Bodo caudatus (Dujardin) Stein

Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen.  If liquid nitrogen storage facilities are not available, frozen ampoules may be stored at or below -70°C for approximately one week.  Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C).  Storage of frozen material at this temperature will result in the death of the culture.

1. One day before thawing the ampule prepare bacterized ATCC medium 802, i.e., inoculate medium with a bacteriological loop of Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831) from a nutrient agar slant (ATCC medium 3).
2. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.  Do not agitate the ampule.
3. Immediately after thawing, aseptically transfer the contents to a T-25 tissue culture flask containing 10 mL of bacterized ATCC Medium 802.
4. Screw the cap on tightly and incubate the tube at 25°C.

1. Prepare bacterized ATCC medium 802.
2. Inoculate a T-25 tissue culture flask containing 10.0 mL of bacterized ATCC medium 802 with 0.1 mL from a culture of predominantly encysted cells.
3. Incubate at 25°C with cap screwed on tightly.
4. For routine maintenance subculture every 10 to 14 days.

1. Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min.
2. Adjust the concentration of cells to 2 x 10⁶ to 10⁷/mL in fresh medium.
3. While cells are centrifuging prepare a 20% (v/v) solution of sterile DMSO in fresh medium.
   1. Add 2.0 mL of DMSO to an ice cold 20 x 150 mm screw-capped test tube.
   2. Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 8.0 mL of ice cold medium.
   3. Invert several times to dissolve the DMSO.
   4. Allow to warm to room temperature.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be 10⁶ to 10⁷ and 10% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
5. Dispense in 0.5 mL aliquots into 1.0 mL to 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.  At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.  Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.  (The cooling rate in this apparatus is approximately -1°C/min.)  
7. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. If frozen material is to be stored at temperatures between -130°C and -70°C the shelf life should be empirically tested, i.e., remove stored material at intervals to determine die-off rate.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the ampule to a level just above the surface of the frozen material. Do not agitate the ampule.
9. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate a T-25 tissue culture flask containing 10 mL of ATCC medium 802 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831).
10. Incubate horizontally with the cap screwed on tightly at 25°C.

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.