Pneumocystis carinii Delanoe and Delanoe

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 ampules 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. 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. Do not leave ampule in water bath after thawed.
2. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected, immunosuppressed rat. Follow the protocol for maintenance in vivo. The course of infection may be longer or shorter than usual depending on percent recovery of the parasite from the frozen state.

1. Seven days prior to thawing the frozen ampule, immunosuppress rats (viral antibody-negative male or female rats weighing 120-140 grams each, any of several strains, e.g., Lewis, Sprague-Dawley, Fischer 344) by administering one injection of 4 mg of methylprednisolone acetate (Upjohn Co., Kalamazoo, MI).
2. On day seven, thaw the frozen ampule rapidly in a 35°C water bath as indicated above.
3. Transfer the thawed contents to a centrifuge tube and add an equal volume of RPMI 1640 medium (GIBCO 31800-022) containing 20% (v/v) heat-inactivated fetal bovine serum.
4. Centrifuge at 1000 x g for 5 minutes, remove supernatant and resuspend the pellet with medium specified in step 3 to a volume such that the final concentration of the P. carinii nuclei is 10⁷-10⁸ per mL (the concentration of the nuclei will be specified on the certificate of analysis shipped with the frozen ampule).
5. Aspirate 0.1 mL of the well-mixed suspension into a 1.0 mL syringe fitted with a three-inch, 20-gauge curved stainless steel animal feeding tube (Popper and Sons, New Hyde Park, NY). Keep filled syringe on ice until ready to inoculate.
6. Lightly anesthetize rats by exposing them briefly to halothane.
7. Suspend anesthetized rats by their upper incisors on a wire loop at the top of a board held at a 60 degree incline. Pull tongue to one side of the lower incisors with a pair of forceps, insert the feeding tube prepared in step 5 and express 0.1 mL of inoculum followed by 0.4 mL of air into the trachea. Note: To assure correct placement of the inoculum into the trachea, direct the feeding tube along the back of the tongue into the larynx while palpating the trachea.
8. Continue weekly injections of 4 mg of methylprednisolone into inoculated rats.
9. Six weeks post-inoculation, check for infection by killing a rat, removing the lungs and preparing impression smears.

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.