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Tetrahymena sp.  拉丁名

(ATCC? PRA-370?)  統(tǒng)一編號

Deposited As Chilodonella uncinata Strain Designations North India strain BP 610 

Biosafety Level 生物安全等級 1 

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.

Isolation 分離基物 Mosquito larvae, Delhi, North India, 1999 

Product Format test tube 

Storage Conditions 保存方法

Frozen Cultures 冷凍物: -70°C for 1 week; liquid N2 vapor for long term storage 

Freeze-dried Cultures 凍干物 : 2-8°C 

Live Cultures 活物 : See Protocols section for handling information

Medium ATCC Medium 802: Sonneborn's Paramecium medium 

Growth Conditions 生長條件

Temperature 培養(yǎng)溫度 : 20°C to 25°C 

Atmosphere: Aerobic Culture system: With Klebsiella pneumoniae subsp. pneumoniae (ATCC 700831) or Enterobacter aerogenes (ATCC 13048). 

Cryopreservation Reagents

Cryoprotective Solution

DMSO, 2.0 mL 

Fresh growth medium w/o bacteria, 8.0 mL 

Harvest and Preservation 

1，Harvest the cells from a culture that is at or near peak density by centrifuging at 650 x g for 5 minutes.,

2，If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to 2 x 106 cells/mL with fresh medium.  If the concentration is too low, centrifuge at 650 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.,

3，While cells are centrifuging prepare a 20% (v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath.  Allow the DMSO to solidify.  Add the required volume of refrigerated medium.  Dissolve the DMSO by inverting the tube several times.   *NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.

4，Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will equal 1 x 106 cells/mL and 10% (v) DMSO. The 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 longer 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 vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. 

If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen.   Alternatively, place the vials in a Nalgene 1°C freezing apparatus.  

6，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.),

7，The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer. 

8，To establish a culture from the frozen state add 0.5 mL bacterized ATCC medium 802 to the frozen ampule and place it in a 35°C water bath.  Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.

9，Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate onto the surface of a 20 x 100 mm petri plate containing ATCC medium 919 (non-nutrient agar) with an overlay of 15.0 mL bacterized ATCC medium 802.,

10，Incubate at 25°C with the cap on loosely.,

11，Once the culture is established, transfer 0.5 mL to a T-25 tissue culture flask containing 10.0 mL of ATCC medium 802 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC 700831) or Enterobacter aerogenes (ATCC 13048).,

12，Incubate with the cap tightly sealed at 25°C. 

Alternative Thawing Procedure,

1，Aseptically add 0.5 mL of sterile exhausted* ATCC medium 802 containing 8% (w) sucrose to the ampule.  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.

2，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.  The cell suspension will pool at the edge of the plate.,

3，Continue to double the volume of the cell suspension at 10 minute intervals by dropwise addition of exhausted ATCC medium 802 containing 4% sucrose (w).  When the volume reaches 16.0 mL place the plate in horizontal position and incubate at 25°C. 

,4，Once the culture has been established subculture into a T-25 flask of bacterized ATCC medium 802 without sucrose. *Previously-bacterized ATCC medium 802 cleared by growth of bacteria (or by growth of ATCC PRA-370 Tetrahymena sp., if available), and filter sterilized. 

Name of Depositor B Das

Chain of Custody ATCC <-- B Das 

References Das BP. Chilodonella uncinata – a protozoa pathogenic to mosquito larvae. Cur. Sci. 85(5): 483-489, 2003.