微信小陈
微信小章| 产品名称： | Entamoeba moshkovskii Tshalaia |
|---|---|
| 商品货号： | TS168083 |
| Strain Designations： | AP-1 |
| 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： | pool in rock crevice two feet above the splash zone; remains of a decaying bird were present in the pool, Appledore Island, ME, 1993 |
| Product Format： | test tube |
| Type Strain： | no |
| Comments： | Ribodeme 1. This strain is maintained in a seawater-based rice starch medium but can be adapted to grow in ATCC medium 1773 or ATCC medium 1171. Entamoeba phylogeny |
| Medium： | ATCC® Medium 1873: Seawater microaerophile medium |
| Growth Conditions： | Temperature: 25.0°C Duration: anaerobic Protocol: ATCCNO: 30131 SPEC: This culture is xenic; i.e., it contains mixed unidentified bacteria, some or all of which serve as food for the amoeba. 1) A growing culture is shipped in a 16 X 125 mm screw-capped test tube filled to within approximately two centimeters of the top with medium, a configuration which enhances survival in transit. 2) Immediately upon receipt of the culture, place it on a 15-degree slant at 35C. Allow the culture to remain undisturbed for at least three hours. 3) Observe the culture with an inverted microscope. Attached trophozoites should be evident. Reduce the volume of the culture to approximately 9 ml. 4) Centrifuge the removed culture fluid at 500 x g for five minutes. Under these conditions any trophozoites in suspension will be pelleted to the bottom of the tube. 5) Inoculate two fresh tubes of ATCC medium 1171 (available from ATCC as item IV-1171) with 0.25 ml of the supernatant derived in step 4 and incubate the tubes at 35C. These tubes will serve as preinoculated bacterized culture tubes. Preinoculation of medium with bacteria prior to subcultivation of Dientamoeba and bacterized Entamoeba strains allows for better growth. 6) Divide the remainder of the supernatant from step 4 into two equal aliquots in 16 X 125 mm screw-capped test tubes. Increase the volume of each tube to approximately 9 ml with fresh ATCC medium 1171. 7) Ice the parent shipped culture for five minutes, invert the tube 20 times and transfer 0.5- and 1.0-ml aliquots to the tubes just set up in step 6. 8) Incubate all cultures at 35C. Transfer cultures when they reach early stationary phase. The transfer interval will depend on the quality of the culture medium used. Inoculate bacterized culture tubes at least one day prior to subcultivation of Dientamoeba and bacterized Entamoeba strains. 9) In general, addition of penicillin G at 75 U/ml and streptomycin at 75 mcg/ml to ATCC medium 1171 may be necessary if the bacterial density is too high. |
| Subcultivation： | Protocol: ATCCNO: 30131 SPEC: This culture is xenic; i.e., it contains mixed unidentified bacteria, some or all of which serve as food for the amoeba. 1) A growing culture is shipped in a 16 X 125 mm screw-capped test tube filled to within approximately two centimeters of the top with medium, a configuration which enhances survival in transit. 2) Immediately upon receipt of the culture, place it on a 15-degree slant at 35C. Allow the culture to remain undisturbed for at least three hours. 3) Observe the culture with an inverted microscope. Attached trophozoites should be evident. Reduce the volume of the culture to approximately 9 ml. 4) Centrifuge the removed culture fluid at 500 x g for five minutes. Under these conditions any trophozoites in suspension will be pelleted to the bottom of the tube. 5) Inoculate two fresh tubes of ATCC medium 1171 (available from ATCC as item IV-1171) with 0.25 ml of the supernatant derived in step 4 and incubate the tubes at 35C. These tubes will serve as preinoculated bacterized culture tubes. Preinoculation of medium with bacteria prior to subcultivation of Dientamoeba and bacterized Entamoeba strains allows for better growth. 6) Divide the remainder of the supernatant from step 4 into two equal aliquots in 16 X 125 mm screw-capped test tubes. Increase the volume of each tube to approximately 9 ml with fresh ATCC medium 1171. 7) Ice the parent shipped culture for five minutes, invert the tube 20 times and transfer 0.5- and 1.0-ml aliquots to the tubes just set up in step 6. 8) Incubate all cultures at 35C. Transfer cultures when they reach early stationary phase. The transfer interval will depend on the quality of the culture medium used. Inoculate bacterized culture tubes at least one day prior to subcultivation of Dientamoeba and bacterized Entamoeba strains. 9) In general, addition of penicillin G at 75 U/ml and streptomycin at 75 mcg/ml to ATCC medium 1171 may be necessary if the bacterial density is too high. |
