For bacterial isolation and culturing, intestinal contents (fecal samples) were collected from all the morbid animals and were shifted to the laboratory immediately. All the collected samples were cultured in reinforced clostridial media (Oxoid, CM0149) and incubated at 37°C for 24 h anaerobically. A loopful from the crude broth culture was streaked onto tryptose sulfite cycloserine agar (TSC, Oxoid, CM0587B) having selective supplement. Incubation was carried out under anaerobic conditions at 37°C for 24 h. The isolated colonies were stained for bacterial morphology. Typical blackened colonies on TSC plates, characteristic beta hemolysis on 5% sheep blood agar, and production of opaque halo around colonies on egg yolk agar confirmed the isolated colonies as C. perfringens. The pure bacterial isolates were further biochemically characterized using API 20A kits (bioMérieux, Marcy l'Étoile, France).
Cm0149
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The CM0149 is a laboratory centrifuge designed for general-purpose applications. It has a maximum speed of 4,000 rpm and can accommodate a variety of sample tubes and rotors. The centrifuge is suitable for separating substances with different densities.
Automatically generated - may contain errors
Lab products found in correlation
Api 20a kit, by bioMérieux (1 mentions)
M nano, by Tecan (1 mentions)
Sheep blood, by Thermo Fisher Scientific (1 mentions)
Agar, by Nacalai Tesque (1 mentions)
Bhi medium, by BD (1 mentions)
Human keratinocyte growth supplement, by Thermo Fisher Scientific (1 mentions)
Ebm 2 basal medium, by Lonza (1 mentions)
Epilife medium, by Thermo Fisher Scientific (1 mentions)
Ebm 2 growth medium, by Lonza (1 mentions)
Cm0325, by Thermo Fisher Scientific (1 mentions)
Cm1127, by Thermo Fisher Scientific (1 mentions)
7 protocols using cm0149
1
Isolation and Identification of C. perfringens
2
Growth Rate Measurement of Bacterial Isolates
3
Evaluating Clostridium Spore Disinfection
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The previously prepared 1 mL aliquots, containing C. sporogenes or C. difficile spores (108–109 CFU/mL), were removed from the freezer and allowed to thaw under chilled conditions. A 1 mL aliquot containing the spore suspension was transferred to 9 mL SDW and vortexed before being added to a test-tube containing 10 mL of the double strength disinfectant agent. SDW was used as the control and 4 test-tubes were prepared for each disinfectant. Each tube was thoroughly vortexed and left on the laboratory bench at a temperature of approximately 18 °C. After 20 and 60 min, 2 test-tubes for each disinfectant and the control were removed, centrifuged (at 7500× g at 4 °C for 10 min), the pellet resuspended in 1.5 mL of SDW and vortexed.
The spores were then resuspended in sterile Eppendorf tubes and put on a heating block (Techne Dri-Block DB-3) for 10 min at 80 °C for C. sporogenes or for 25 min at 60 °C for C. difficile to remove any vegetative cells. The ability of a given disinfectant to reduce the target spore concentration was evaluated in suspension tests in SDW and in broth RCM (reinforced clostridial medium (RCM; Oxoid, (CM0149))) or BHI broth, respectively, for C. sporogenes and C. difficile to study the efficacy of these agents in the presence of low concentrations of organic matter.
The spores were then resuspended in sterile Eppendorf tubes and put on a heating block (Techne Dri-Block DB-3) for 10 min at 80 °C for C. sporogenes or for 25 min at 60 °C for C. difficile to remove any vegetative cells. The ability of a given disinfectant to reduce the target spore concentration was evaluated in suspension tests in SDW and in broth RCM (reinforced clostridial medium (RCM; Oxoid, (CM0149))) or BHI broth, respectively, for C. sporogenes and C. difficile to study the efficacy of these agents in the presence of low concentrations of organic matter.
4
Reviving Propionibacterium freudenreichii for Experiments
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Propionibacterium freudenreichii (DSM 2027) was obtained from German collection of Microorganisms and Cell Cultures GmbH, Leibniz Institute DSMZ, Germany. The bacteria were freeze-dried and packaged in a double glass vial. To revive the freeze-dried bacterium cells, the bacteria were grown at 37 °C for 3 days in an anaerobic chamber using anaerogen gas packs (Thermo Fisher Scientific, Waltham, MA, USA) using reinforced clostridial media (RCM) broth (CM0149 Oxoid, Basingstoke, UK) and tryptic soy agar (TSA) enriched with sheep blood (Oxoid, Basingstoke, UK). The P. freudenreichii were washed twice with PBS and centrifuged at 5000× g. After that, bacteria were suspended at a concentration of 1 × 109 CFU in PBS to create live bacterial inoculum. The cell-free supernatant was prepared by filtering the bacterial culture broth via a 0.22 µm polyethersulfone (PES) membrane and stored in single use aliquots at −20 °C until needed.
5
Isolation of Oral and Gut Bacteria
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To isolate Streptococcus salivarius, human saliva was plated onto an autoclaved reinforced clostridial medium (CM0149, Oxoid, Basingstoke, Hants, UK) with 1% agar (Nacalai Tesque, Kyoto, Japan) and incubated at 37°C under aerobic conditions for 1 day. In addition, a single colony was subcultured in brain heart infusion (BHI) medium (BD Co., USA) and stored as a stock culture at −80°C with 16% (vol vol−1) glycerol.
