Babesia microti (Franca) Reichenow (ATCC^® PRA-398^™)

1. Prepare a 30% (v/v) sterile glycerol solution in Alsever's solution.
2. Draw approximately 0.5 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 into the syringe from a host animal that has reached or is near peak parasitemia.  If clotting occurs during extraction of blood, insufficient heparin was used.  
3. Mix the heparinized blood with the 30% glycerol solution in a 2:1 ratio.  If any clotting has occurred do not use.  After mixing, the final concentration of cryoprotectant solution will be 10% (v/v).  The mixture should be placed in a 4°C ice bath.  The time from the mixing of the cell preparation and glycerol stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
4. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryovials (special plastic vials for cryopreservation).  Filled ampules should be placed in a 4°C ice bath.  Do not immerse ampules to the level of the vial cap.
5. Plunge ampules from 4°C into liquid nitrogen.  The frozen preparations may be stored in a mechanical freezer until needed, however storage in either the vapor or liquid phase of a nitrogen refrigerator is recommended for the longest viability.
6. To thaw a frozen ampule, place in a 35°C water bath, until thawed (2-3 min).  Immerse the ampule just sufficient to cover the frozen material.  Do not agitate the ampule.
7. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected hamster.  Follow the protocol for maintenance in vivo.  The course of infection may be longer or shorter than usual depending on recovery of the parasite from the frozen state.

Gray J, et al. Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils. J Clin Microbiol 40(4): 1259-1263, 2002. PubMed: 11923342

Piesman J, et al. Development of Babesia microti sporozoites in adult Ixodes dammini. Int J Parasitol 16(4): 381-385, 1986. PubMed: 3744675

Ryan R, et al. Diagnosis of babesiosis using an immunoblot serologic test. Clin Diagn Lab Immunol 8(6): 1177-1180, 2001. PubMed: 11687460

Wilson M, et al. Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani. Exp. Parasitol. 149: 24-31, 2015. PubMed: 25500215

Nucleotide (GenBank) : AF510194.1 Babesia microti strain GI 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence

Nucleotide (GenBank) : AF231348.1 Babesia microti strain GI nuclear small subunit ribosomal RNA gene, partial sequence