Babesia microti (Franca) Reichenow

1. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.  Do not agitate the ampule.  Do not leave ampule in water bath after it is thawed.
2. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected hamster.  Follow the protocol for maintenance of the culture below.  The course of infection may be longer or shorter than usual depending on recovery of the parasite from the frozen state.

Yaeger's Anticoagulant
Sodium citrate: 1.33 g
Citric acid: 0.47 g
Dextrose: 3.00 g
Sodium heparin: 0.20 g
Glass distilled H₂O to: 100.00 mL

1.  Inoculate up to 0.5 mL of infected blood intraperitoneally into a hamster using facility approved methods.   
2. Monitor the infection daily or at 2-day intervals by examination of blood films stained with 5% Giemsa solution.   
3. Count the number of infected red blood cells (rbc) versus the total number of red cells under oil immersion and determine the % parasitemia: % parasitemia = infected rbc / rbc X 100. A minimum of 500 red blood cells should be counted. (Note that a red blood cell infected with multiple parasites is counted as a single infected cell.)   
4. When the level of parasitemia is ≥ 10% the strain should be passaged. Normally this would occur 1-3 weeks post-inoculation, but the rate of infection may vary considerably. (Note that the level of parasitemia before the host will succumb will vary with the strain used. Monitoring parasitemia as described above will alert the experimenter as to when the strain should be passaged.)   
5. To passage the strain, collect blood from the infected hamster using cardiac puncture or other facility approved method using a syringe and suitable anticoagulant.       
6. Anesthetize the animal by a facility approved method for anesthesia. Collect blood by cardiac puncture or other facility approved blood collection method.
7. Add approximately 0.05 - 0.1 mL of anticoagulant solution (Yaeger's or heparin, etc.) to a syringe.      
8. Transfer blood to a collection tube containing and additional 0.05 – 0.1 mL anticoagulant per mL of anticipated blood collection. Mix the blood and anticoagulant by gentle repeated inversion of the tube to prevent clotting of the blood.
9. If necessary, blood may be diluted with Alsever’s solution.      
10. Inject up to 0.5 mL of the infected blood suspension into uninfected hamster(s) to passage or expand the infection as needed. Monitor parasitemia and passage as needed.  

1. Prepare a 30% (v/v) sterile glycerol solution in Alsever's solution.   
2. Draw approximately 0.05 mL of anticoagulant solution (Yaeger's or heparin, etc.) into a syringe and move it back and forth over the length of the syringe, several times. Remove all air bubbles. Draw blood by cardiac puncture or other facility approved blood collection method from a host animal that has reached desired parasitemia. Transfer blood to a collection tube containing additional anticoagulant (0.05 – 0.1 mL per mL of anticipated blood collection). If clotting occurs during extraction of blood, insufficient heparin was used. Mix the heparinized blood with the 30% glycerol solution in a 2:1 ratio to obtain a final concentration of cryoprotectant of 10% (v/v). Mix slowly by inversion and place the mixture on ice. The freezing process should start 15 to 30 minutes following the addition of the heparinized blood to the cryoprotectant solution.
3. Dispense 0.5 mL aliquots of blood suspension into 1.0 to 2.0 mL sterile plastic screw-capped cryovials. Place the vials in a controlled rate freezing unit. From room temperature, cool the vials at -1C°/min to -40°C. If the freezing unit can compensate for the heat fusion, maintain rate at -1°C/min through this phase. At -40°C, plunge the vials into liquid nitrogen. Alternatively, place the vial in a Mr. Frosty freezing container. Place the container at -80°C for minimum 4 hours and then plunge vials into liquid nitrogen.
4. To thaw a frozen ampule, place in a 35°C ± 2°C water bath, until thawed (2-3 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the ampule.   
5. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected animal. Follow the protocol for in vivo propagation and maintenance in the Product Information Sheet. The course of infection may be longer or shorter than usual depending on percent recovery of the parasite from the frozen state.

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