Propionibacterium acnes isolates were cultured anaerobically in GAM Broth (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) in an air-tight container containing an Anaero-Pack (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) at 37°C for 3 days. Cultures were diluted to an OD595 of 0.01 (corresponding to 2 × 107 colony-forming units/ml) with fresh GAM Broth (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 1% glucose (GAMG Broth). Aliquots (200 μl) of this bacterial suspension were added to the wells of a 96-well flat-bottomed polystyrene plate and incubated anaerobically for 3 days at 37°C. Cell growth of P. acnes isolates was evaluated by measuring the absorbance at 595 nm. After removal of the supernatants, biofilms formed on the bottom of the wells were stained with 0.1% crystal violet for 10 min and subsequently washed twice with 200 μl of phosphate-buffered saline (PBS). Next, 200 μl of ethanol was added to the wells to extract crystal violet and the absorbance at 595 nm was measured using an Infinite 200 PRO Microplate Reader (Tecan, Mannedorf, Switzerland).
Gam broth
Manufactured by Nissui Pharmaceutical
Sourced in Japan
GAM broth is a culture medium used for the cultivation and growth of anaerobic bacteria. It provides the necessary nutrients and growth conditions to support the proliferation of anaerobic microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Anaeropack, by Mitsubishi (2 mentions)
Gam agar, by Nissui Pharmaceutical (2 mentions)
Mrs broth, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (2 mentions)
Bbg9 1, by Nissui Pharmaceutical (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Infinite 200 pro microplate reader, by Tecan (1 mentions)
Dneasy powersoil kit, by Qiagen (1 mentions)
Rnase a, by Merck Group (1 mentions)
Mrs broth, by BD (1 mentions)
L cysteine hydrochloride, by Fujifilm (1 mentions)
Dcode system, by Bio-Rad (1 mentions)
Linoleic acid, by Fujifilm (1 mentions)
Chloroform d, by Merck Group (1 mentions)
Wakogel c 300 hg, by Fujifilm (1 mentions)
1 1 diphenyl 2 picrylhydrazyl, by Tokyo Chemical Industry (1 mentions)
β carotene, by Fujifilm (1 mentions)
Benzalkonium chloride, by MP Biomedicals (1 mentions)
37 protocols using gam broth
1
Quantification of P. acnes Biofilm Formation
2
Cultivation and Preparation of Bacteria for Lifespan and Behavioral Assays
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Escherichia coli OP50 was grown on tryptone soya broth (TSB) and tryptone soya agar (TSA) (Nissui Pharmaceutical, Tokyo, Japan) at 37°C. Similarly, Bacillus
subtilis NBRC3134 and Staphylococcus aureus NBRC13276 were cultured using TSB and TSA. B. infantis ATCC15697 was cultured using GAM broth (Nissui) and
TOS propionate agar (Yakult Pharmaceutical Industry, Tokyo, Japan). Clostridium butyricum MIYAIRI 588 (CBM 588), which was kindly provided by Miyarisan Pharmaceutical, was
cultured using GAM broth and GAM agar (Nissui Pharmaceutical, Japan). Cultivated bacteria were scraped and weighed. Aliquots (100 mg wet weight) of bacteria were suspended in 0.5 ml of M9 buffer
(5 mM potassium phosphate, 1 mM CaCl2, and 1 mM MgSO4) and used in the lifespan assays; aliquots at 66.7 mg/ml were used in the behavioral assays.
subtilis NBRC3134 and Staphylococcus aureus NBRC13276 were cultured using TSB and TSA. B. infantis ATCC15697 was cultured using GAM broth (Nissui) and
TOS propionate agar (Yakult Pharmaceutical Industry, Tokyo, Japan). Clostridium butyricum MIYAIRI 588 (CBM 588), which was kindly provided by Miyarisan Pharmaceutical, was
cultured using GAM broth and GAM agar (Nissui Pharmaceutical, Japan). Cultivated bacteria were scraped and weighed. Aliquots (100 mg wet weight) of bacteria were suspended in 0.5 ml of M9 buffer
(5 mM potassium phosphate, 1 mM CaCl2, and 1 mM MgSO4) and used in the lifespan assays; aliquots at 66.7 mg/ml were used in the behavioral assays.