For isolation of Limosilactobacillus fermentum, a human faecal sample was cultured in a medium containing 20 g L−1 of young barley leaf extract (Genryoya, Osaka, Japan) and 0.5 g L−1 of L‐cysteine hydrochloride and cultured at 37°C under anaerobic condition (N2: 80%, H2: 10%, CO2: 10%) for 7 days. This culture solution was mixed with the same medium, containing 1.5 g L−1 of agar. A single colony was picked from this pour plate, subcultured in reinforced clostridial medium and stored as a stock culture at −80°C with glycerol (16% [v/v]).
S. salivarius and L. fermentum were precultured overnight in BHI and Difco Lactobacilli MRS medium (Becton, Dickinson), respectively. At 0 h of cultivation, 4 × 104 cells mL−1 of S. salivarius or L. fermentum was administrated into the in vitro model. Control was set without any administration. Cultivation was conducted for 72 h.
For isolation of Limosilactobacillus fermentum, a human faecal sample was cultured in a medium containing 20 g L−1 of young barley leaf extract (Genryoya, Osaka, Japan) and 0.5 g L−1 of L‐cysteine hydrochloride and cultured at 37°C under anaerobic condition (N2: 80%, H2: 10%, CO2: 10%) for 7 days. This culture solution was mixed with the same medium, containing 1.5 g L−1 of agar. A single colony was picked from this pour plate, subcultured in reinforced clostridial medium and stored as a stock culture at −80°C with glycerol (16% [v/v]).
S. salivarius and L. fermentum were precultured overnight in BHI and Difco Lactobacilli MRS medium (Becton, Dickinson), respectively. At 0 h of cultivation, 4 × 104 cells mL−1 of S. salivarius or L. fermentum was administrated into the in vitro model. Control was set without any administration. Cultivation was conducted for 72 h.
6
Culturing and Co-culturing Skin Cells
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Primary normal human epidermal keratinocytes were cultured at 37°C in 5% CO2 in Epilife medium supplemented with human keratinocyte growth supplement (Gibco, USA). Human microvascular endothelial cells (HMVECs) were cultured at 37°C in 5% CO2 in EBM-2 basal medium supplemented with EBM-2 growth medium (Lonza, USA).
M. furfur (ATCC 12078) was cultured at 30°C on Difco YM agar supplemented with 1% olive oil. S. epidermidis (Staphylococcus epidermidis, ATCC 12228) was cultured at 37°C on Difco tryptic soy agar. C. acnes (Cutibacterium acnes, ATCC 6919) was cultured at 37°C on forced clostridial medium (CM0149; Oxoid) with 2% agar. To induce hypoxia, a BD GasPakTM EZ Pouch was used. All the media were sterilized by autoclaving at 121°C for 15 min.
Organisms were harvested by centrifugation, and the pellet was suspended in the corresponding media. The organisms were heat-killed by incubation at 80°C for 3 min, and then co-cultured with normal human epidermal keratinocytes or human microvascular endothelial cells for 24 h at a density of 1 × 105 cells/mL. To induce allergic environments, recombinant thymic stromal lymphopoietin (TSLP) (50 ng/mL) or IL-4 (50 ng/mL) was used.
M. furfur (ATCC 12078) was cultured at 30°C on Difco YM agar supplemented with 1% olive oil. S. epidermidis (Staphylococcus epidermidis, ATCC 12228) was cultured at 37°C on Difco tryptic soy agar. C. acnes (Cutibacterium acnes, ATCC 6919) was cultured at 37°C on forced clostridial medium (CM0149; Oxoid) with 2% agar. To induce hypoxia, a BD GasPakTM EZ Pouch was used. All the media were sterilized by autoclaving at 121°C for 15 min.
Organisms were harvested by centrifugation, and the pellet was suspended in the corresponding media. The organisms were heat-killed by incubation at 80°C for 3 min, and then co-cultured with normal human epidermal keratinocytes or human microvascular endothelial cells for 24 h at a density of 1 × 105 cells/mL. To induce allergic environments, recombinant thymic stromal lymphopoietin (TSLP) (50 ng/mL) or IL-4 (50 ng/mL) was used.
7
Microbial Quality Assessment of Smoked Fish
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Smoked fish sample preparation and tenfold decimal dilutions were according to ISO/6887-1 (2017). The assessment of the microbial quality includes enumeration of the total aerobic plate count using plate count agar (Oxoid CM0325) and incubation at 35 °C for 48 hr (Dale Morton, 2001), the total mesophilic anaerobic sporeformers bacterial count using Reinforced Clostridial Agar "RCM" (Oxoid CM0149) and anaerobic incubation in a gas pack system at 35 ºC for 48 h (Scott et al., 2001) . Incubation of Baird-Parker agar plates (Oxoid, CM1127) at 37 ºC for 48 hours was used for enumeration of the presumptive Staph. count (Bennett and Ga, 2016), while incubation of Sabaroud dextrose agar (M063 HIMEDIA, Germany) for 5 days at 25 ºC was used for counting the total mold counts (Beuchat and Cousin, 2001) . The average count for each sample was reported as log 10 Colony-Forming Units/g sample (log 10 CFU/g).
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