Helgoeca nana Leadbeater

Media: ATCC^® medium 2417 Marine Choanoflagellate medium (augmented with two sterile rice grains added to culture flask) inoculated with Klebsiella pneumoniae subsp. pneumoniae (ATCC 700831)

Alternate media: ATCC^® medium 1405 HESNW medium, ATCC Medium 1361 Marine Flagellate medium (both augmented with two sterile rice grains added to culture flask and inoculated with Klebsiella pneumoniae subsp. pneumoniae (ATCC 700831)

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 ampules 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.

Note: Care must be taken to maintain this organism at 4°C at all times. All media used should be pre-chilled to 4°C. The culture may be briefly examined under the microscope at room temperature but every effort should be made to keep the organism at 4°C (e.g. transport culture on ice to and from the incubator)

1. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.  Do not agitate the ampule.  Care must be taken Do not leave ampule in water bath after it is thawed.
2. Add the thawed contents to a T-25 flask containing 10 ml of  of chilled  ATCC medium 2417 (augmented with two sterile rice grains added to culture flask)  bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC 700831).
3. Immediately incubate flask at 4°C with the cap tightly sealed. 

Subculture every two  to three  weeks to a fresh T-25 flask of bacterized medium in the following manner:

1. Vigorously agitate the flask and aseptically transfer 0.5 ml from a growing culture to a T-25 tissue culture flask containing 10.0 ml of ATCC medium 2417 (augmented with two sterile rice grains added to culture flask)  bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC® 700831)
2. Incubate flask at 4°C with the cap on tightly.

Cryoprotective Solution
DMSO, 1.5 ml
Fresh growth medium w/o bacteria, 8.5 ml

Note: Care must be taken to maintain this organism at 4°C at all times.  Media and cell suspension should be kept on ice during entire process.

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.  Chill to 4°C.
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 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.  Do not leave in water bath after thawing. Aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 ml chilled ATCC medium 2417 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^®  700831).
9. Incubate at 4°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 2417.
11. 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.

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

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