Plasmodium falciparum Welch

Storage and Culture Initiation

The following directions for recovery from the frozen state must be carefully followed if a culture is to be successfully established.

1. Place the frozen vial in a 37^°C water bath until mixture is completely thawed.
2. Aseptically transfer the contents to a 50 ml sterile conical tube.
3. Slowly add 1 volume (0.1 ml) 12% Sodium Chloride solution dropwise via a 1ml syringe to 5 volumes sample (0.5 ml) and agitate continuously.
4. Allow the mixture to stand for 5 mins. at room temperature.
5. Slowly add 5 ml 1.8% Sodium Chloride dropwise via a larger syringe and allow to stand at room temperature for 2 mins.
6. Add 5 ml of 0.9% Sodium Chloride / 0.2% Glucose solution as in step 5.
7. Centrifuge for 5 min. at 1500rpm, remove supernatant.
8. Wash pellet in 20 ml incomplete medium.
9. Centrifuge for 5 min at 1500 rpm, remove supernatant.
10. Resuspend pellet in 8ml complete medium in tissue culture flask and gently aerate culture with gas mixture of 5% CO₂, 5% O₂ and 90% N₂ using a sterile, cotton plugged Pasteur pipet.
11. Smear if required (see below).

* Medium Formulation:

NOTE: Outdated blood and serum cannot be used to cultivate this strain. Use only fresh blood and serum.

Making a Blood Smear:

1. Aseptically transfer 0.5–1ml of mixed culture with a sterile pipet into an eppendorf tube.
2. Spin down the eppendorf tube at high speed and aspirate the supernatant.
3. Mix the pellet and place a drop of the suspension on a glass slide. Spread the drop into a thin film with the edge of another glass slide. Air dry for 3 mins. at room temperature.
4. Fix air-dried blood film by rinsing with methyl alcohol. Air dry for 3 mins. at room temperature.
5. Stain blood films in 5% Giemsa solution for 15 mins. Rinse with distilled water, air dry.
6. Observe using light microscopy at 100X magnification to determine parasitemia of culture.

Only young cells (rings) can be frozen in glycerolyte medium** because their membranes are more robust.

1. Centrifuge ring stage culture for 5 min at 1800rpm 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 (see below) to 3 volumes pellet dropwise via a syringe as follows:
   1. Add the first volume of glycerolyte and allow the tube to stand for 5 mins. at room temperature.
   2. Add the remaining 4 volumes of glycerolyte and gently agitate.
5. Aliquot mixture into Nunc screwtop freezing vials and store at -80^°C overnight.
6. Plunge vials into liquid nitrogen (-196^°C) the next day and store in liquid nitrogen or liquid nitrogen vapor.

** To formulate glycerolyte medium, combine the following with distilled water to 100 ml:  57.00g glycerol, 1.60g sodium lactate (C₃H₅NaO₃), 30.00mg potassium chloride (KCl), 1.38g sodium dihydrogen phosphate (NaH₂PO₄). Mix well and adjust pH to 6.8 using concentrated NaOH and/or HCl. Autoclave to sterilize, and store at 4^°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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