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微信小章| 产品名称： | Balamuthia mandrillaris Visvesvara et al. |
|---|---|
| 商品货号： | TS147126 |
| Strain Designations： | CDC:V451 |
| Biosafety Level： | 2
Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. |
| Isolation： | Brain tissue from 6 year old female human, New York |
| Product Format： | frozen |
| Type Strain： | yes |
| Comments： | Genotyping based on SSU rDNA and mitochondrial rDNA |
| Medium： | ATCC® Medium 2222: Cell Cultivation Medium for Parasites |
| Growth Conditions： | Temperature: 35-37.0°C Growth condition: Grown on Vero cells ATCC CCL-81 |
| Cryopreservation： | 1.xa0xa0 To harvest the Balamuthia culture, detach cysts by scraping the inside bottom surface of the flask with a sterile cell scraper. 2.xa0xa0 Transfer the cyst suspension to 15 ml plastic centrifuge tubes. Centrifuge at approximately 800 x g for 5 min. 3.xa0xa0 Remove all but 0.5 ml of the supernatant from each tube, resuspend the cyst pellets, and pool them to a single tube. 4.xa0xa0 Adjust the concentration of cysts to 2.0 - 4.0 x 105 cysts/ml with fresh medium or PBS. xa0xa0xa0xa0xa0 NOTE: If the concentration of cysts is too low, centrifuge at 800 x g for 5 min and resuspend in the volume of fresh medium or PBS required to yield the desired concentration. 5.xa0xa0 Prepare a cryoprotective solution containing 15% (v/v) DMSO and 6% (v/v) HIFBS in fresh medium or PBS. 6.xa0xa0 Mix the cyst preparation and cryoprotective solution in equal portions. The final concentration will be 1.0 - 2.0 x 105 cysts/ml, 7.5% DMSO, and 3% HIFBS. The time from the mixing of the cyst preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min. 7.xa0xa0 Dispense in 0.5 ml aliquots to 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation). 8.xa0xa0 Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1 C/min through heat of fusion. 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.xa0xa0 Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator. 10.xa0xa0xa0xa0xa0xa0xa0xa0xa0 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.xa0 Do not agitate the ampule.xa0 Do not leave ampule in water bath after thawed. 11.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC CCL-81 cells and 10 ml ATCC 30-2003 with 3% (v/v) HIFBS. 12.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture. 13.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Incubate in a 35-37°C CO2 incubator with the cap screwed on tightly. |
| Name of Depositor： | GS Visvesvara |
| References： | Booton GC, et al. Genotyping of Balamuthia mandrillaris based on nuclear 18S and mitochondrial 16S rRNA genes. Am. J. Trop. Med. Hyg. 68: 65-69, 2003. PubMed: 12556151 Schuster FL, et al. Under the radar: balamuthia amebic encephalitis. Clin. Infect. Dis. 48: 879-887, 2009. PubMed: 19236272 |