1. Harvest cells from a culture which is at or near peak density by centrifugation at 800-1000 x g for 5 min.
2. Adjust concentration of cells to between 2 x 10⁷ and 2 x 10⁸ cells/mL in fresh medium. xa0If the cell concentration is too low, centrifuge at 800-1000 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.
3. While cells are centrifuging, prepare a 10% (v/v) solution of sterile DMSO in fresh medium (broth).xa0 The DMSO solution when first prepared will warm up due to chemical heat. xa0The solution should be allowed to return to room temperature prior to use.
4. Mix the cell preparation and the DMSO solution in equal portions. xa0The final concentration will be 10⁷ – 10⁸ cells/mL and 5% (v/v) DMSO. xa0The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process should be no less than 15 min and no more than 30 min.
5. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the ampules in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.)xa0 If freezing unit can compensate for the heat of fusion, maintain rate atxa0-1°C/min through heat of fusion.xa0 At -40°C, plunge ampules into liquid nitrogen.
7. Store in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state, place an ampule in a 35°C water bath (2-3 min). Immerse the vial just sufficiently to cover the frozen material. Do not agitate the vial.
9. Remove the vial from the water bath immediately after thawing.xa0 Aseptically transfer the entire contents of the ampule into a T-25 tissue culture flask containing 10.0 mL complete medium.xa0 Incubate with the cap tightly sealed at 20-25°C.xa0
10. Maintain as described above.xa0

Schwarz RS, et al. Characterization of Two Species of Trypanosomatidae from the Honey Bee Apis mellifera: Crithidia mellificae Langridge and McGhee, and Lotmaria passim n. gen., n. sp. J. Eukaryot. Microbiol. doi: 10.1111/jeu.12209, 2015. PubMed: 25712037

Nucleotide (GenBank) : KJ713353 Lotmaria passim clone 277-5+6b(x5) glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene, partial cds

Nucleotide (GenBank) : KJ713354 Lotmaria passim clone 277-5+6f glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene, partial cds

Nucleotide (GenBank) : KJ713355 Lotmaria passim clone 277-5+6g(x5) glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene, partial cds

Nucleotide (GenBank) : KJ713356 Lotmaria passim clone 277-5+6n glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene, partial cds

Nucleotide (GenBank) : KJ713377 Lotmaria passim clone 278-5+6a_(x13) small subunit ribosomal RNA gene, partial sequence

Nucleotide (GenBank) : KJ713378 Lotmaria passim clone 278-5+6i small subunit ribosomal RNA gene, partial sequence