Mouse cytomegalovirus (MCMV-K181) was obtained from C. Benedict (La Jolla Institute for Allergy & Immunology). Mice were infected intra-peritoneally with 1×105 PFU diluted in 300 ul PBS. S. paucimobilis was obtained from M. Kronenberg (La Jolla Institute for Allergy & Immunology). S. paucimobilis cultured in Tryptic Soy Broth (BD) at 37°C were collected at a mid-log phase and washed with PBS. Mice were inoculated intravenously with ~1×109 colony forming units diluted in 200 ul PBS. Streptococcus pneumoniae URF918 (clinical isolate, serotype 3) was obtained from M. Kronenberg (La Jolla Institute for Allergy & Immunology). S. pneumoniae cultured in Todd-Hewitt broth (BD) at 37°C were collected at a mid-log phase and then washed with PBS. Mice were inoculated intravenously with ~1×107 colony-forming units diluted in 200 ul PBS. Salmonella typhimurium (SL1344) was obtained from S. McSorley (University of California, Davis). S. typhimurium cultured in Todd-Hewitt broth (BD) at 37°C were collected at a mid-log phase and then washed with PBS. Mice were inoculated intravenously with ~1×106 colony-forming units diluted in 200 ul PBS. As a control for all infections, 2 ug of αGalCer diluted in 200 ul of PBS was injected intravenously, and spleen and liver were harvested 2-4 hours later.
Todd hewitt broth
Manufactured by BD
Sourced in United States, Japan, Germany, France, Canada
Todd-Hewitt broth is a microbiological culture medium used for the cultivation and isolation of streptococci and other fastidious organisms. It provides nutrients and growth factors essential for the development of these bacteria.
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Lab products found in correlation
Yeast extract, by BD (26 mentions)
Erythromycin, by Merck Group (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Sheep blood, by BD (6 mentions)
Pqe30, by Qiagen (5 mentions)
Spectinomycin, by Fujifilm (5 mentions)
Blood agar plate, by BD (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Sheep blood agar, by Hardy Diagnostics (4 mentions)
Tryptic soy broth (tsb), by BD (4 mentions)
Ec 14, by BD (3 mentions)
Ec 14, by Fujifilm (3 mentions)
Chloramphenicol, by Merck Group (3 mentions)
Ampicillin, by Fujifilm (3 mentions)
Trypticase soy agar, by BD (3 mentions)
Kanamycin, by Merck Group (3 mentions)
Bacto agar, by BD (3 mentions)
Miniphoto 518r, by TAITEC (2 mentions)
Luria bertani lb, by Merck Group (2 mentions)
Ampicillin, by Merck Group (2 mentions)
141 protocols using todd hewitt broth
1
Murine Infections with Multiple Pathogens
2
Growth and Transformation of S. pneumoniae
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Growth studies were conducted using S. pneumoniae strain TIGR4 and mutants generated in that background (Tettelin et al., 2001 (link)). Broth cultures were routinely grown at 37°C in Todd-Hewitt Broth (Becton Dickinson) supplemented with 0.2% (w/v) yeast extract (Becton Dickinson) (THY). C medium with 5% yeast extract (pH 8; C + Y) was used for transformations (Lacks and Hotchkiss, 1960 (link)). S. pneumoniae were also grown at 37°C and 5% CO2 on tryptic soy agar (TS; Becton Dickinson) spread with 5,000 U of catalase (Worthington Biochemical) and TS plates supplemented with 5% sheep blood (Becton Dickinson). Transformants were selected on TS plates containing streptomycin (200 μg/mL) or kanamycin (500 μg/mL).
3
Growth Conditions for Bacterial Strains
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The bacterial strains and plasmids used in this study are described in Table 1 . Escherichia coli was grown overnight at 37°C in Luria–Bertani broth (also known as LB broth, Difco) with gentle aeration, or on Luria-Bertani medium containing 1.5% w/v agar (LB agar). Kanamycin sulfate and ampicillin were added to these media when appropriate, each at a final concentration of 100 µg ml−1. S. mutans was grown as standing overnight cultures at 37°C and 5% CO2 in Todd-Hewitt broth (Becton Dickinson) supplemented with 0.3% w/v yeast extract (THYE). Kanamycin (700 µg ml−1) was added to THYE to maintain selection for the S. mutans GMS905, GMS906 and GMS907 fusion strains. S. mutans GMS584 and SmuvicK, isogenic mutants of the wild-type UA159 strain, were grown in THYE supplemented with erythromycin at a final concentration of 10 µg ml−1, when needed.
