Pneumocystis murina

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. 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 it is thawed.
2. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected, immunosuppressed mouse.  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.

When the frozen ampule arrives, store it as indicated above until ready to use. The following directions for establishing an infection have been adapted and modified from: Boylan, C.J. and W.L. Current. 1992. Improved rat model of Pneumocystis carinii pneumonia: induced laboratory infections in  Pneumocystis-free animals. Infect. Immun. 60: 1589-1597. They must be followed carefully to assure success.

1. Up to two weeks* prior to thawing the frozen ampule, immunosuppress mice (viral antibody-negative male or female mice, any strain, e.g., C57, ICR, BALB/c) by administering dexamethasone at a concentraion of 4 mg/liter to their drinking water. (IVX Animal Health, Inc., St. Joseph, MO).
   *Note: Immunosuppression of mice may alternatively begin on the same day as the first inoculation with Pneumocystis.
2. 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. murina nuclei is 10⁷ to 10⁸ per mL (the concentration of the nuclei will be specified on the certificate of analysis shipped with the frozen ampule).
5. Aspirate 0.05 mL of the well-mixed suspension into a 1.0 mL syringe (optionally fitted with a 20-gauge stainless steel animal feeding tube; Popper and Sons, New Hyde Park, NY). Keep filled syringe on ice until ready to inoculate.
6. Lightly anesthetize mice by exposing them briefly to isoflurane.
7. Perform intranasal inoculation using the syringe prepared in step 5 by expressing 0.05 mL of inoculum onto the nares. Since mice are obligate nose-breathers, the Pneumocystis suspension will immediately be inhaled.
8. Continue administration of dexamethasone to inoculated mice as indicated above.
9. Six weeks post-inoculation, check for infection by killing a mouse, removing the lungs and preparing impression smears.

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