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.
M17 broth
Manufactured by BD
Sourced in United States
M17 broth is a microbiological culture medium used for the growth and cultivation of streptococcus bacteria. It provides the necessary nutrients and growth factors required for the proliferation of these bacterial species.
Automatically generated - may contain errors
Lab products found in correlation
Bbl gaspak system, by BD (3 mentions)
Brain heart infusion broth, by Thermo Fisher Scientific (2 mentions)
Reinforced clostridial broth, by BD (2 mentions)
Tryptic soy broth (tsb), by BD (2 mentions)
Lb lennox broth, by Thermo Fisher Scientific (2 mentions)
Staphylococcus aureus, by BD (2 mentions)
M17 agar, by BD (2 mentions)
U 2810 spectrophotometer, by Hitachi (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Erythromycin, by Merck Group (2 mentions)
Pqe30, by Qiagen (1 mentions)
Todd hewitt broth, by BD (1 mentions)
Bhi broth, by BD (1 mentions)
Ampicillin, by Fujifilm (1 mentions)
Luria bertani lb, by Merck Group (1 mentions)
Escherichia coli strain top10, by Thermo Fisher Scientific (1 mentions)
Xl10 gold, by Qiagen (1 mentions)
Gifu anaerobic medium, by Nissui Pharmaceutical (1 mentions)
Anaero pouch anaero, by Mitsubishi (1 mentions)
Agmatine sulfate, by Merck Group (1 mentions)
25 protocols using m17 broth
1
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.
2
Constructing Mock Bacterial Community from Bovine Milk
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Bacterial strains representing species commonly found in bovine milk were used to construct a mock bacterial community (Table 1 ). Each bacterial strain was grown in standard laboratory culture medium with negative controls for that species and harvested at early stationary phase by centrifugation at 13,000 × g for 2 min. The laboratory culture media were as follows: Bacillus subtilis , Pseudomonas fluorescens , and Escherichia coli , LB (Lennox broth; Thermo Fisher Scientific); Enterococcus faecalis and Streptococcus agalactiae , brain heart infusion broth (Thermo Fisher Scientific); Staphylococcus aureus , tryptic soy broth (Becton Dickinson); Corynebacterium bovis , tryptic soy broth (Becton Dickinson) with 0.1% Tween 80; Lactococcus lactis , M17 broth (Becton Dickinson) with 0.5% glucose; and Clostridium tyrobutyricum , reinforced clostridial broth (Becton Dickinson). All strains were incubated at 37°C, with the exception of B. subtilis , L. lactis , and P. fluorescens , which were incubated at 30°C. B. subtilis , C. bovis , E. faecalis , E. coli , and P. fluorescens were grown under aeration (250 rpm).
3
C. elegans as Pathogen Infection Model
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Lactococcus cremoris strain FC (a stock strain of Fujicco Co., Kobe, Japan) was used as a test food source for C. elegans and was aerobically cultured in M17 broth (Becton, Dickinson, Franklin Lakes, NJ, USA) or M17 agar (Becton, Dickinson) with 0.5% lactose at 25°C for 48 h. Salmonella enterica subsp. enterica serovar Enteritidis strain SE1, originally isolated from a diarrheal specimen, was used as a Gram-negative pathogen. Staphylococcus aureus strain 96-55-17A was used as a Gram-positive pathogen. These pathogenic bacteria were reported as infection models using C. elegans (56 (link)) and were grown on tryptone soya agar at 37°C for 24 h.
4
Cultivation of Dairy-associated Bacteria
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Bacterial strains from species commonly found in milk and other dairy products were selected for a BCMC (Table 1
5
Anaerobic Culture of Thermophilic Bacteria
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Bacteria were cultured anaerobically with AnaeroPouch-Anaero (Mitsubishi Gas Chemical, Tokyo, Japan) at 37°C for 18 h in the medium described in Table 1 . Bacteria were washed twice with phosphate-buffered saline (PBS; pH 7.4) and suspended in PBS so that the optical density at 600 nm was 10.0 using a U-2810 spectrophotometer (Hitachi, Tokyo, Japan). Heat-treated bacteria were obtained by incubating at 75°C for 1 h.
Bacteria and medium used in this study.
| Strain | Medium |
|---|---|
| L. bulgaricus 2038 | MRS* |
| S. thermophilus 1131 | LM17** |
| L. bulgaricus JCM 1002T | MRS |
| L. bulgaricus ME-876 | MRS |
| S. thermophilus NCIMB 8510T | LM17 |
| Lactobacillus paragasseri ME-879 | MRS |
| Lactobacillus paragasseri ME-880 | MRS |
| Lacticaseibacillus paracasei ME-881 | MRS |
| Lacticaseibacillus paracasei ME-882 | MRS |
| Lactococcus lactis subsp. lactis ME-883 | MRS |
| Bifidobacterium bifidum ME-884 | GAM*** |
| Bifidobacterium longum ME-885 | GAM |
| Propionibacterium freudenreichii ME-886 | GAM |
*de Man Rogosa Sharpe broth (Becton Dickinson, Cockeysville, MD, USA).
