The five oral bacterial strains included S. mutans strain ATCC 700,610, S. sobrinus strain 6715, Bifidobacterium dentium (B. dentium) strain ATCC 27,534, Lactobacillus acidophilus (L. acidophilus) strain ATCC 4356 and (A) naeslundii strain ATCC 12,104. Strains of (B) dentium ATCC 27,534 (order No. ATCC 27,534) and L. acidophilus ATCC 4356 (order No. ATCC 4356) and genomic DNA of S. mutans ATCC 700610D (order No. ATCC 700610-5) and A. naeslundii ATCC 12104D (order No. ATCC 12104-5) were directly purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). S. sobrinus strain 6715 was purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). The selected stains were incubated under the anaerobic conditions at 37 °C using mediums of brain heart infusion (Becton Dickinson, NJ, USA), tryticase phytone glucose (Hopebio, Qingdao, China) and lactobacilli MRS (Becton Dickinson, NJ, USA). Genomic DNA preparations from each strain, except S. mutans and A. naeslundii, were obtained using the method above and then purified.
Lactobacilli mrs
Manufactured by BD
Sourced in United States
Lactobacilli MRS is a selective culture medium used for the isolation and enumeration of Lactobacillus species from food and other sources. It provides the necessary nutrients and growth factors to support the growth of Lactobacillus species while inhibiting the growth of other bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Brain heart infusion, by BD (1 mentions)
L cysteine hcl, by Kanto Chemical (1 mentions)
Anaerogentm, by Thermo Fisher Scientific (1 mentions)
Lactobacilli mrs broth, by BD (1 mentions)
Itaq dna polymerase kit, by Bio-Rad (1 mentions)
Tryptic soy, by BD (1 mentions)
Nutrient broth, by BD (1 mentions)
Uv vis spectrophotometer, by Unico (1 mentions)
7 protocols using lactobacilli mrs
1
Oral Bacterial Strain Cultivation and DNA Extraction
2
Isolation and Genomic Analysis of Bifidobacterium
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Three pieces of filter, with a diameter of 47 mm, were first placed on a TOS propionate agar plate, and were then removed, followed by incubation at 37 °C for 48 hours under anaerobic conditions. Twelve to sixty-three colonies per sample (see Supplementary Table 1 ) were randomly selected and were examined by colony PCR using a primer set specific for the genus Bifidobacterium, as based on 16S ribosomal RNA gene sequence (g-Bifid-F: 5′-CTCCTGGAAACGGGTGG-3′; g-Bifid-R: 5′-GGTGTTCTTCCCGATATCTACA-3′)20 (link). Each identified bifidobacterial isolate was cultivated in Difco Lactobacilli MRS (Becton Dickinson, NJ) supplemented with 0.05% L-cysteine HCl (Kanto Chemical, Tokyo, Japan) at 37 °C for 16 hours under anaerobic conditions before DNA extraction. A total of 669 strains were isolated and assessed by random amplified polymorphic DNA (RAPD) analysis to detect (and, in case they appeared to be same, discard) clonal isolates obtained from the same sample. In this manner, 98 distinct isolates were selected for genome sequencing.
3
Isolation and Cultivation of Lactic Acid Bacteria from Kimchi
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Thirty-one lactic acid bacteria strains were used (Table 1 ), which were isolated from kimchi in South Korea [16 ]. One hundred microliters of 20% glycerol stock of each strain was cultured in 10 mL Lactobacilli MRS (de Man, Rogosa and Sharpe) broth (Becton, Dickinson and Company, Franklin lakes, NJ, USA) and incubated anaerobically at 37 °C for 24 h, using an anaerobic pack (AnaerogenTM, Oxoid, Hampshire, UK). Then, 100 μL of the culture medium was transferred into fresh 10 mL Lactobacilli MRS broth (Becton, Dickinson and Company) and incubated at 37 °C for 24 h. The cultured medium was precipitated by centrifuge at 1912× g and 4 °C for 15 min, and the cell pellets were washed twice with phosphate-buffered saline (PBS; pH 7.4; KH2PO4 0.2 g, Na2HPO4 1.5 g, NaCl 8.0 g, KCl 0.2 g/distilled water 1 L). The final cell pellets were suspended in 10 mL of PBS to obtain 9–10 log CFU/mL.
