Subulatomonas tetraspora

1. One day before thawing the ampule, prepare bacterized ATCC medium 1989, i.e. inoculate medium with a bacteriological loop of Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831™) or Enterobacter aerogenes (ATCC^® 13048™) from a nutrient agar slant (ATCC medium 3).
2. 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.
3. Immediately after thawing, aseptically transfer contents to a 16 x 125 mm screw-capped test tube containing 13 mL of bacterized ATCC Medium 1989.  Screw the cap on tightly and incubate upright at 25°C.

1. Prepare bacterized ATCC medium 1989.
2. When the culture is at or near peak density, rub the surface of the tube with a sterile cotton swab, and agitate the swab to dislodge the adherent cells.  Invert gently 10 times to distribute cells evenly.
3. Transfer approximately 0.25 mL to a 16 x 125 mm screw-capped test tube containing 13 mL of fresh, bacterized ATCC medium 1989. 
4. Screw the cap on tightly and incubate upright at 25°C.

1. Harvest the cells from a culture that is at or near peak density by centrifuging at 850 x g for 5 minutes.
2. If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 10⁶ and 2 x 10⁷cells/mL with fresh medium.  If the concentration is too low, centrifuge at 850 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 between 10⁶ and 10⁷ cells/mL and 10% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to 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 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. 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 an ampule in a water bath set at 35°C (2-3 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the vial.
9. Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 13 mL of fresh, bacterized ATCC medium 1989 in a 16 x 125 screw-capped test tube.  Incubate upright at 25°C.

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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