ATCC? Number:50614?
Organism: Blastocystis hominis Brumpt
Designations: WBY
Depositors: CH Zierdt
Biosafety Level:2
Growth Conditions: ATCCmedium 1671: Blastocystis egg mediumTemperature: 25.0°C Duration: anaerobic Protocol: ATCCNO: 50177 SPEC: The following directions for recovery from the frozen preparation must be carefully followed if a culture is to be successfully established: 1. Upon receipt, it is necessary to store the frozen ampule at temperatures of -135C or lower for at least 48 hours prior to thawing. The ampule may be stored in a mechanical refrigerator set at -60 to -70C if the storage time does not exceed 48 hours. The ampule should be removed from the dry-ice when stored in either a mechanical or nitrogen refrigerator. 2. After the frozen ampule is placed in storage, prepare a single tube of reduced ATCCmedium 1671 in the manner outlined in step 1 under Maintenance of Strain and Subculturing below. 3. When the medium is ready for inoculation, thaw the ampule in a 35C water bath without agitation (do not fully immerse ampule) and remove vial immediately after the preparation has become completely liquid. 4. Aseptically, gently lower a sterile Pasteur pipette from which the air has been expelled to the bottom of the liquid in the ampule and slowly aspirate the entire contents into the pipette. Be careful to minimize agitation of the fluid and do not introduce air bubbles at any time. These organisms are sensitive to oxygen. 5. Add the cell suspension to the bottom of the liquid overlay of the reduced tube of ATCCmedium 1671. Avoid expulsion of air bubbles from the tip of the pipette. 6. With the cap of the test tube loosened (1 full turn) place it in an anaerobic jar which contains a BBL GasPak (one anaerobic system GasPak per BBL GasPak 100 anaerobic culture jar). Close the vessel securely and incubate at 35C. Maintenance of Strain and Subculturing: Subculture at 2-3 day intervals. 1. Prior to inoculation, reduce (lower redox potential) complete ATCCmedium 1671 by placing the tubes with caps on loosely (1 full turn) in an anaerobic jar containing a BBL GasPak (one anaerobic system GasPak per BBL GasPak 100 anaerobic culture jar) for at least 48 hours. Note: The palladium catalyst in the GasPak jar should be replaced biweekly with fresh catalyst. 2. To subculture, remove the growing cultures without agitation from the anaerobic jar and immediately screw the caps down tightly. The strain grows at the bottom of the liquid overlay as a dense mass of cells. 3. Carefully introduce a sterile Pasteur pipette aseptically through the liquid overlay - air interface (avoid expulsion of air bubbles) and move the tip of the pipette to the cell mass, aspirate approximately one third of the mass into the pipette. Tighten the cap immediately unless the culture is to be promptly placed in the anaerobic jar. 4. Place the freshly inoculated culture into the anaerobic jar with the cap loosened (1 full turn), prepare the GasPak and quickly seal the jar.
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Classification: KINGDOM: Chromista