Bdellovibrio exovorus

1. Have a 24 hour host culture ready. The host, BAA-2331™, can be purchased from ATCC^®. Alternatively, the host may be recovered from the Bdellovibrio exovorus preparation by streaking a drop on ATCC^® medium #36 plate and incubating at 30°C. Once growth of the host has been obtained, a colony of the host can be picked and inoculated into ATCC^® #36 broth and incubated at 30°C.
2. The Bdellovibrio can be propagated in a variety of ways. Open the FD vial according to the enclosed instructions.
   1. Propagation in Broth: Inoculate 0.5 mL of a 24 hour host culture into 5 - 10 mL ATCC^® medium 2814. Take 0.5 mL of this culture and add it to the vial to rehydrate the FD pellet. Add the entire content the vial back into the host culture. Incubate at 30°C with shaking
   2. Double Layer Technique: Warm up plates of #2813, bottom agar, by placing them at 30°C for approximately one hour.
      1. Melt tubes of top agar (2.5 – 3.0 mL agar / tube) by boiling and then placing the tubes in a 45°C water bath.
      2. Once the top agar has cooled to 45°C, add 0.3 mL host (BAA-2331™) and 0.1 – 0.2 mL of Bdellovibrio exovorus (BAA-2330™), mix by rolling the tube between your hands and pour onto the bottom agar plate. Cover the bottom agar with the top agar by swirling the plate. Let the plate sit at room temperature until the top agar hardens. Incubate the inverted plates at 30°C.
3. After 24 to 48 hours, growth of the Bdellovibrio can be detected in the broth by a decrease in optical density. When examined by phase contrast microscope, the host cells are large, curved rods that are motile and appear singly and in pairs and clumps. The Bdellovibrio are small vibrio shaped cells that are very motile. As the Bdellovibrio continue to propagate, their densities should increase and the density of the host will decrease.
4. After 3 to 4 days of growth, plaques should appear in the top agar. Using a sterile microscope slide, one edge of the slide can be used to scrape the top agar off. The top agar can be placed in a sterile conical centrifuge tube and fresh #2814 broth can be added. Vortex, then let the particulates settle. Bdellovibrio can be detected under phase microscope [see step 3 above].
5. Bdellovibrio can be maintained for one week by storage at 4°C. Bdellovibrio can be stored long term by concentrating the cell by centrifugation then resuspending in maintenance medium with either 10% glycerol or 5% DMSO. Distribute the cells into cryovials, and store at a minimum of -80°C.

We have had difficulty detecting plaques using the double layer technique.  Despite not detecting plaques, when the top layer has been harvested, Bdellovibrio have been detected in large quantities (almost pure) when examined by phase.

We have had great success recovering Bdellovibrio using the broth technique.

Additional information on this culture is available on the ATCC^® web site at www.atcc.org.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.