Pneumocystis carinii Delanoe and Delanoe|376178 |BNCC

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Pneumocystis carinii Delanoe and Delanoe

Growth conditions	Mouse in vivo culture
Subculture procedure	1. Seven days prior to thawing the frozen ampule,immunosuppress rats (viral antibody-negative maleor 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(GIBC0 31800-022) containing 20% (v/v) heat-inactivated fetal bovine serum.4. Centrifuge at 10o0 x g for 5 minutes, remove supernatant and resuspend the pellet with mediumspecified in step 3 to a volume such that the final concentration of the P carinii nuclei is 107-108per mL (the concentration of the nuclei will be specified on the certificate of analysis shipped withthe frozen ampule).5. Aspirate O.1 mL of the well-mixed suspension into a 1.0 mL syringe fitted with a three-inch,20-gaugecurvedstainlessteelanimalfeedingtube(PopperandSon,NewHydePark,Ny.Keepflledsyringe 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 60degree incline. Pull tongue to one side of the lower incisors with a pair of forceps, insert thefeeding tube prepared in step 5 and express 0.1 mL of inoculum followed by 0.4 mL of air into thetrachea. Note: To assure correct placement of the inoculum into the trachea, direct the feedingtube 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 preparingimpression smears.
Storage conditions	2-8°C
Sharing mode	Public welfare sharing