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.
1. Vigorously agitate the flask and aseptically transfer 0.5 ml from a growing culture to a T-25 tissue culture flask containing 10.0 ml of ATCC medium 802 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831)
2. Incubate flask at 25°C with the cap loosened one half turn.
DMSO 2.0 ml
Fresh growth medium w/o bacteria 8.0 ml
2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 500 x g for 5 min.
3. Adjust the concentration of cells at least 2 x 106/ml in fresh medium.
4. Allow the concentrated cells to remain undisturbed for approximately 1 hour. Make sure that the concentrated suspension has plenty of air, i.e., the surface to volume ratio of the suspension should be high. This resting period allows the cells to repair membrane damage that occurs during centrifugation.
5. Mix the cell preparation and the cryoprotective solution in equal portions.
6. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. 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
8. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
9. To establish a culture from the frozen state place the vial in a 35°C water bath. Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 ml ATCC medium 802 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831).
10.Incubate at 25°C with the cap loosened one half turn.
11.Once the culture is established, vigorously agitate the flask
and aseptically transfer 0.5 ml to 10.0 ml of bacterized ATCC medium 802.
12.Follow the protocol for maintenance of culture.
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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While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.
This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.
Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.
Smirnov, AV, et al. Correct identification of species makes the amoebozoan rRNA tree congruent with morphology for the order Leptomyxida Page 1987; with description of Acramoeba dendroida n. g., n. sp., originally misidentified as 'Gephyramoeba sp.'. Eur J Protistol 44:35-44, 2008. PubMed: 17905574