inoculum Preparation for Broths
2
Anaerobic Microbial Susceptibility Assay
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The MIC assay for anaerobic growth conditions was performed to the same procedure as the standard aerobic MIC assay described above with the following exceptions:
All steps were performed in a COY type B anaerobic chamber with the anaerobic atmosphere controlled by the introduction of 10%CO2/5% H2 in N2CoA gas mix, catalyst Stak-Pak and O2-H2 gas analyzer, with H2 levels kept at ~2% for the duration of the assay. Brain Heart Infusion (BHI) (OXOID CM1135B) media with 1% cysteine to further promote an anaerobic environment was used in replacement of MHB, and this broth was incubated in the anaerobic chamber for 24 h prior to use to allow sufficient atmosphere exchange. All the plates were covered and incubated at 37 °C for 48 h. MICs were determined as the lowest concentration showing no visible growth by eye.
All steps were performed in a COY type B anaerobic chamber with the anaerobic atmosphere controlled by the introduction of 10%CO2/5% H2 in N2CoA gas mix, catalyst Stak-Pak and O2-H2 gas analyzer, with H2 levels kept at ~2% for the duration of the assay. Brain Heart Infusion (BHI) (OXOID CM1135B) media with 1% cysteine to further promote an anaerobic environment was used in replacement of MHB, and this broth was incubated in the anaerobic chamber for 24 h prior to use to allow sufficient atmosphere exchange. All the plates were covered and incubated at 37 °C for 48 h. MICs were determined as the lowest concentration showing no visible growth by eye.
3
Pneumococcal Strain Culture and Antibiotic Use
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The strains used in this study are listed in SI Appendix, Table S1 . Clinical isolates of S. pneumoniae expressing 84 CPS types were collected from hospitals and research institutes (69 (link)). Unless otherwise specified, S. pneumoniae was grown in brain heart infusion broth (BHI; ThermoFisher Scientific CM1135B) or on tryptic soy agar-II plates supplemented with 5% (v/v) sheep blood (blood agar; Biomed Diagnostics, 221261) at 37 °C in 5% CO2. Antibiotics were purchased from Sigma-Aldrich and used at final concentrations of 250 mg/mL for kanamycin (Kan) and 300 mg/mL for streptomycin.
4
Isolation and Preservation of H. pylori
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The biopsy specimens maintained in BHI broth (CM1135B, Thermo Fisher Scientific) were ground using a sterile glass rod and homogenized. Twenty microliters of this material was inoculated onto Columbia blood agar base (CM0331B, Thermo Fisher Scientific) enriched with 10% defibrinated horse blood and Dent supplement (SR0147E, Thermo Fisher Scientific). The inoculated plates were incubated at 37 °C in a microaerobic atmosphere (Anaerocult C, Merck Millipore, Darmstadt, Germany) for 7–10 days. At the end of the incubation period, the
H. pylori-suspect colonies (S type, gram-negative, and oxidase-, catalase-, and urease-positive) were assessed and their pure cultures were prepared [16,17]. The pure cultures of the isolates were stored in BHI broth supplemented with 15% glycerol at –84 °C for further use in molecular analyses and antibacterial susceptibility tests.
H. pylori-suspect colonies (S type, gram-negative, and oxidase-, catalase-, and urease-positive) were assessed and their pure cultures were prepared [16,17]. The pure cultures of the isolates were stored in BHI broth supplemented with 15% glycerol at –84 °C for further use in molecular analyses and antibacterial susceptibility tests.
5
Supernate Cytotoxicity Assay with Antibody Neutralization
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