4
Isolation and Cultivation of Lactobacillus and Streptococcus Strains
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L. brevis strains were isolated using lactobacilli MRS (BD Bioscience, USA). Lactobacillus rhamnosus GG (Cell Biotech, Ltd., Korea) was used as a commercial control strain and obtained from the Korean Collection for Type Cultures (Korea). All Lactobacillus strains were cultured in MRS broth at 37°C for 24 h.
S. mutans KCTC 5124, KCTC 5458, and KCTC 5316 were obtained from the Korean Collection for Type Cultures. The strains were cultured in brain heart infusion broth (BHI, BD Bioscience, USA) supplemented with 3% sucrose at 37°C for 24 h and used as oral pathogenic bacteria.
S. mutans KCTC 5124, KCTC 5458, and KCTC 5316 were obtained from the Korean Collection for Type Cultures. The strains were cultured in brain heart infusion broth (BHI, BD Bioscience, USA) supplemented with 3% sucrose at 37°C for 24 h and used as oral pathogenic bacteria.
5
Multimodal Microbiome Analysis Protocol
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MAT and mucosal scrapings were homogenized by standing mortar and pestle (Fisher Scientific) in sterile PBS using aseptic techniques. Samples were serially diluted and plated on the following media with 1.2% agar in both aerobic and anaerobic conditions: chocolate blood (CBA;BD), Lactobacilli MRS (BD), brain heart infusion media (BHI; Sigma-Aldrich, USA), Brucella (BRU; Hardy Diagnostics) with 0.5% pyruvate, 0.5% taurine and 0.05% ammonium iron citrate, reinforced Clostridial media (RCM; BD), Bacteroides bile esculin (Anaerobe Systems) and Sabouraud dextrose (SAB; Hardy Diagnostics) with and without the addition of olive oil post-inoculation. BHI, BRU and RCM were supplemented with 5 mg/L hemin (BeanTown Chemical) and 0.5 mg/L vitamin K (Alfa Aesar). All the plates were incubated at 37°C except for SAB, which was cultured at room temperature. Distinct colony forming units were re-steaked at day 4 and 7 post-incubation. Colony PCR was performed with full length 16S or ITS primers (Key Resources Table). Amplification was carried out using the iTaq DNA polymerase kit (Bio-Rad). Amplicons were submitted to Laragen for Sanger sequencing. Sequence traces were examined in FinchTV v1.4, and the resultant trimmed reads were identified by Microbial BLAST.
6
Bacterial Growth Kinetics in Leaf Infusions
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Scaffolds were removed from the pressure vessel, aseptically opened in a sterile tissue culture hood and washed with DI water for 24 h. Leaves were cut in half and placed in Erlenmeyer flasks. Each flask was filled with 25 mL of one of four different bacterial broths, Tryptic Soy (BD), Nutrient Broth (BD), Brain Heart Broth (Sigma) or Lactobacilli MRS (BD). Turbidity was measured after 72 h incubation under shaking at 35 °C by a UV/VIS Spectrophotometer (Unico, USA) at 600 nm wavelength.
7
Bacterial Strains for Gene Cloning and Riboflavin Production
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All the bacterial strains used in this study are listed in Table S1 . E. coli XL1-blue was used as a host for the gene cloning and maintenance of plasmids. L. citreum CB2567 [38 (link)] was used as a major host for the development of the CRISPRi system and riboflavin production. E. coli was cultivated in a Luria–Bertani (LB) medium (tryptone 10 g/L, yeast extract 5 g/L, and sodium chloride 10 g/L, purchased as premixed media from BD, Franklin Lakes, NJ, USA) at 37 ℃ with shaking (200 rpm). L. citreum CB2567 was cultivated in Lactobacilli MRS (De Man, Rogosa and Sharpe) medium (proteose peptone no. 3 10 g/L, beef extract 10 g/L, yeast extract 5 g/L, dextrose 20 g/L, polysorbate 80 1 g/L, ammonium citrate 2 g/L, sodium acetate 5 g/L, magnesium sulfate 0.1 g/L, manganese sulfate 0.05 g/L, and dipotassium phosphate 2 g/L, purchased as premixed media from BD) at 30 ℃ with shaking (200 rpm). Ampicillin (Amp, 100 mg/L) was added for the selection and cultivation of E. coli. Chloramphenicol (Cm, 10 mg/L) and erythromycin (Em, 10 mg/L) were used for the selection and cultivation of L. citreum.
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