Gaspak ez anaerobe container system sachets

All samples were diluted in 1:10 phosphate-buffered saline and placed in a water bath for 10 minutes at 80°C to kill the non spore-forming bacteria. The processed samples were sub-cultured in cooked meat broth media (Oxoid, Cambridge, UK) for 24 hours under strict anaerobic conditions for enrichment of CP. After that, these inoculated broth tubes were directly streaked onto sterile freshly prepared blood agar plates (nutrient agar; Oxoid, UK that were supplemented with 5% sterile defibrinated sheep blood and Neomycin sulfate in a concentration of 200 μg/ml). All cultivated blood agar plates were then incubated in anaerobic jars at 37°C for 24 hours using anaerobe sachets (BD GasPak™ EZ Anaerobe Container System Sachets) to produce the strict conditions of anaerobiosis, which must be provided for vitality and viability of CP. Further identification tests for the recovered CP isolates including colony morphology, Gram staining, and biochemical tests (catalase, sugar fermentation, and gelatin liquefaction) were performed. In addition, the lecithinase and lipase activities of these recovered CP isolates on egg yolk agar medium were also examined. In addition, for toxinotyping of the recovered CP isolates, a toxin-antitoxin neutralization test was performed and interpreted according to Quinn et al. (2002) .

Nematodes were grown in anaerobic chambers. We seeded NGM plates with synchronized C. elegans or H. mephisto L1 larvae from overnight hatching in M9 (at 20°C for C. elegans and 37°C for H. mephisto), allowing a few minutes to dry, and then placing the plates into 2L chambers (Cat # 260002) with BD GasPak EZ anaerobe container system sachets (Cat # 260678) to create an anaerobic environment. Nematodes were cultured at either 20°C or 37°C depending on the species in standard incubators, for the indicated time (2 or 9 days) before opening and measuring viability. Survival was counted by washing the worms off the plates and exposing them to Sytox Orange dye (Thermo Fisher Cat # S11368), diluted 1:1000 in M9, for 15 minutes. Worms were then collected by centrifugation (400g for 2 minutes), pipetted onto 2% agarose-M9 pads, coverslipped, and sealed with clear nail polish prior to measurement of the dead animals using an Olympus BX61 fluorescence microscope set to the TRITC channel. Worm lengths (live animals only) were measured at the same time using CellSense v. 2.3 software.