Anophryoides soldoi Small and Lynn
ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.
ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
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 in a 35°C water bath, until thawed (2-3 min). Immerse the ampule just sufficient to cover the frozen material. Do not agitate the ampule.
2. Immediately after thawing, add 0.5 to 1 ml of media to vial and let stand 10 minutes.
3. Aseptically transfer the entire contents to a single 16 x 125 mm screw-capped test tube and gently add 4 ml of ATCC Medium 1651 broth. Incubate the tube on a 15o horizontal slant with the cap screwed on loosely (loosened one half turn) at 25°C.
2. Aseptically transfer a 0.1ml aliquot to 5 ml of fresh medium in a 16 x 125 mm screw-capped test tube.
3. Screw caps on loosely (loosened one-half turn) and incubate on a 15° horizontal slant at 25°C.
4. Subculture every 3-5 days.
1. Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min.
2. Adjust the concentration of cells to 2 x 106 - 2 x 107/ml in fresh medium.
3. While cells are centrifuging prepare a 22% (v/v) solution of sterile DMSO in fresh medium.
4. Mix the cell preparation and the 22% DMSO in equal portions. Thus, the final concentration will be 106 - 107 cells/ml and 11% (v/v) DMSO. The time from the mixing of the cell preparation and methanol stock solution to the beginning of the freezing process should be no less than 5 min and no greater than 15 min.
5. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge 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
7. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.
8. 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.
9. Immediately after thawing, aseptically transfer contents to a 16 x 125 mm screw-capped test tube containing 5 ml of ATCC Medium 1651.
10. Incubate the tube on a 15° horizontal slant with the cap screwed on loosely (loosened one half turn) at 25°C.
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.
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Gomez-Saladin E, Small E. Prey induced transformation of Miamiensis avidus strain Ma/2 by a soluble factor. J. Eukaryot. Microbiol. 40: 550-556, 1993.