**M17 broth (Becton Dickinson) supplemented with 1% lactose.
***Gifu anaerobic medium (Nissui Pharmaceutical, Tokyo, Japan).
6
Bacterial Strain Cultivation and Cell Line Maintenance
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Bacterial strains used in this study are listed in Table 1 . Bacterial strains were either purchased from American Type Culture Collection (ATCC, United States) or provided by National University Hospital, Singapore. Prior to assays, all bacteria, except for Enterococcus faecalis, were grown overnight to stationary phase at 37°C in Luria Bertani (LB) broth (Novagen, Germany). The overnight cultures were 10-fold diluted in fresh cation-adjusted Mueller Hinton II broth (MHBII) (Becton Dickinson, United States) and grown for additional few hours at 37°C to obtain mid-log phase culture. E. faecalis strains were grown in M17 broth (Becton Dickenson, United States) for both overnight and mid-log phase culturing. Human keratinocyte cell line, HaCaT, was purchased from Creative Bioarray (United States) and cultured in Dulbecco’s Modified Eagle’s medium with 4.5 g/L glucose, 2 mM L-glutamine, and 10% fetal bovine serum.
7
Isolation and Characterization of Dairy Bacteriophages
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Bacterial strains and bacteriophages are listed in Table 2 . All L. lactis strains were grown at 30 °C in sterile 11 % nonfat dry milk or in M17 broth (Becton Dickinson) supplemented with 0.5 % lactose (all strains except IL1403) or glucose (IL1403). Preparation of bacteriophage lysates and standard plaque assays were performed as described by Terzaghi & Sandine (1975) . Single-plaque isolates were purified from commercial whey samples and high-titre lysates passed through a 0.45 µm filter and stored at 4 °C. Phages M5938, M5939 and M5940 were isolated at approximately the same time period in 2010 from three distinct geographical locations in the USA while D4410 and D4412 were isolated in Europe in 2003. Phages M6162, M6165 and M6202 were isolated from industrial whey samples from three distinct geographical locations in the USA and Canada in 2012. Phage typing was performed using the multiplex PCR method as described by Labrie & Moineau (2000 (link)). Adsorption assays were performed as described by Sanders & Klaenhammer (1980) (link).
8
Culturing and Heat-Killing Probiotic Bacteria
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L. bulgaricus and S. thermophilus were cultured anaerobically in de Man Rogosa Sharpe broth (Becton Dickinson, Cockeysville, MD, USA) and M17 broth (Becton Dickinson, Cockeysville, MD, USA) supplemented with 1% lactose, respectively, at 37 °C for 18 h. For in vitro cytokine production assays, bacteria were washed twice with phosphate-buffered saline (PBS; pH 7.4) and suspended in PBS so that the optical density at 600 nm was 2.0 using U-2810 spectrophotometer (Hitachi, Tokyo, Japan). Then, bacteria were heat-killed at 75 °C for 1 h.
All bacteria used in this study are the property of Meiji Co., Ltd.
All bacteria used in this study are the property of Meiji Co., Ltd.
9
Enzymatic Reactions with Radiolabeled Fatty Acids
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The 15-methyl palmitic acid was purchased from Cayman Chemicals and all the other fatty acids, antibiotics, phospholipase A2, ortho-nitrophenyl-β-galactoside, and agmatine sulfate were purchased from Sigma-Aldrich. The media were purchased from Fisher Scientific. The DNA polymerase, restriction endonuclease, and T4 ligase were purchased from New England Biolabs. Sodium[1-14C]acetate (specific activity, 57.0 mCi/mmol) and [1-14C]stearic acid (specific activity, 53.0 mCi/mmol) were provided by Moravek, Inc, and the [1-14C]oleic acid (specific activity, 55 mCi/mmol) was purchased from American Radiolabeled Chemicals. Ni-NTA resin was from Invitrogen, and the DNA purification kits were from Qiagen. Silver nitrate silica gel thin layer plates were purchased from Analtech and M17 Broth was from Becton Dickenson. All the other reagents were of the highest available quality. Oligonucleotide primers were synthesized by Integrated DNA Technologies, and DNA sequencing was performed by ACGT, Inc.
10
Activation and Enumeration of Probiotic Strains
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Levilactobacillus brevis NPS-QW-145 and 3 different Lactobacillus delbrueckii ssp. bulgaricus strains (Table 1) were activated in Difco lactobacilli de Man, Rogosa, and Sharpe (MRS) broth (Becton, Dickinson and Co.), and Strep. thermophilus strain 1275 was cultivated in M17 broth (Becton, Dickinson and Co.) . Working cultures were propagated 3 times consecutively using 1% inoculation in the previously listed media (MRS or M17) at 37°C for 16 h. Cell counts of the working cultures were enumerated on MRS or M17 agar plates using the plate counting method before inoculation in reconstituted skim milk (RSM). The initial cell counts for both L. brevis 145 and Strep. thermophilus were ~1 × 10 9 cfu/mL.
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