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Tetrahymena tropicalis Nanney and McCoy
Tetrahymena tropicalis Nanney and McCoy
規(guī)格:
貨期:
編號:B235372
品牌:Mingzhoubio

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產(chǎn)品名稱 Tetrahymena tropicalis Nanney and McCoy
商品貨號 B235372
Strain Designations TC-3
Application
Identification of Tetrahymena species using PCR/RFLP analysis of rDNA
Biosafety Level 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
freshwater, irrigation ditch, near E. Mante, Mexico, 1954
Product Format test tube
Type Strain no
Comments
III
Identification of Tetrahymena species using PCR/RFLP analysis of rDNA
Tetrahymena from Mexico, Panama, and Columbia
Ribosomal RNA introns
unique position of the degenerating macronucleus
Medium ATCC® Medium 357: Tetrahymena medium
Growth Conditions
Temperature: 25.0°C
Duration: axenic
Cryopreservation
RM-9 Media for cryopreservation of Tetrahymena

Proteose Peptone (Difco 0120) ??????????????????????????????????? 5.0 g

Tryptone ???????????????????????????????????????????????????????????????????????????? 5.0 g

K2HPO4??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? 0.2 g

Glucose ????????????????????????????????????????????????????????????????????????????? 1.0 g

Liver extract??????????????????????????????????????????????????????????????????????? 0.1 g

Glass distilled water???????????????????????????????????????????????????????? 1.0 L

Dissolve components in glass distilled H2O and autoclave.

Dryl?s Salt Solution

0.1 M NaH2PO4 .? 3H20????????????????????????????????????????????????????????????????????????????? 10.0 ml

0.1 M Na2HPO4 . ?7H20????????????????????????????????????????????????????????????????????????????? 10.0 ml

0.1 M Sodium citrate . 2H20 ????????????????????????????????????????? 15.0 ml

0.1 M CaCl2 .? 2H20????????????????????????????????????????????????????????? 15.0 ml

Distilled water?????????????????????????????????????????????????????????????? 950.0 ml

Add the first 3 components to the distilled H2O and mix thoroughly.

Add the CaC12 ?solution and mix thoroughly.

(Adding the solutions in the order indicated will avoid the precipitation of Ca salts.)

1.? Transfer tetrahymena from usual growth medium to RM-9 medium and allow to grow to near peak density.

2.?? Harvest cells from a culture by centrifugation at 300 x g for 2 min.??????????

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

????? medium.

4.?? While cells are centrifuging, prepare a 22% (v/v) sterile

solution of sterile DMSO in fresh medium.

a) Add 2.2 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 7.8 ml of ice cold medium;

c) Invert several times to dissolve the DMSO;

d) Allow to warm to room temperature.

5.?? Add a volume of the DMSO solution equal to the cell

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

????? intervals. Thus, the final concentration of the preparation

????? will equal 11% (v/v) DMSO and 106 cells /ml.

6.?? Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic

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

7.?? Place the ampules in a controlled rate freezing unit. The

cooling cycle should be initiated no less than 15 min and no longer than 60 min after the addition of the DMSO to the cell preparation. 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? -50°C ampules are plunged into liquid nitrogen.

8.?? Store in the vapor or liquid phase of a nitrogen

????? refrigerator.

9.?? To establish a culture from the frozen state aseptically add 0.5 ml sterile Dryl's Salt Solution to an ampule. Immediately place the ampule 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.

10. Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 5.0 ml of fresh medium in a 16 x 125 mm screw-capped test tube with a slightly loosened cap. Incubate at 25°C.

CRYOPRESERVATION:

Alternative Thawing Procedure

?1.? Aseptically? add 0.5 ml of sterile modified PYNFH medium (ATCC Medium 1034) containing 8% (w/v) 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 adding ATCC medium 1034) containing 4% sucrose (w/v).  When the volume reaches 16.0 ml place the plate in horizontal position and incubate at 25°C. 

4.?? On the following day, gently remove the cell suspension for the plate and transfer to a T-25 tissue culture flask.  Note the volume of the suspension and add a volume of fresh medium containing 4% sucrose equal to the volume of  the cell suspension.  Incubate the culture at 25°C.

5.?? After culture has been established subculture into fresh

????? normal medium without sucrose. 

Name of Depositor DL Nanney, EM Simon
Chain of Custody
ATCC <<--DL Nanney, EM Simon<<--Univ. Michigan <<--- A.M. Elliott/ R.E. Hayes
Year of Origin 1954
References

Elliott AM, Hayes RE. Tetrahymena from Mexico, Panama, and Columbia, with special reference to sexuality. J. Protozool. 2: 75-80, 1955.

Elliott AM, et al. Tetrahymena pyriformis from several Pacific Islands and Australia. J. Protozool. 11: 370-378, 1964.

Simon EM, Doerder FP. The unique position of the degenerating macronucleus in Tetrahymena tropicalis. J. Protozool. 28: 203-205, 1981.

Jerome CA, Lynn DH. Identifying and distinguishing sibling species in the Tetrahymena pyriformis complex (Ciliophora, Oligohymenophora) using PCR/RFLP analysis of nuclear ribosomal DNA. J. Eukaryot. Microbiol. 43: 492-497, 1996. PubMed: 8976607

Sogin ML, et al. Phylogenetic evidence for the acquisition of ribosomal RNA introns subsequent to the divergence of some of the major Tetrahymena groups. EMBO J. 5: 3625-3630, 1986. PubMed: 3830129

Nanney DL, et al. Comparison of sequence differences in a variable 23S rRNA domain among sets of cryptic species of ciliated protozoa. J. Eukaryot. Microbiol. 45: 91-100, 1998. PubMed: 9495037

Cross References

Nucleotide (GenBank) : M98023 Tetrahymena tropicalis 16S ribosomal RNA gene, intron.

Nucleotide (GenBank) : X56168 T.tropicalis gene for small subunit ribosomal RNA (16S like rRNA).

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