Entamoeba moshkovskii Tshalaia (ATCC® 50415)

Strain Designations: AP-1  /  Depositor: SA Schaffer  /  Biosafety Level: 1

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Strain Designations AP-1
Biosafety Level 1
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 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

DMSO                                                                                                   1.0 ml

2.5 M Sucrose                                                    0.8 ml

L-Cysteine/Ascorbic Acid Solution                         0.2 ml

CPMB-2 Base Solution                                    6.0 ml

Heat-inactivated bovine serum                      2.0 ml

CPMB #2 Basal Solution 

Casein Digest Peptone (BBL)                      40.0 g

Yeast Extract                                                                              20.0 g

K2HPO4                                                                1.0 g

KH2PO4                                                                0.6 g     

NaCl                                                                    2.0 g

Distilled water                                                    1.0 L

(Autoclave the solution)

L-Cysteine/Ascorbic Acid Solution

L-Cysteine-HCL                                                1.0 g

Acorbic Acid                                                       0.1 g

Distilled water                                                  10.0 ml

Add 9.0 ml of distilled water to a 20 ml beaker and dissolve the first two components.  While stirring, adjust the pH to 7.2 with 10N NaOH (approximately 0.7 ml).  Adjust final volume to 10 ml with distilled water and filter sterilize.

1.   Harvest cells from several cultures which are in the late logarithmic to early stationary phase of growth.  Place culture vessels on ice for 10 min.

2.   Invert tubes 20 times and centrifuge at 200 x g for 5 min.        

3.   While cells are centrifuging, prepare the cryoprotective solution. 

a)  Place the DMSO in a 16 x 125 mm screw-capped tube and ice until solidified.

b)  Add 0.8 ml of  the 2.5 M Sucrose solution, remove from ice and invert until the DMSO is liquefied.  Return to ice bath.

c) Add 0.2 ml of the L-Cysteine/Ascorbic Acid solution to the DMSO solution and mix.

d)  Add 6.0 ml of the CPMB #2 Basal solution and mix.

e)  Add 2.0 ml heat-inactivated bovine serum and mix.

4.   Resuspend the cell pellets and pool to a final volume of approximately 10 ml with the supernatant.  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.  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.   After the cell concentration is adjusted, centrifuge as in step 2.

6.   Remove as much supernatant as possible and determine the volume removed.

7.   Resuspend the cell pellet with a volume of the cryoprotective solution equal to the volume of  the supernatant removed.  Invert the tube several times to obtain a uniform cell density.

8.   Dispense 0.5 ml aliquots into 1.0 - 2.0 ml plastic sterile cryules (special plastic vials for cryopreservation).

9.   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.

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).  Immerse the vial just sufficient to cover the frozen material.  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.  Observe the culture daily and transfer when many trophozoites are observed.

Name of Depositor SA Schaffer
Chain of Custody
ATCC <<--SA Schaffer<<--T.A. Nerad
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

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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