Hexamita inflata Dujardin (ATCC® 50268)

Organism: Hexamita inflata Dujardin  /  Depositor: TK Sawyer

Permits and Restrictions

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Deposited As Hexamita sp.
Strain Designations AZ-4
Biosafety Level 1
Isolation
fibrous plant roots and soil from small pond, Desert Museum, Tucson, AZ, 1991
Product Format frozen
Type Strain no
Comments
Structure/phylogeny of glyceraldehyde-3-phosphate dehydrogenase genes
Small subunit ribosomal RNA
Growth Conditions
Temperature: 25.0°C
Duration: anaerobic
Protocol: ATCCNO: 50268 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: 50268 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

1.   Harvest the cells from a culture that is at or near peak density by centrifuging at 850 x g for 5 minutes.

2.   If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 106 and 2 x 107 cells/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.

3.   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.

4.   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.

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.  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 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.

9.   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 TK Sawyer
Chain of Custody
ATCC <<--TK Sawyer<<--T.A. Nerad
Year of Origin 1991
References

Rozario C, et al. Primary structure and phylogenetic relationships of glyceraldehyde-3-phosphate dehydrogenase genes of free-living and parasitic diplomonad flagellates. J. Eukaryot. Microbiol. 43: 330-340, 1996. PubMed: 8768438

Leipe DD, et al. Small subunit ribosomal RNA+ of Hexamita inflata and the quest for the first branch in the eukaryotic tree. Mol. Biochem. Parasitol. 59: 41-48, 1993. PubMed: 8515782

Chen N, et al. A dynein heavy chain homologue gene in Hexamita inflata. Gene 208: 83-87, 1998. PubMed: 9479053

Cross References

Nucleotide (GenBank) : L07836 Hexamita inflata small subunit (16S-like) ribosomal RNA sequence.

Nucleotide (GenBank) : AF022888 Hexamita inflata dynein heavy chain homolog (hdyhh) gene, complete cds.

Nucleotide (GenBank) : U40817 Hexamita inflata glyceraldehyde-3-phosphate dehydrogenase gene, partial cds.

Nucleotide (GenBank) : L07836 Hexamita inflata small subunit (16S-like) ribosomal RNA sequence.

Nucleotide (GenBank) : AF022888 Hexamita inflata dynein heavy chain homolog (hdyhh) gene, complete cds.

Nucleotide (GenBank) : U40817 Hexamita inflata glyceraldehyde-3-phosphate dehydrogenase gene, partial cds.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation