Crithidia fasciculata Leger
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.
2. Transfer approximately 0.10 ml to a fresh tube containing 5 ml of fresh ATCC medium 355.
3. Incubate upright at 25°C with caps screwed on tightly.
4. Transfer every 14 days.
1. Prepare a 10% (v/v) sterile DMSO solution in fresh ATCC Medium 355.
2. Transfer a culture at peak density to centrifuge tubes and centrifuge at 525 x g for 5 minutes.
3. Remove the supernatant and resuspend the cells in ATCC medium 355 to a concentration of 2 x 106 to 2 x 107 cells/ml.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 106 and 107 cells/ml and 5% (v/v) DMSO.
5. Distribute the cell suspension in 0.5 ml aliquots into 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation). The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
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 -1°C/min.)
7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the vial.
9. Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 5 ml of fresh ATCC medium 355 in a 16 x 125 mm screw-capped test tube. Incubate upright at 25°C with caps screwed on tightly.
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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Raether W, et al. Adaption of amoebae-Crithidia-cultures (Entamoeba histolytica) to axenic conditions of cultivation in TP-S-1-medium of Diamond 1968 (author's transl). Z. Parasitenkd. 42: 279-291, 1973. PubMed: 4360330
Clark CG. Riboprinting: A tool for the study of genetic diversity in microorganisms. J. Eukaryot. Microbiol. 44: 277-283, 1997. PubMed: 9225441
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