The P. gingivalis JCM12257 and A. actinomycetemcomitans JCM8577 isolates used in this study were obtained from the Japan Collection of Microorganisms (RIKEN BioResource Center, Tsukuba, Ibaraki, Japan). P. gingivalis was grown anaerobically on modified GAM agar (Nissui Pharmaceutical Co., Ltd, Ueno, Tokyo, Japan) for 3 days at 37 °C. A. actinomycetemcomitans was grown on modified GAM agar for 24 h at 37 °C in an atmosphere of 5% CO2 in air. The initial inocula were adjusted to 1 × 107 colony forming units (CFUs)/mL.
Modified gam agar
Manufactured by Nissui Pharmaceutical
Sourced in Japan
Modified GAM agar is a culture medium used for the isolation and enumeration of anaerobic bacteria. It is a modification of the original Gifu Anaerobic Medium (GAM) agar, which is widely used in microbiology laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Anaeropack kenki, by Mitsubishi (1 mentions)
Modified gam broth, by Nissui Pharmaceutical (1 mentions)
Porphyromonas gingivalis atcc 33277, by American Type Culture Collection (1 mentions)
Fusobacterium nucleatum atcc 25586, by American Type Culture Collection (1 mentions)
Streptococcus sanguinis atcc 10556, by American Type Culture Collection (1 mentions)
Streptococcus mutans atcc 25175, by American Type Culture Collection (1 mentions)
Lb broth, by BD (1 mentions)
Columbia blood agar, by BD (1 mentions)
Lactobacilli mrs agar, by BD (1 mentions)
4 protocols using modified gam agar
1
Cultivation of Oral Pathogens P. gingivalis and A. actinomycetemcomitans
2
Genomic DNA Extraction from CP118
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The genomic DNA of CP118 was manually extracted from the strain cultured on modified GAM agar (Nissui Pharmaceutical Co.) supplemented with 5% sheep blood at 37°C for 24 hr under anaerobic
conditions according to the methods described by Okamoto et al. [37 (link)]. In brief, CP118 was suspended in TE (10 mM Tris–HCl [pH 8.0] and
1 mM EDTA [pH 8.0]), treated with lysozyme and mutanoysin, and lysed by 10% sodium dodecyl sulfate. The lysate was then treated with phenol, phenol-chloroform-isoamyl alcohol (25:24:1)
(PCI), and chloroform once, three times, and twice, respectively. Nucleic acid was precipitated and washed by ethanol and resuspended in sterile H2O. After the RNase treatment and
additional extraction with PCI and chloroform, bacterial DNA was precipitated again, rinsed with ethanol, and dissolved in 10 mM Tris-HCl (pH 8.5).
conditions according to the methods described by Okamoto et al. [37 (link)]. In brief, CP118 was suspended in TE (10 mM Tris–HCl [pH 8.0] and
1 mM EDTA [pH 8.0]), treated with lysozyme and mutanoysin, and lysed by 10% sodium dodecyl sulfate. The lysate was then treated with phenol, phenol-chloroform-isoamyl alcohol (25:24:1)
(PCI), and chloroform once, three times, and twice, respectively. Nucleic acid was precipitated and washed by ethanol and resuspended in sterile H2O. After the RNase treatment and
additional extraction with PCI and chloroform, bacterial DNA was precipitated again, rinsed with ethanol, and dissolved in 10 mM Tris-HCl (pH 8.5).
3
Culturing Oral Bacterial Strains
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Brain-Heat Infusion (BHI) agar was purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Modified GAM broth and modified GAM agar were purchased from Nissui Pharmaceutical Co., LTD (Tokyo, Japan). Porphyromonas gingivalis ATCC 33277, Fusobacterium nucleatum ATCC 25586, Streptococcus sanguinis ATCC 10556, and Streptococcus mutans ATCC 25175 were purchased from the American Type Culture Collection (Manassas, VA). P. gingivalis and F. nucleatum were grown on GAM broth and GAM agar, and S. sanguinis and S. mutans were grown on BHI broth and BHI agar in an anaerobic chamber with Anaeropack Kenki (Mitsubishi Gas Chemical Company, Tokyo, Japan) at 37 °C.
4
Isolating Bacteria from Honey Samples
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For isolation of bacteria from honey, 20 ml of each honey sample was diluted to 50% (v/v) with sterile water and centrifuged at 11,000 rpm for 45 min. The supernatant was removed and the sediment was suspended with 1 ml of sterile water. One hundred microliters of each suspension was plated on Columbia blood agar (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) containing 5% sheep blood (CSA), KSBHI agar (Arai et al., 2012 (link)), Lactobacilli MRS agar (Becton, Dickinson and Company) and modified GAM agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 5% sheep blood (GAMSA). For selective isolation of TS-resistant bacteria, all agar media used for the isolation were supplemented with 1 μg/ml of TS. CSA was incubated at 35°C under air plus 5% CO2 conditions for two days, and the other agar media were incubated at 35°C under anaerobic conditions for four days. Colonies grown on the agar media were subcultured for purification. Pure-cultured bacterial isolates were suspended in LB broth (Becton, Dickinson and Company) containing 30% glycerol and stored at −80°C until use.
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