testing

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.

River Cam at Kings College, Cambridge, England, 1990

This strain has a unique DNA sequence of the D2 region of the 23S rDNA gene.

Intermediate between Colpidium and Glaucoma

Temperature: 25.0°C

Duration: axenic

Protocol: ATCCNO: 50402 SPEC: Loosen cap and aseptically transfer 0.25 ml of culture to fresh medium (16 x 125 mm screw-capped test tube containing 5 ml of ATCC medium 1034 available from ATCC as item 327-X). Place both tubes with caps loosened one full turn in an upright position in a 25C incubator. Aseptically transfer every 2 weeks.

Protocol: ATCCNO: 50402 SPEC: Loosen cap and aseptically transfer 0.25 ml of culture to fresh medium (16 x 125 mm screw-capped test tube containing 5 ml of ATCC medium 1034 available from ATCC as item 327-X). Place both tubes with caps loosened one full turn in an upright position in a 25C incubator. Aseptically transfer every 2 weeks.

1. xa0xa0Harvest cells from a culture at or near peak growth by centrifugation at 300 x g for 2 min.xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0

2.xa0xa0 Adjust concentration of cells to 2 x 10⁶/ml in fresh

xa0xa0xa0xa0xa0 medium.

3.xa0xa0 While cells are centrifuging, prepare a 13% (v/v) solution of sterile DMSO in fresh medium.

a) Add 1.3 ml of DMSO to an ice cold 20 x 150 mm screw-capped test tube;

b) Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 8.7 ml of ice cold medium;

c) Invert several times to dissolve the DMSO;

d) Allow to warm to room temperature.

4.xa0xa0 Add a volume of the DMSO solution equal to the cell

xa0xa0xa0xa0xa0 suspension volume but add in 3 equal aliquots at 2 min

xa0xa0xa0xa0xa0 intervals. Thus, the final concentration of the preparation

xa0xa0xa0xa0xa0 will equal 6.5% (v/v) DMSO and 10⁶ cells /ml.

5.xa0xa0 Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic

xa0xa0xa0xa0xa0 screw-capped cryules (special plastic vials for xa0xa0xa0xa0xa0 cryopreservation).

6.xa0xa0 Place the vials in a controlled rate freezing unit.xa0 Use the following cooling cycle: From room temperature cool at

-10°C/min to the heat of fusion; from the heat of fusion toxa0xa0

-40°C, cool at -1°C/min.xa0xa0 At -40°C plunge into liquid nitrogen.xa0 The cooling cycle should be initiated no less than 15 and no more than 30 minutes after the addition of DMSO to the cell preparation.

7.xa0xa0 Store in the vapor or liquid phase of a nitrogen

xa0xa0xa0xa0xa0 refrigerator.

8.xa0xa0 To establish a culture from the frozen state, asepticallyxa0 add 0.5 ml of sterile modified PYNFH medium (ATCC Medium 1034) containing 8% (w/v) sucrose to the frozen ampule.xa0 Immediately, place in a 35°C water bath, until thawed. Immerse the ampule just sufficient to cover the frozen material. Do not agitate the ampule.

9.xa0xa0 Immediately after thawing, aseptically remove the contents of the ampule and gently add the material to the edge of a 20 x 100 mm petri plate containing ATCC Medium 919 (non-nutrient agar) and position on a 15 degree slant. xa0The cell suspension will pool at the edge of the plate.

10.Continue to double the volume of the cell suspension at 10 minute intervals by adding ATCC medium 1034 containing 4% sucrose (w/v). xa0When the volume reaches 16.0 ml place the plate in a horizontal position and incubate at 25°C.xa0

11.xa0xa0xa0xa0xa0xa0xa0xa0xa0 On the following day, gently remove the cell suspension for the plate and transfer to a T-25 tissue culture flask. xa0Note the volume of the suspension and add a volume of fresh medium without sucrose equal to the volume of xa0the cell suspension. xa0Incubate the culture at 25°C.

12.xa0xa0xa0xa0xa0xa0xa0xa0xa0 After culture has been established subculture into fresh

xa0xa0xa0xa0xa0 medium without sucrose.xa0