3
Culturing Bifidobacterium bifidum G9-1
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BBG9-1 (Bifidobacterium bifidum G9-1; Biofermin R&D Center, Kobe, Japan)11 (link) was obtained from the Culture Collection of Biofermin Pharmaceutical Co., Ltd. and cultured at 37 °C for 18 h in GAM Broth (Nissui Pharmaceutical. Co., Ltd., Tokyo, Japan) supplemented with 0.7% glucose and 0.1% Tween 80. The bacterial cells were recovered by centrifugation at 3000 × g for 15 min.
4
Anaerobic Fecal Bacteria Isolation and Identification
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Fresh fecal samples were collected on Days 1 and 16. These samples were suspended in PBS and plated onto 5% horse blood-supplemented BL agar and CPLX agar [21 ]; these plates were incubated anaerobically for 2–3 days at 37°C. Colonies were selected by Gram staining and cellular morphology, and inoculated in GAM broth (#05422, Nissui Pharmaceutical, Tokyo, Japan) supplemented with 1% glucose, or modified GAM agar (#05426, Nissui Pharmaceutical, Tokyo, Japan). Subsequently, cultured bacteria were examined by FISH by using a Bifidobacterium-specific probe, Bif153 (S1 Table ). Single colonies were picked and streaked three times to obtain pure cultures, and the isolates were stored at -80°C.
5
Porphyromonas gingivalis Growth Conditions
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Porphyromonas gingivalis strains used in this study are listed in Table 1 , and were grown anaerobically at 37°C in a modified GAM broth (Nissui, Tokyo, Japan) or on Brucella HK agar plates (Kyokuto Pharmaceutical Industrial, Tokyo, Japan), supplemented with 5% rabbit blood. The following antibiotic concentrations were used, as appropriate: 20 μg/ml erythromycin, 0.5 μg/ml tetracycline, and/or 10 μg/ml ampicillin. E. coli DH5α and BL21 (DE3) strains were grown aerobically at 37°C in 2× YT medium (Becton Dickinson Japan, Tokyo, Japan) with 100 μg/ml ampicillin, 200 μg/ml erythromycin, or 10 μg/ml tetracycline.
6
Isolation and Genomic Analysis of Clostridium perfringens Strains
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The five bec-harbouring C. perfringens strains (OS1, TS1, O13–19, A18–256, and A19–1) sequenced in this study were isolated in Japan. OS1, TS1 [17 (link)], and A18–256 were isolated from faecal specimens of patients during distinct C. perfringens foodborne outbreaks in 2009, 2010, and 2018, respectively; O13–19 was isolated from a faecal specimen of a patient during a non-C. perfringens foodborne outbreak in 2013; and A19–1 was isolated from an oyster in 2019.
All strains were anaerobically cultured in GAM broth (Nissui) at 37 °C, and then harvested by centrifugation at 3000 rpm for 20 min and subsequent collection of the cell pellet. Genomic DNA was extracted from the resuspended cells using the DNeasy PowerSoil Kit (QIAGEN).
All strains were anaerobically cultured in GAM broth (Nissui) at 37 °C, and then harvested by centrifugation at 3000 rpm for 20 min and subsequent collection of the cell pellet. Genomic DNA was extracted from the resuspended cells using the DNeasy PowerSoil Kit (QIAGEN).
7
Anaerobic cultivation of gut bacteria
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B. wexlerae (JCM 17041), B. vulgatus (JCM 5826), P. copri (JCM 13464), F. prausnitzii (JCM 31915), and B. faecihominis (JCM 31056) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. All bacterial strains were cultured anaerobically at 37 °C by using an anaerobic chamber (Bactron 300, Toei Kaisha, Ltd, Tokyo, Japan). For oral administration into mice, B. wexlerae was cultured anaerobically in reinforced clostridial medium (BD Difco, Franklin Lakes, NJ, USA) at 37 °C for 48 h until OD600 = 1.0–1.5. Cultures were stored as 0.5-ml aliquots at –80 °C until use for oral administration into mice. For measurement of starch, SCFAs, and metabolites and Raman spectroscopic analysis, B. wexlerae, B. vulgatus, P. copri, and F. prausnitzii were cultured in clostridial reinforced medium (BD Difco) at 37 °C for 48 h; at inoculation, OD600 was approximately 0.05 for all strains (B. wexlerae, 1.0; B. vulgatus, 1.1; P. copri, 0.8; and F. prausnitzii, 0.9). For the cross-feeding assay, B. faecihominis was cultured anaerobically at 37 °C in GAM broth (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) supplemented without or with B. wexlerae culture supernatant, succinate acid, sodium lactate, or sodium acetate; the pH of the medium was adjusted to 7.0.
