Willaertia magna De Jonckheere et al.

Storage and Culture Initiation

Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen.  If liquid nitrogen storage facilities are not available, frozen ampoules may be stored at or below -70°C for approximately one week.  Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C).  Storage of frozen material at this temperature will result in the death of the culture.

1. To thaw a frozen ampule, place it in a 35°C water bath until thawed (2-3 min).  Immerse the ampule enough to cover only the frozen material.  Do not agitate the ampule.
2. Immediately after thawing, aseptically transfer the entire contents to a plate of ATCC medium 997.  Distribute the material evenly over the plate using a spread bar.
3. Wrap the entire edge of the plate with parafilm and incubate upright at 25°C.  Trophozoites should be seen within 2-3 d.

1. Streak an ATCC medium 997 plate with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831?) or Enterobacter aerogenes (ATCC® 13048?) and incubate at 35°C overnight.
2. Remove an agar block (~5 mm²) with trophozoites from the edge of an agar plate culture and invert the block at the edge of the freshly bacterized plate.
3. Wrap the entire edge of the plate with parafilm and incubate upright at 25°C.
4. Repeat steps 1-3 at 10-14 d intervals.

Note: a monoxenic amoeba culture can be established in this manner using any suitable bacterial food source.

Cryoprotective Solution
DMSO, 1.5 ml
Page's Balanced Salt Solution (or similar), 8.5 ml

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 which is at or near peak density by adding 5 ml ATCC medium 1323 (Page's Balanced Salt Solution) and washing cells into suspension.  Rub the surface of the plate with a spread bar to detach adhering trophozoites.
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, aseptically transfer the contents of the ampule to the center of a fresh plate of ATCC medium 997.  Distribute the material evenly over the plate using a spread bar.
9. Wrap the entire edge of the plate with parafilm and incubate upright at 25°C.
10. Follow the protocol for maintenance of culture.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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