Bacteria were grown in sterile Todd-Hewitt broth (THB, Hardy Diagnostics, Santa Maria, CA, United States). WT + pDCerm, Δess + pDCerm, and Δess + pDCerm:ess GBS strains were grown in THB supplemented with 5 μg/mL erythromycin (Thermo Fisher Scientific, Waltham, MA, United States) or Tryptic Soy Agar (TSA) with 5% sheep’s blood (BD BBL, Franklin Lakes, NJ, United States).
Sheep s blood
Manufactured by BD
Sourced in United States
Sheep's blood is a laboratory material derived from sheep. It is used as a culture medium or component in various microbiological and diagnostic laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Trypticase soy agar, by BD (2 mentions)
Erythromycin, by Thermo Fisher Scientific (1 mentions)
Columbia agar, by BD (1 mentions)
Macfarland densimat, by bioMérieux (1 mentions)
E test strip, by bioMérieux (1 mentions)
Campygen, by Thermo Fisher Scientific (1 mentions)
Mccda, by Thermo Fisher Scientific (1 mentions)
Vitek 2, by bioMérieux (1 mentions)
9 protocols using sheep s blood
1
Growth of Group B Streptococcus Strains
2
Detailed Cultivation of Vibrio cholerae and Streptococcus pneumoniae
3
Bacterial Growth Assessment Protocol
4
Culturing and Characterizing H. pylori Strains
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Human GECs N87 and AGS were obtained from the American Type Culture Collection (ATCC) and the GEC line HGC-27 was obtained from RIKEN, The Institute of Physical and Chemical Research, Japan. These cell lines were maintained in RPMI 1640 with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. Immortomouse stomach epithelium (ImSt) cells were derived from C57/Bl6 and were maintained in media as described by Whitehead et al. [26 (link)]. H. pylori strains 51B and 26695 as well as their corresponding isogenic cagA and cag PAI mutants were described previously [27 (link),28 (link)]. H. pylori strains were grown on tryptic soy agar (TSA) plates supplemented with 5% sheep’s blood (Becton Dickinson, San Jose, CA) or on blood agar plates with 2.5 μg/ml of chloramphenicol (Technova, Hollister, CA) to maintain cagA- [28 (link)] and cag PAI- strains at 37°C under microaerophilic conditions. H. pylori strain Sydney strain 1 (SS1) and PM-SS1 (pre-mouse SS1) [29 (link)] were used to infect mice. These strains were provided by Drs. J. Pappo (Astra) and Richard Peek (Vanderbilt Univ.), respectively.
5
Antimicrobial Susceptibility Testing
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Susceptibility to penicillin, erythromycin, and tetracycline was determined using E-test strips (AB Biodisk, Solna, Sweden) graduated from 0.016 to 256 μg mL−1. Bacterial suspensions were prepared in saline from overnight cultures on blood agar to a density of 0.5 McFarland Standard. A swab from this suspension was used to inoculate Mueller-Hinton plates supplemented with 5% sheep’s blood (Becton Dickinson, Cockeysville, MD). E-strips were then placed onto the plates whereupon they were incubated at 35°C in 5% CO2 for 24 h. Minimal inhibitory concentrations (MICs) were read as the intersection between the ellipse edge and strip. Intermediate E-test MICs were adjusted to the next highest doubling-dilution value.
6
Aerobic Bacterial Viability Assay
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Presence of viable bacteria was assessed for one sample collected from several sampled locations. Samples were thawed and 5 mL of each were placed into sterile 50-mL conical tubes. Immediately after, 45 mL of PBS was added before being vortexed. After mixing, 0.1 mL was removed and serially diluted in PBS. Each dilution was then plated on trypticase soy agar with 5% sheep’s blood (Becton Dickson, Franklin Lakes, NJ) and incubated at 37 °C for 24 h before counting colony forming units (CFUs) to determine total aerobic bacterial counts.
7
Campylobacter Detection in Human Feces
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A total of 681 human faecal samples originating from 269 households were cultured at NIPH in Cambodia. In addition, 200 randomly selected duplicate samples were cultured at the Clinical Microbiology Laboratory at UU. A diagram of the analytical procedure used for detection of Campylobacter in faecal samples from humans is shown in Fig. 2A. Samples were thawed and gently vortexed before faecal material was spread on agar plates. Culturing at NIPH was performed on Campylobacter agar base with antimicrobic supplement Blaser and 10% sheep's blood (Becton, Dickinson and Company, Sparks, MD, USA). The plates were incubated in a microaerobic atmosphere (GasPak EZ Gas Generating Pouch Systems; Becton, Dickinson and Company) at 42 °C for 48 h. Campylobacter jejuni type strain (ATCC 81146) and C. coli type strain (ATCC 43474) were used as controls. Culture at UU was performed on modified charcoal-cefoperazone-deoxycholate agar plates (mCCDA) (Oxoid, Basingstoke, UK). The plates were incubated in a microaerobic atmosphere (CampyGen; Oxoid) at 42 °C for 48 h. The type strain C. jejuni (81-176) was used as a control. Identification of Campylobacter was based on typical microscopical appearance (curviform bacteria exhibiting spin movement) and positive reactions to catalase and oxidase.
8
Antibacterial Susceptibility of S. aureus
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Seven S. aureus strains were used in this study. Further details are shown in Table 1 . Prior to use, each strain, kept at −80 °C, was transferred onto Tryptic Soy Agar (TSA) with 5% sheep’s blood (BD, Breda, the Netherlands) plate, grown overnight and inoculated into MH broth at 37 °C with agitation (150 rpm). The antibacterial susceptibility of the strains was tested using a Vitek® II (bioMérieux); the antimicrobial susceptibility test was analyzed according to the CLSI guidelines32 , 57 .
9
Quantifying Biofilm Bacteria on Catheters
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96-well microtiter plates containing biofilms were prepared as described above. After the 24-h incubation, the culture supernatant from each well was gently harvested. Biofilms were then washed with 200 μL of PBS with 0.1% Triton X-100 and repeatedly pipetted to disperse biofilm. This low amount of surfactant was verified to have no effect on bacterial viability (data not shown). The dispersed biofilm and harvested supernatant were each serially diluted in PBS and plated on trypticase soy agar containing 5% sheep’s blood (BD and Co.) for incubation, 24 h at 37 °C. After incubation, colonies on each plate were counted to allow CFU quantification. Dilutions containing 20–200 colonies were used unless near the limit of quantitation. Catheter segments were incubated as described above and then transferred to tubes containing 1 mL of PBS with 0.1% Triton X-100 for dispersion by sonication. Dispersed bacteria were plated and enumerated as described.
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