Babesia duncani

1. Prepare the Puck's Saline Glucose (PSG) Medium, mix well, adjust pH to 7.2, and adjust the volume to 1 L with distilled, deionized water. Filter sterilize using a 0.22 μm filter and store at 4°C.
2. Prepare the PSG+G solution, mix well, filter sterilize using a 0.22 μm filter, and store at 4°C.
3. Aseptically, wash donor blood three times by centrifugation at 600 to 800 × g for 15 minutes at 4°C in RPMI 1640 medium.
4. After each wash, aseptically remove the supernatant, consisting of the plasmid and buffy (leukocyte) layers located on the top of the RBC (erythrocyte) pellet.
5. After the last wash, aseptically resuspend human erythrocytes in sterile PSG+G solution at a concentration of 50% erythrocytes. The human erythrocytes in PSG+G solution may be stored at 4°C until use for a maximum of two weeks.

1. Place the frozen vial in a 35°C to 37°C water bath until the culture is completely thawed. Transfer the vial to a biological safety hood and wipe the outside surface of the vial with 70% ethanol. 
2. Immediately after thawing, aseptically transfer the contents of the vial to a sterile 50 mL conical centrifuge tube using at 1 mL pipette.
3. Add dropwise a 12% sodium chloride (NaCl) solution to reach approximately a 1:5 ratio of NaCl to cell mixture (approximately 0.2× the original culture volume). Allow the vial to incubate for 5 minutes at room temperature.
4. Using a 10 mL pipette, add dropwise while shaking 10 volumes of a 1.6% NaCl solution (10:1 ratio of NaCl to original culture volume).
5. Centrifuge at 625 × g for 5 minutes. Remove the supernatant, leaving approximately 0.5 to 1 mL of supernatant in the tube. Resuspend the cells by gently swirling the tube.
6. Add dropwise while shaking 10 volumes of growth medium. Centrifuge at 625 × g for 5 minutes and carefully remove the supernatant.
7. Add 5 mL of growth medium (warmed to 37°C) and transfer the culture to a non-vented cap 25-mm² cell culture flask (T-25).
8. For continuous culture, add uninfected donor red blood cells (RBCs) to a 5% hematocrit (HT) suspension every 2 to 3 days.
9. Gently aerate the cultures with a 93% N₂, 5% CO₂, and 2% O₂ gas mixture through a sterile, cotton-plugged Pasteur pipet and then quickly tighten the cap. Incubate the flask at 37°C.
10. Monitor the parasitemia daily by microscopic examination of blood films stained with a Giemsa solution.

1. To determine parasitemia of the culture, prepare thin smears of 3 μL to 5 μL of cell culture samples on microscopic slides, fix in methanol and allow to air dry. Stain with a 5% Giemsa solution, allowing the slides to incubate in the stain for 40 minutes. Prepare fresh Giemsa solution on a daily basis.
2. Examine the slides under a microscope at 1000× magnification for the presence of intracellular parasite forms.
3. Count the number of infected RBCs versus the total number of RBSs under oil immersion and determine the % parasitemia.
   % parasitema = (infected RBC/Total RBC) × 100
   Note: For a more accurate determination of parasitemia, a minimum of 500 RBCs should be counted.

1. Carefully remove the flask with infected culture from the 37°C incubator without disturbing the RBCs and place it inside a biosafety cabinet.
2. Carefully remove the supernatant with a sterile, unplugged Pasteur pipet under vacuum, or using a sterile pipette. Remove as much of the supernatant as possible without removing the cells.
3. Remove a small cell sample for microscopic examination by Giemsa staining.
4. To the culture flask, gently add prewarmed (37°C) sterile growth medium and uninfected donor RBCs, as needed, for a total of 5% hematocrit. mix the medium and the cells inside the flask by gentle swirling.
5. Aerate the culture flask with a 93% N₂, 5% CO₂, 2% O₂ gas mixture through a sterile pipette, tighten the cap, and incubate the flask in a 37°C incubator.
   Note: for rapid increase of parasitemia, changing of the culture medium daily is required for Babesia-infected erythrocyte cultures. Subculture should be performed when the culture is stable and parasitemia reaches 6%.

1. Harvest Babesia cultures from multiple flask using sterile pipettes and transfer the cell suspensions to 15 mL or 50 mL plastic centrifuge tubes. Cultures should be well established and growing vigorously with a parasitemia ≥ 6%.
2. Centrifuge at 625 × g for 5 minutes at room temperature.
3. Wash the pellet once with 10 or more volumes of incomplete DMEM/F12 medium. Centrifuge the cell suspension at 625 × g for 5 minutes. Remove most of the supernatant, leaving enough supernatant to resuspend the pellet. Estimate the volume of the pellet.
4. To the volume of packed red blood cells, slowly add dropwise one volume of cold (4°C) Glycerolyte 57 solution (or equivalent). Allow to incubate for 5 minutes at room temperature.
5. Add dropwise an additional 4 volumes of cold Glycerolyte 57 solution to the pellet and mix well.
6. Dispense 0.5 mL aliquots into 1 to 2 mL sterile plastic screw-capped vials for cryopreservation.
7. Place the vials in a controlled-rate freezing unit. From room temperature, cool the vials 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 this phase. At -40°C, plunge vials into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing container. Place the container at -80°C for 1 to 2 days and then plunge vials into liquid nitrogen.
8. Store the frozen vials in either the vapor or liquid phase of a nitrogen refrigerator (-130°C or colder).

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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