4
Cultivation and Protein Quantification of E. coli EC-14
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Escherichia coli EC-14 (Shionogi, Osaka, Japan) was isolated as the clinical isolate strain. In pre-cultivation, EC-14 stock solutions with 50% glycerol were mixed with THY medium consisting of Todd–Hewitt Broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and 0.2% yeast extract (Becton, Dickinson and Company) in the ratio of 1:100 and incubated at 37 °C for 12–14 h with shaking. After incubation, EC-14 (1.2 × 108 CFU/100 µL) was seeded in Dulbecco’s modified Eagle’s medium (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), which does not contain amino acids (amino-acid-free DMEM) and incubated at 37 °C with shaking, for the experiments. Bacterial protein was measured using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
5
Streptococcus pneumoniae Pneumonia Mouse Model
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Streptococcus pneumoniae (S. pneumoniae; serotype 19, ATCC 49619) was purchased from American Type Culture Collection. Bacteria were grown overnight at 37°C in 5% CO2 on blood agar plates, 5% sheep blood in tryptic soy agar (ThermoFisher). 10–20 colonies were then suspended in Todd‐Hewitt Broth (Becton Dickinson) supplemented with 17% (v/v) Fetal Bovine Serum (ThermoFisher) and incubated at 37°C with shaking at 225 rpm for several hours until an OD600 0.3 was reached as previously described (D'Alessio,2018 ). The media was distributed into 1 ml aliquots and flash‐frozen in liquid nitrogen before storage at −80°C (D'Alessio, 2018 ). Pneumonia was induced by intratracheal instillation of the thawed bacterial suspension at a dose of 2 µl/g mouse body weight. Colony‐forming units (CFU) in bacterial suspensions were subsequently determined by plating serial dilutions of the bacterial suspension on blood agar plates. The range of CFUs was 4.79–7.54 × 106 CFU/mouse.
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Streptococcus pneumoniae (S. pneumoniae; serotype 19, ATCC 49619) was purchased from American Type Culture Collection. Bacteria were grown overnight at 37°C in 5% CO2 on blood agar plates, 5% sheep blood in tryptic soy agar (ThermoFisher). 10–20 colonies were then suspended in Todd‐Hewitt Broth (Becton Dickinson) supplemented with 17% (v/v) Fetal Bovine Serum (ThermoFisher) and incubated at 37°C with shaking at 225 rpm for several hours until an OD600 0.3 was reached as previously described (D'Alessio,
6
GAS Growth Conditions and Optimization
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For in vitro growth, GAS strains were grown in Todd-Hewitt broth (Becton, Dickinson [BD]) supplemented with 0.2% yeast extract (THY medium). This medium was used (i) because it is a standard medium for GAS growth used by many investigators and (ii) because there is no medium that adequately mimics in vivo conditions. THY medium was supplemented with spectinomycin (150 μg ml−1) as needed. Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37˚C. LB medium was supplemented with spectinomycin (50 μg ml−1), as needed. Trypticase soy agar supplemented with 5% sheep red blood cells (Becton, Dickinson and Co.) was also used for growth of GAS strains. Spectinomycin dihydrochloride pentahydrate was purchased from Sigma. Gap electroporation cuvettes (2-mm gap size) were purchased from BTX Harvard apparatus. Growth experiments were performed as described previously (93 (link)). Locus tag equivalence between strains MGAS5005 and MGAS2221 is shown in Table S1J in the supplemental material.
7
Plasmid-based Study of Streptococcal Toxic Shock Syndrome
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The plasmids used in this study are described in Table 2 . STSS criteria were based on those proposed by the Working Group on Severe Streptococcal Infections22 (link). The clinical isolates from STSS and non-invasive (non-STSS) infections were collected during 1973–2008 as previously reported3 (link). Escherichia coli DH5α was used as a host for plasmid construction and was grown in liquid of Luria-Bertani medium with shaking or on agar plates at 37 °C. GAS was cultured in Todd-Hewitt broth (Becton Dickinson, Tokyo, Japan) supplemented with 0.5% yeast extract (THY media) without agitation or on Columbia Agar supplemented with 5% sheep blood (Becton Dickinson). Cultures were grown at 37 °C in 5% CO2. When required, antibiotics were added to the medium at the following final concentrations: kanamycin (Km), 25 μg/mL for E. coli and 200 μg/mL for GAS; spectinomycin (Sp), 25 μg/mL for E. coli and GAS. The growth of GAS was turbidimetrically monitored at 600 nm using MiniPhoto 518R (Taitec, Tokyo, Japan).
8
Pneumococcal Capsular Polysaccharide Binding Assay
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VWF binding analyses were performed with seven different pneumococcal strains including the laboratory strains: R6, R800, TIGER4 (serotype 4), D39 (serotype 2), and serotype 35A, and the clinical isolates of serotypes 23F and 12F. The strains differ in the amount of polysaccharide capsule as indicated in results. Red fluorescent protein (RFP)-expressing serotype 35A pneumococci were generated essentially as described in Kjos et al. (2015 (link)). This serotype was also used in all cell culture infection assays, microfluidic analyses and in vivo zebrafish infections. For cell culture infection and binding analyses, pneumococci were cultured on Columbia blood agar (Becton Dickinson) to mid-log phase (OD600 of 0.35) at 37°C and 5.0% CO2 or in Todd-Hewitt broth (Becton Dickinson) supplemented with 1.0% yeast extract (THY).