| Cryopreservation： | CPMB-5 Cryoprotective Solution DMSOxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.0 ml 2.5 M Sucrose xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 0.8 ml L-Cysteine/Ascorbic Acid Solutionxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 0.2 ml CPMB-2 Base Solutionxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 6.0 ml Heat-inactivated bovine serumxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 2.0 ml CPMB #2 Basal Solutionxa0 Casein Digest Peptone (BBL)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 40.0 g Yeast Extractxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 20.0 g K2HPO4xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.0 g KH2PO4xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 0.6 gxa0xa0xa0xa0xa0 NaClxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 2.0 g Distilled waterxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.0 L (Autoclave the solution) L-Cysteine/Ascorbic Acid Solution L-Cysteine-HCLxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.0 g Acorbic Acidxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 0.1 g Distilled waterxa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 10.0 ml Add 9.0 ml of distilled water to a 20 ml beaker and dissolve the first two components.xa0 While stirring, adjust the pH to 7.2 with 10N NaOH (approximately 0.7 ml).xa0 Adjust final volume to 10 ml with distilled water and filter sterilize. 1.xa0xa0 Harvest cells from several cultures which are in the late logarithmic to early stationary phase of growth.xa0 Place culture vessels on ice for 10 min. 2.xa0xa0 Invert tubes 20 times and centrifuge at 200 x g for 5 min.xa0xa0xa0xa0xa0xa0xa0xa0 3.xa0xa0 While cells are centrifuging, prepare the cryoprotective solution.xa0 a)xa0 Place the DMSO in a 16 x 125 mm screw-capped tube and ice until solidified. b)xa0 Add 0.8 ml ofxa0 the 2.5 M Sucrose solution, remove from ice and invert until the DMSO is liquefied.xa0 Return to ice bath. c) Add 0.2 ml of the L-Cysteine/Ascorbic Acid solution to the DMSO solution and mix. d)xa0 Add 6.0 ml of the CPMB #2 Basal solution and mix. e)xa0 Add 2.0 ml heat-inactivated bovine serum and mix. 4.xa0xa0 Resuspend the cell pellets and pool to a final volume of approximately 10 ml with the supernatant.xa0 Make a determination of the cell density and adjust the concentration of the cells between 5 x 105/ml - 1 x 106/ml using fresh medium.xa0 If the cell concentration is below 5 x 106/ml, centrifuge the cell suspension and resuspend the pellet in a volume the will yield the desired concentration. 5.xa0xa0 After the cell concentration is adjusted, centrifuge as in step 2. 6.xa0xa0 Remove as much supernatant as possible and determine the volume removed. 7. xa0 Resuspend the cell pellet with a volume of the cryoprotective solution equal to the volume ofxa0 the supernatant removed.xa0 Invert the tube several times to obtain a uniform cell density. 8.xa0xa0 Dispense 0.5 ml aliquots into 1.0 - 2.0 ml plastic sterile cryules (special plastic vials for cryopreservation). 9.xa0xa0 Place vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion.xa0 At -40°C plunge ampules into liquid nitrogen. 10.Store ampules in a liquid nitrogen refrigerator until needed. 11.To establish a culture from the frozen state, place an ampule in a 35°C water bath, until thawed (2-3 min).xa0 Immerse the vial just sufficient to cover the frozen material.xa0 Do not agitate the ampule. 12.Transfer contents of thawed ampule to a 16 x 125 mm screw-capped test tube containing 13 ml of ATCC medium 1978. 13.Screw cap on tightly and incubate at a 15° horizontal slant at 25°C.xa0 Observe the culture daily and transfer when many trophozoites are observed. |
| Name of Depositor： | SA Schaffer |
| Chain of Custody： | ATCC < |
| Year of Origin： | 1993 |
| References： | Clark CG, Diamond LS. Intraspecific variation and phylogenetic relationships in the genus Entamoeba as revealed by riboprinting. J. Eukaryot. Microbiol. 44: 142-154, 1997. PubMed: 9109261 |