TSD-288 ™
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
Resuspend encapsulated prey bacteria cell pellet (e.g., K. pneumoniae ATCC 43816) to a concentration of 1 × 109 CFU/mL (optical density at 600 nm of ~2) in sterile 25 mM HEPES complete buffer (1L HEPES supplemented with 3.33 ml of 0.6 M MgCl2.7H2O and 6 ml of 0.5 M CaCl2·2H2O).
Defined or complex media can not support growth of Bdellovibrio reynosensis. It must be co-cultured with encapsulated prey cells. The prey cells, such as K. pneumoniae (ATCC 43816), are grown in rich media, centrifuged, and the cell pellet resuspended in HEPES complete buffer. Then co-culture with B. reynosensis (from glycerol stock, or a fresh plaque taken from a soft agar lawn of prey cells) with the prey cells in HEPES buffer. Incubate 1-3 days at 20-30°C with aeration. Monitor lysis of prey cells and appearance of small, motile B. reynosensis cells by Dark Field or Phase Contrast microscopy.
Depositor suggests to incubate 1-3 days at 20-30°C with aeration. The resulting lysate can be stored for up to a month at 4 °C. The lysate can be subcultured indefinitely, though it it unknown if B. reynosensis will mutate during repeated passaging. Consecutive subcultures can be done every 1-3 days. Alternatively the co-culture can be used to produce plaques in a DNB soft agar or soft agarose thin layer containing 5 × 109 CFU/mL of encapsulated prey. DNB soft agar or agarose is 5 g Agar or Agarose, 0.8 g Nutrient Broth (BD Difco™ Nutrient Broth, catalog number BD 234000) in 1L H2O; autoclave; cool to 55°C before use. A plaque can then be excised with a pasteur pipet and used to seed a fresh co-culture of prey cells.
Remove residual prey cells by clarifying, then filtering a fresh lysate. Clarify by centrifugation at 3,000 RCF for 2 minutes in a microfuge tube, or for larger volumes such as 50 mL, centrifuge at 2,500 RCF for 10 minutes. Pour off and save supernatant in a new tube. Filter the supernatant through a 0.45 µm pore size filter, saving the filtrate. A 0.45 µm pore size allows passage of B. reynosense but not larger prey cells such as K. pneumoniae. For small volumes, use a 4 mm diameter syringe filter (e.g., MilliporeSigma™ Millex™ Syringe Filters with PE Housing - PVDF Membrane - 4mm Diameter, catalog number SLHVR04NL). Larger diameter syringe filters will not allow passage of B. reynosensis. For larger volumes use a larger diameter 0.45 µm pore size PVDF membrane filter (e.g., MilliporeSigma™ Steriflip™ Sterile Disposable Vacuum Filter Units, catalog number SE1M003M00). Add glycerol to the filtrate to 20% (v/v) and mix well prior to freezing in cryovials.
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