Entamoeba histolytica Schaudinn

1. One day before thawing a frozen ampule inoculate two tubes of ATCC medium 1171 with the bacterial flora only.  Incubate the tubes on a 15° horizontal slant at 35°C.  If the specific bacterial flora associated with this culture are not available, skip this step and proceed to step 2.
2. On the following day combine 4.1 mL ATCC medium 1171 with 0.9 mL of HIBS (heat-inactivated bovine serum) to produce 5 mL of medium enriched with 20% serum.  Invert gently several times to mix.  Note:  If bacterized medium 1171 was prepared in step 1, use the bacterized medium in place of fresh medium for faster recovery/growth.
3. Remove the frozen ampule from liquid nitrogen and flame gently at the base of the cap.  Remove the cap and aseptically add 0.5 mL of the serum-enriched medium prepared in step 2.  Place in a 35°C water bath until thawed (2-3 min).  Note:  Manipulations of the ampule before placing in the water bath should be done as quickly as possible to avoid warming of the contents at a suboptimal rate.
4. Transfer contents of the thawed ampule to a one-dram screw-capped vial (vial holds approximately 4.0 mL).
5. Add 2.5 mL of serum-enriched medium prepared in step 2 to the vial in dropwise fashion.  Tighten the cap and incubate on a 15° horizontal slant at 35°C for 2-3 hours.
6. Ice the vial for 10 minutes, then invert gently 10 times. Centrifuge the vial at 100-200 x g for 5 min.
7. Aspirate the supernatant leaving approximately 0.5 mL.  Note:  Do not aspirate the pelleted material. 
8. Replace the supernatant with 3.0 mL of the bacterized medium 1171 prepared in step 1 if available; otherwise use fresh medium 1171 (i.e., not serum-enriched).
9. Incubate the vial on a 15° horizontal slant at 35°C with the cap screwed on tightly.  Observe the culture daily and transfer when many trophozoites are observed (i.e., early stationary phase).

1. Ice a test tube culture at or near peak density for 10 minutes, invert 20 times and aseptically transfer a 0.1 and 0.3 mL aliquot to a fresh tube of ATCC medium 1171.
2. Screw cap on tightly and incubate on a 15° horizontal slant at 35°C.  Transfer when many trophozoites are observed (i.e., early stationary phase).

Notes:  In general, better growth can often be achieved by using media pre-inoculated with bacteria.  Addition of penicillin G at 75 U/mL and streptomycin at 75 µg/mL to ATCC Medium 1171 may be necessary if the bacterial density in the culture is too high.  Inoculate fresh tubes of media with bacteria at least one day prior to subcultivation and prior to addition of antibiotics, if used.  Also, nutrients in ATCC medium 1171 may be diluted through use of an osmotically-balanced saline buffer such as Dulbecco's PBS (ATCC 30-2200) with the medium.  Mixtures in the range of 1:1 to 3:1 (medium to buffer) usually work well to control or limit bacterial growth in cultures where other methods are not sufficient.

1. Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.
2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 800 x g for 5 min.
3. Adjust the concentration of cells to at least 2 x 10⁶/ml in fresh medium.
4. Mix the cell preparation and the cryoprotective solution in equal portions.
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. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place the vial in a 35°C water bath.  Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.   Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 ml ATCC medium 802 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831™) or Enterobacter aerogenes (ATCC^® 13048™).
9. Incubate at 25°C with the cap screwed on tightly.
10. Once the culture is established, vigorously agitate the flask and aseptically transfer 0.5 ml to 10.0 ml of bacterized ATCC medium 802. Follow the protocol for maintenance of culture.

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