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.
L cysteine hcl
Manufactured by Kanto Chemical
Sourced in Japan
L-cysteine HCl is a fine, crystalline powder that is a source of the amino acid cysteine. It is water-soluble and used as a component in various biochemical applications and research.
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Lab products found in correlation
4 protocols using l cysteine hcl
1
Isolation and Genomic Analysis of Bifidobacterium
2
Bifidobacterium longum isolation and characterization
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Three hundred ninety three of the 453 assessed fecal samples (nearly 87%) were confirmed to contain B. longum subsp. longum DNA employing PCR and a subspecies-specific primer pair20 (link), of which 177 were selected to retrieve bifidobacteria by cultivation on TOS propionate agar (Eiken Chemical, Tokyo, Japan) supplemented with 50 mg/l mupirocin (Merck KGaA, Darmstadt, Germany). Following incubation at 37 °C for 48 hours under anaerobic conditions, ten colonies per sample were randomly selected and were examined by colony PCR using the above mentioned species-specific primer set. Each identified B. longum subsp. longum 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. In this manner a total of 162 B. longum subsp. longum strains were isolated, of which a proportion (based on age representation) was selected for sequencing (see below).
3
Anaerobic Nurmi-type Culture Preparation
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VL broth or NB was used as the basal culture medium for preparing Nurmi-type cultures. VL broth contained 10 g Bacto Tryptone (BD Difco), 3 g Lab-Lemco powder (Oxoid), 5 g Bacto Yeast Extract (BD Difco), 2.5 g glucose, 5 g NaCl, 0.4 g l -cysteine HCl (Kanto Chemical Co., Inc., Tokyo, Japan), and 0.6 g agar in 1 liter of distilled water adjusted to pH 7.2 using 1 N NaOH (Barnes and Impey, 1970 ). NB contained 10 g Bonito extract (Wako Pure Chemical Industries, Ltd., Tokyo, Japan), 10 g peptone (Wako), 2 g NaCl, 5 g K2HPO4, and 0.8 g agar (Wako) in 1 liter of distilled water adjusted to pH 7.0 using a 1 N NaOH solution. Other broth cultures were prepared with or without Cys. Cys was added to NB (NB+Cys) and not to VL broth, which usually contains Cys in its original formulation (VL−Cys). The final Cys concentration in each broth was 2 mM (Barnes and Impey, 1970 ). Each Nurmi-type culture was started by inoculating 1 ml of mixed CE source into 100 ml of each medium followed by incubation at 37°C for 24 h under anaerobic conditions without agitation, using the AnaeroPak method (Mitsubishi Gas).
4
Bifidobacterial Strain Cultivation and Preparation
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The bacterial strains used in this study and general information about these strains are listed in Table 1 . The bifidobacterial strains were obtained from stock cultures maintained in the Morinaga Culture Collection (MCC; Morinaga Milk Industry Co., Ltd., Zama, Japan) and the American Type Culture Collection (ATCC, VA). Each strain 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 h under anaerobic conditions before DNA extraction. The microorganisms were collected by centrifugation, washed once with sterile saline, resuspended in an equivalent volume of sterile saline, and used as seed cultures for fermentation studies.
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