Plasmodium falciparum Welch
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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.
In vitro culture in human erythrocyte
1. Remove flask with infected culture from 37oC incubator and place onto flask warmer in biological safety hood.
2. Carefully aspirate the medium with a sterile unplugged Pasteur pipet attached to a vacuum line. Remove as much fluid as possible without taking the cells.
3. Aseptically add sterile warm (37oC) completed medium to the flask (~8ml to a T-25, ~25ml to a T-75, etc.). Mix and smear as required to determine parasitemia (see below).
4. Add washed RBCs as necessary to obtain desired haematocrit and parasitemia.
5. Gently mix and aerate culture with gas mixture of 5% CO2, 5% O2 and 90% N2 using a sterile, cotton plugged Pasteur pipet. Quickly tighten cap of the flask and place in 37oC incubator until the next medium change.
1. Centrifuge ring-stage culture for 5 mins. at 1000 x g in 50 ml centrifuge tube.
2. Aspirate supernatant using sterile Pasteur pipet.
3. Resuspend pellet gently in remaining supernatant.
4. Slowly add 5 volumes of glycerolyte medium to 3 volumes pellet dropwise via a syringe as follows:
A. Add the first volume of glycerolyte and allow the tube to stand for 5 mins. at room temperature.
B. Add the remaining 4 volumes of glycerolyte and gently agitate.
5. Aliquot mixture into Nunc screw-capped freezing vials and place in a Nalgene 1C cooling apparatus. Place the apparatus at -80°C overnight and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.).
6. Plunge vials into liquid nitrogen (-196oC) the next day and store in liquid nitrogen or liquid nitrogen vapor.
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Nguyen-Dinh P, Trager W. Plasmodium falciparum in vitro: determination of chloroquine sensitivity of three new strains by a modified 48-hour test. Am. J. Trop. Med. Hyg. 29: 339-342, 1980. PubMed: 6992605