Hartaetosiga balthica (Wylezich and Karpov) Carr, Richter and Nitsche

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.  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 ATCC medium 1525 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831™) or Enterobacter aerogenes (ATCC^® 13048™).
3. Incubate with the cap tightly sealed at 20-25°C.

Subculture at peak density (approximately every 10-14 d) to a fresh T-25 flask of fresh medium in the following manner:

1. Vigorously agitate the flask (or scrape the flask bottom using a sterile cell scraper) and aseptically transfer 0.5-1.0 mL to a T-25 tissue culture flask containing 10 mL complete medium.  If an organism cannot be easily suspended using agitation alone, rub the surface of the flask with a sterile cotton swab, cell scraper, or a rubber policeman before agitation.
2. Incubate with the cap tightly sealed at 20-25°C.

1. To achieve the best results, set up cultures with several different inocula (i.e., 0.5 mL, 1.0 mL, and 2.0mL).  Harvest cultures and pool when the culture that received the lowest inoculum is at or near peak density.  Use a sterile cotton swab, cell scraper, or a rubber policeman to detach adherent organisms
2. Adjust the concentration to approximately 1 x 10⁷ cells/mL by centrifugation at 700-800 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration. 
3. While cells are centrifuging prepare a 20% (v/v) solution of sterile DMSO as follows:  Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath.  Allow the DMSO to solidify.  Add the required volume of refrigerated medium.  Dissolve the DMSO by inverting the tube several times.
   Note: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be approximately 5 x 10⁶ cells/mL and 10% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process should be no less than 15 min and no longer than 30 min.
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 the vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.  At -40°C plunge 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. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
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 1525 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831™) or Enterobacter aerogenes (ATCC^® 13048™).
9. Incubate with the cap tightly sealed at 20-25°C.
10. Once the culture is established, 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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