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soil from aviary at base of tree, Tucson Desert Museum, Tuscon, AZ, 1991

This strain is tentatively placed in this genus because the cells superficially resemble members of Spongomonas based upon features discernable at the light microscopical level.

The strain will probably be assigned to a new genus.

Temperature: 25.0°C

Protocol: ATCCNO: 50397 SPEC: Store vial in refrigerator until ready to open it. Open sealed outer glass vial according to instructions provided with the vial. Rehydrate the material by adding 0.5 ml of ATCC medium 802 bacterized with Enterobacter aerogenes ATCC 13048. (Bacterize the medium by inoculating it with a loopful of bacteria from an agar slant at least 24 hours prior to inoculatiion with the flagellate.) Aseptically remove the shredded filter paper pellet with sterile forceps and add a T-25 tissue flask containing 10 ml of bacterized medium. Aseptically remove the remainder of the fluid from the glass vial and add it to the T-25 flask. Screw the cap on tightly and incubate the flask at 25C. Transfer every 21-28 days by vigorously agitating the flask and aseptically transferring 0.1 ml to a fresh flask of bacterized medium.

Protocol: ATCCNO: 50397 SPEC: Store vial in refrigerator until ready to open it. Open sealed outer glass vial according to instructions provided with the vial. Rehydrate the material by adding 0.5 ml of ATCC medium 802 bacterized with Enterobacter aerogenes ATCC 13048. (Bacterize the medium by inoculating it with a loopful of bacteria from an agar slant at least 24 hours prior to inoculatiion with the flagellate.) Aseptically remove the shredded filter paper pellet with sterile forceps and add a T-25 tissue flask containing 10 ml of bacterized medium. Aseptically remove the remainder of the fluid from the glass vial and add it to the T-25 flask. Screw the cap on tightly and incubate the flask at 25C. Transfer every 21-28 days by vigorously agitating the flask and aseptically transferring 0.1 ml to a fresh flask of bacterized medium.

1.?? Harvest cysts from several cultures in stationary phase of growth.? Detach cysts using a sterile cell scraper or cotton swab.

2.?? Aseptically transfer the cyst suspension to 15 ml plastic centrifuge tubes.

3.?? Centrifuge at ~800 x g for 5 min.

4.?? While cysts are centrifuging, prepare a 20% solution of DMSO in bacterized ATCC Medium 802.? Cool on ice.

5.?? Remove the supernatant and pool the cell pellets to one-half the final volume desired with fresh growth medium.

6.?? Combine the cell suspension with an equal volume of 20% DMSO cryoprotectant solution (prepared in step 4) to yield a final concentration of 10% DMSO.

7.?? Dispense in 0.5 ml aliquots to 1.0-2.0 ml Nunc vials (special plastic vials for cryopreservation).

8.?? Place the vials in a controlled rate freezing unit.? From room temperature cool at -1°C/min to -40°C.? At -40°C, plunge ampules 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.). ?

9.?? Store ampules in a liquid nitrogen refrigerator until needed.

10.????????? To establish a culture from the frozen state, place a frozen ampule in a 35°C water bath just enough to cover the frozen material.? Allow the ampule to thaw completely (2-3 min).

11.????????? Immediately after thawing, aseptically remove the contents and transfer to a T-25 tissue culture flask containing 10 ml of fresh? ATCC medium 802 inoculated with Enterobacter aerogenes (ATCC 13048).

12.Screw the cap on tightly and incubate at 25°C.? Subculture every 10-15d.??????

ATCC <<--TK Sawyer<<--T.A. Nerad