8
Anaerobic Growth of Lactobacillus Strains
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Lactobacillus spp. (L. jensenii Ich 2023-1, L. iners 22-2590, L. gasseri Ich 2023-1, L. crispatus 8-1, L. crispatus 14-2), Limosilactobacillus spp. (L. vaginalis Ich9595, L. reuteri YB1506, L. fermentum YB1839) strains were cultured anaerobically (6% H2, 20% CO2, 74% N2 at 37°C) with shaking in GAM broth (Nissui Pharmaceutical Co., Ltd. 05422) containing supernatants of CBM 588 culture solution of GAM broth (Nissui Pharmaceutical Co., Ltd.) at 0, 1, 5, and 10%. Colony measurements were performed after 24 h incubations (n = 4 per group). To assess bacterial concentrations, samples were obtained and serially diluted in normal saline. Aliquots of the diluted samples were plated for quantitative culture. Brucella HK nutrient agar plates (100-mm diameter) were used for quantitative determinations (Kyokuto Pharmaceutical Industrial Co., Ltd). The colony counts were read after 48 to 72 h of incubation anaerobically at 37°C.
9
Nuclease Treatment of Heat-Killed Lactic Acid Bacteria
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Lactic acid bacteria were purchased from the Japan Collection of Microorganisms (JCM) or isolated from fermented foods (Table S1 in Supplementary Material). Pediococcus acidilactici strain K15, Lactobacillus plantarum ATCC14197T, Lactobacillus pentosus ATCC8041T, and Lactococcus lactis subsp. lactis ATCC19435T were cultured at 30°C for 24 h in MRS broth (BD). Lactobacillus delbrueckii subsp. bulgaricus ATCC11842T and Lactobacillus rhamnosus ATCC53103T (LGG) were cultured at 37°C for 24 h in MRS broth. Four strains of Bacteroides sp. were cultured at 37°C for 24 h in GAM broth (Nissui Pharmaceutical Co. Ltd.). Then, they were heat-killed at 95°C for 10 min, washed twice with saline, and suspended in saline. For the nuclease treatment of heat-killed bacteria, RNase A (from bovine pancreas, Sigma) treatment was performed under low salt conditions (10 mM Tris–HCl, pH 8.0) or high salt conditions (10 mM Tris–HCl, 0.3 M NaCl, pH 8.0) at 37°C for 2 h. RNase A-treated bacteria were washed twice with each buffer and used for subsequent experiments.
10
Wheat Bran Fermentation by Lactobacillus
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WB powder (0.5 g) was added to a test tube containing 5 mL of DW and autoclaved at 121 °C for 15 min. Lactobacillus johnsonii Wheat-1 (LC586157) and L. reuteri Wheat-12 (LC586168) strains isolated from the ICR mic were pre-cultured in 3 mL of GAM broth (Nissui Pharmaceutical Co., Ltd.) at 37 °C for 48 h. The pre-culture (0.05 mL) was then inoculated into the 10% (w/v) WB suspension (n = 3) and incubated at 37 °C for 7 d. The pH of the cultures was measured on days 1, 2, 4, and 7. The fermented sample at 4 days of incubation was filtered through a 0.45-μm pore syringe filter (DISMIC 13CP045AN; Toyo Roshi Kaisha Ltd., Tokyo, Japan) and subjected to high-performance liquid chromatography (HPLC) under the following conditions: column, ICSep ICEORH-801 (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); operating temperature, 35 °C; elution, 0.01 mol/L sulphuric acid (H2SO4); and flow rate, 0.8 mL/min. The eluted compounds were detected using a refractive index detector.
Total phenolic compound content (TPC), ferric reducing power, and superoxide anion (O2–) radical scavenging capacity in WB fermented for 4 days were determined as previously reported (Kuda et al., 2021 ).
Total phenolic compound content (TPC), ferric reducing power, and superoxide anion (O2–) radical scavenging capacity in WB fermented for 4 days were determined as previously reported (Kuda et al., 2021 ).
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