9
Standardizing Pneumococcal Strain Growth
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Twelve pneumococcal strains were selected to represent different genetic backgrounds and serotypes (Table 1 ). S. pneumoniae were grown in Todd Hewitt broth containing 0.2% yeast extract (both from Becton Dickinson, CA) (THY). All S. pneumoniae strains were stored at -80°C and the bacterial cells were picked using a sterile swab and patched on a 5% sheep's blood agar plate and incubated in 5% CO2 for 16 hr. A single colony was picked into 5 mL of THY medium and grown to mid exponential phase (OD600 = 0.25 to 0.6), glycerol was added to a 15% v/v ratio and then fast frozen and stored at −80°C as a starter culture. S. pneumoniae starter cultures were thawed and used to inoculate 5 mL of THY medium, and they were grown at 37°C to mid log phase and four samples OD (0.2 to 0.6 OD600) taken were used for plotting calibration curves for each strain. The exact CFUs were enumerated by a serial dilution plating method. Bacterial CFUs used in the following experiments were determined based on their respective calibration curve.
10
Cultivation and Antibiotic Selection of Bacterial Strains
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S. sanguinis strain SK36 (kindly provided by Dr. Kilian) [27] and its derivatives were routinely cultured in Todd-Hewitt broth (TH, Becton Dickinson, NJ, USA) at 37°C. For a deoxyribonuclease (DNase) assay using agar plates, S. sanguinis strains as well as S. oralis NCTC 11427T/SK23 [27] , S. mutans MT8148 [28] (link), S. salivarius HHT [29] (link), S. parasanguinis ATCC 903 [27] , and S. sobrinus MT10186 [30] (link) were cultured in Brain Heart Infusion (BHI) broth (Becton Dickinson). The Escherichia coli strain TOP10 (Life Technologies, CA, USA) served as a host for derivatives of pSET6s and pAT18 [31] (link), [32] (link). The E. coli strain XL10-gold (Stratagene, CA, USA) was utilized as a host for the pQE30 derivatives (Qiagen, Germany). E. coli strains were cultured in Luria-Bertani (LB, Sigma Aldrich, MO, USA) medium at 37°C with constant agitation. Lactococcus lactis NZ9000 (kindly provided by Dr. Poolman) and its derivatives were grown in M17 broth (Becton Dickinson) containing 0.5% glucose (M17G, Wako, Japan) at 28°C. To select mutant strains, antibiotics were added to the media at the following concentrations: ampicillin (Wako); 100 µg/ml for E. coli, chloramphenicol (Sigma Aldrich); 10 µg/ml for E. coli and 5 µg/ml for S. sanguinis, and erythromycin (Sigma Aldrich); 150 µg/ml for E. coli and 1 µg/ml for L. lactis.
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S. sanguinis strain SK36 (kindly provided by Dr. Kilian) [27] and its derivatives were routinely cultured in Todd-Hewitt broth (TH, Becton Dickinson, NJ, USA) at 37°C. For a deoxyribonuclease (DNase) assay using agar plates, S. sanguinis strains as well as S. oralis NCTC 11427T/SK23 [27] , S. mutans MT8148 [28] (link), S. salivarius HHT [29] (link), S. parasanguinis ATCC 903 [27] , and S. sobrinus MT10186 [30] (link) were cultured in Brain Heart Infusion (BHI) broth (Becton Dickinson). The Escherichia coli strain TOP10 (Life Technologies, CA, USA) served as a host for derivatives of pSET6s and pAT18 [31] (link), [32] (link). The E. coli strain XL10-gold (Stratagene, CA, USA) was utilized as a host for the pQE30 derivatives (Qiagen, Germany). E. coli strains were cultured in Luria-Bertani (LB, Sigma Aldrich, MO, USA) medium at 37°C with constant agitation. Lactococcus lactis NZ9000 (kindly provided by Dr. Poolman) and its derivatives were grown in M17 broth (Becton Dickinson) containing 0.5% glucose (M17G, Wako, Japan) at 28°C. To select mutant strains, antibiotics were added to the media at the following concentrations: ampicillin (Wako); 100 µg/ml for E. coli, chloramphenicol (Sigma Aldrich); 10 µg/ml for E. coli and 5 µg/ml for S. sanguinis, and erythromycin (Sigma Aldrich); 150 µg/ml for E. coli and 1 µg/ml for L. lactis.
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