Vector borne research

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

raccoon, Procyon lotor, Laurel, MD, 1955

Host specificity of ribosomal DNA variation

Temperature: 25.0°C

Duration: axenic

Protocol: ATCCNO: 11745 SPEC: See general instructions for thawing and storage of frozen material before proceeding. Add thawed contents to a single 16 x 125 mm glass screw-capped test tube of the appropriate medium. Incubate the culture vertically with the cap screwed on tightly. It is essential to establish cultures initially in small volumes. Once established, the culture can be scaled up to larger volumes. Vigorously agitate the culture and aseptically transfer 0.1 ml of culture to a fresh tube of medium weekly.

Protocol: ATCCNO: 11745 SPEC: See general instructions for thawing and storage of frozen material before proceeding. Add thawed contents to a single 16 x 125 mm glass screw-capped test tube of the appropriate medium. Incubate the culture vertically with the cap screwed on tightly. It is essential to establish cultures initially in small volumes. Once established, the culture can be scaled up to larger volumes. Vigorously agitate the culture and aseptically transfer 0.1 ml of culture to a fresh tube of medium weekly.

1.xa0xa0 Harvest cells from several cultures in very late logarithmic to early stationary phase of growth.xa0 Vigorously agitate to suspend the cells.

2.xa0xa0 Aseptically transfer the cell suspension to 15 ml plastic centrifuge tubes.

3.xa0xa0 Centrifuge at ~800 x g for 5 min.

4.xa0xa0 While cells are centrifuging, prepare a 10% solution of DMSO in complete ATCC Medium 1029.xa0 Cool on ice.

5.xa0xa0 Remove the supernatant and pool the cell pellets to the final volume desired with fresh growth medium.

6.xa0xa0 Combine the cell suspension with an equal volume of 10% DMSO cryoprotectant solution (prepared in step 4) to yield a final concentration of 5% DMSO.

7.xa0xa0 Dispense in 0.5 ml aliquots to 1.0-2.0 ml Nunc vials (special plastic vials for cryopreservation).

8.xa0xa0 Place the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 At -40°C, plunge ampules into liquid nitrogen.xa0 Alternatively, place the vials 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. (The cooling rate in this apparatus is approximately -1°C/min.). xa0

9.xa0xa0 Store ampules in a liquid nitrogen refrigerator until needed.

10.xa0xa0xa0xa0xa0xa0xa0xa0xa0 To establish a culture from the frozen state, place a frozen ampule in a 35°C water bath just enough to cover the frozen material.xa0 Allow the ampule to thaw completely (2-3 min).

11.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Immediately after thawing, aseptically remove the contents and transfer to a T-25 tissue culture flask containing 10 ml of fresh complete ATCC medium 1029.

12.Screw the cap on tightly and incubate at 20-25°C.xa0xa0xa0 Observe the culture daily and transfer when numerous trophozoites are observed.xa0xa0xa0xa0xa0xa0xa0xa0xa0