For preparation of inoculum, tested isolates were cultured anaerobically at 37 °C for 20 to 24 h with Mueller-Hinton broth (MHB; Difco Becton Dickinson and company, Sparks, MD, USA) containing 0.001% pyridoxal hydrochloride (Wako) and the bacterial cell suspensions were adjusted to yield about 5 × 105 CFU/mL. MICs for the Abiotrophia and Granulicatella strains were determined using the microdilution broth method with MHB containing 2.5% lysed horse blood (Strepto hemo supplement ‘Eiken’, Eiken Chemical Co., Ltd., Tokyo, Japan) and 0.001% pyridoxal hydrochloride, according to the consensus guideline from the CLSI for fastidious bacteria [12 (link)]. Briefly, the antimicrobial agents (100 µL/well) were diluted on 96-well round bottom plates (Sumilon, Sumitomo Bakelite Co., Ltd., Tokyo, Japan) in serial two-fold with the supplemented MHB, and 5 µL of the bacterial inoculum was added to each well. The plates were incubated at 35 °C in anaerobic condition for 20 h. The MIC values were defined as the lowest concentrations of antimicrobial agents that completely inhibited the bacterial growth in the microdilution wells, detected by unaided eyes. The strain of S. pneumoniae ATCC 49619 was used for quality control testing, and all MIC values for the strain were within the acceptable limits.
Pyridoxal hydrochloride
Manufactured by Fujifilm
Sourced in Japan
Pyridoxal hydrochloride is a chemical compound that serves as a versatile laboratory reagent. It is a water-soluble vitamin B6 derivative used in various analytical and biochemical applications. The core function of pyridoxal hydrochloride is to act as a cofactor for enzymatic reactions and to facilitate the study of metabolic processes.
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3 protocols using pyridoxal hydrochloride
1
Antimicrobial Susceptibility Testing for Fastidious Bacteria
2
A. defectiva Anaerobic Culturing
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A. defectiva ATCC was cultured in Todd–Hewitt broth (THB; Difco Laboratories. Detroit. Mich.) supplemented with 10 μg/mL of pyridoxal hydrochloride (Wako Pure Chemicals, Tokyo, Japan) and 200 μg/mL of L-cystein hydrochloride (Kanto Chemicals, Tokyo, Japan) at 37 °C for 48 h under anaerobic conditions. In some experiments, the bacteria were cultured with 37 kBq of [methyl-3H]-thymidine (PerkinElmer Japan, Yokohama, Japan) per mL of THB containing pyridoxal and l -cysteine for radioactive labeling.
3
Isolation and Cultivation of Oral Bacteria
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Bacterial strains and culture condition G. adiacens ATCC 49175, A. defectiva ATCC 49176, and their two isolates, G. adiacens a-1 and a-2 and A. defectiva d-1 and d-2, were extracted from the dental plaque of healthy human volunteers and stored at -70° for use in this study (Tajika S, Dent J Iwate Med Uni, 21, [271] [272] [273] [274] [275] [276] [277] [278] [279] [280] [281] [282] [283] [284] [285] 1996) . To isolate G. adiacens and A. defectiva from dental plaque samples, colonies demonstrating bacteriolytic activity on Todd-Hewitt (Difco Laboratories, Detroit, MI, USA) agar plates containing heat-killed Micrococcus luteus cells were isolated after incubation at 37°C for 24 h from oral specimens. These isolates were then identified as G. adiacens or A. defectiva after performing biochemical tests and using the PCR-RFLP method [9] . Bacteria were cultured in Todd-Hewitt broth (THB; Difco Laboratories) supplemented with 10 µg/mL pyridoxal hydrochloride (Fujifilm Wako Pure Chemicals, Tokyo, Japan) and 200 µg/mL l-cysteine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and were maintained at 37°C for 24 h. In other experiments, bacteria were cultured with 37 kBq of [methyl-3 H]-thymidine (PerkinElmer Japan, Yokohama, Japan) per mL of THB containing pyridoxal and l-cysteine for radioactive labeling.
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