| 產品名稱 |
Hexamita pusilla Klebs |
| 商品貨號 |
B172533 |
| Strain Designations |
Bermuda |
| 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 |
moist soil near pond at Bermuda Biological Station, Bermuda, 1992 |
| Product Format |
frozen |
| Type Strain |
no |
| Comments |
Phylogeny based on SSU rRNA gene sequence |
| Medium |
ATCC® Medium 1773: Hexamita medium
|
| Growth Conditions |
Temperature: 25.0°C Duration: anaerobic Protocol: ATCCNO: 50336 SPEC:This strain is routinely shipped as a frozen stabilate. Thaw the ampule, aseptically transfer the material to a 16 x 125 mm screw-capped test tube containing 12 ml of bacterized ATCC medium 1773, screw the cap on tightly, and incubate on a 15 degree slant at 25C. Trophozoites should be active on the following day. Within 7-10 days most of the cells will encyst. The cysts remain viable for at least one month. However, to ensure viability, subculture every 14-21 days. To subculture, rub the surface of the tube with a sterile cotton swab, gently invert 10 times, aseptically transfer a 0.25-ml aliquot to a fresh tube of bacterized ATCC medium 1773, and incubate as above. This strain has been cultivated with a non-pathogenic strain of Enterobacter aerogenes ATCC-13048 as a food source. Other unidentified bacteria are also present. Note: ATCC medium 1773 is bacterized by inoculating with a partial bacteriological loopful of a non-pathogenic strain of K. pneumoniae subsp. pneumoniae ATCC-700831 (other strains of Klebsiella will probably work equally well) from an ATCC medium 3 slant approximately 24 hours before using. There is currently no known commercial source of rice starch that is suitable for inclusion in ATCC medium 1773. However, purified rice starch of a suitable nature can be prepared (Diamond, L.S. Lumen dwelling protozoa: Entamoeba, trichomonads, and Giardia. In: Jensen, J.B. ed., In vitro cultivation of protozoan parasites. Boca Raton, FL: CRC Press; 1983:p. 75).
|
| Subcultivation |
Protocol: ATCCNO: 50336 SPEC:This strain is routinely shipped as a frozen stabilate. Thaw the ampule, aseptically transfer the material to a 16 x 125 mm screw-capped test tube containing 12 ml of bacterized ATCC medium 1773, screw the cap on tightly, and incubate on a 15 degree slant at 25C. Trophozoites should be active on the following day. Within 7-10 days most of the cells will encyst. The cysts remain viable for at least one month. However, to ensure viability, subculture every 14-21 days. To subculture, rub the surface of the tube with a sterile cotton swab, gently invert 10 times, aseptically transfer a 0.25-ml aliquot to a fresh tube of bacterized ATCC medium 1773, and incubate as above. This strain has been cultivated with a non-pathogenic strain of Enterobacter aerogenes ATCC-13048 as a food source. Other unidentified bacteria are also present. Note: ATCC medium 1773 is bacterized by inoculating with a partial bacteriological loopful of a non-pathogenic strain of K. pneumoniae subsp. pneumoniae ATCC-700831 (other strains of Klebsiella will probably work equally well) from an ATCC medium 3 slant approximately 24 hours before using. There is currently no known commercial source of rice starch that is suitable for inclusion in ATCC medium 1773. However, purified rice starch of a suitable nature can be prepared (Diamond, L.S. Lumen dwelling protozoa: Entamoeba, trichomonads, and Giardia. In: Jensen, J.B. ed., In vitro cultivation of protozoan parasites. Boca Raton, FL: CRC Press; 1983:p. 75).
|
| Cryopreservation |
- Harvest the cells from a culture that is at or near peak density by centrifuging at 850 x g for 5 minutes.
- If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 106 and 2 x 107cells/ml with fresh medium. If the concentration is too low, centrifuge at 850 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
- While cells are centrifuging prepare a 20% (v/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.
- Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 106 and 107 cells/ml and 10% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
- Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
- 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. 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.)
- The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
- To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the vial.
- Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 12 ml of fresh ATCC medium 1773 in a 16 x 125 screw-capped test tube. Incubate on a 15° horizontal slant at 25°C.
|
| Name of Depositor |
DA Munson |
| Year of Origin |
1992 |
| References |
Cavalier-Smith T, Chao EE. Molecular phylogeny of the free-living archezoan Trepomonas agilis and the nature of the first eukaryote. J. Mol. Evol. 43: 551-562, 1996. PubMed: 8995052
Chen N, et al. A dynein heavy chain homologue gene in Hexamita inflata. Gene 208: 83-87, 1998. PubMed: 9479053
|
| Cross References |
Nucleotide (GenBank) :
U53120
Trepomonas agilis 18S ribosomal RNA gene.
|
