| Cryopreservation |
M9 buffer
KH2PO4 3.0 g
Na2HPO4 6.0 g
NaCl 5.0 g
MgSO4 (1M) 1.0 ml
Distilled H2O 1.0 L
Autoclave 15 min. to sterilize.
See www.atcc.org for ATCC medium formulations.
An infectious extract of Nematocida-infected worms is prepared and cryopreserved as follows:
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Harvest infected nematodes when most worms are filled with spores. Using a balanced saline buffer solution such as ATCC medium 1323, M9 buffer, or a PBS solution, wash nematode worms into suspension and transfer to a 15-ml centrifuge tube.
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Centrifuge at 1500 x g for 30 sec, rinse 3 times with distilled water, let sit for 1 hr, then rinse again 2 times with distilled water.
- Reduce volume of supernatant to ~1 ml, resuspend pelleted worms and transfer to a 2-ml microcentrifuge tube. Add ~500 µl silicon carbide beads (BioSpec Products, Inc. cat. 11079110sc) to the tube and vortex for 1 min, repeating four to five times.
- Filter the worm lysate is through a 5 µm filter (Millipore) attached to a syringe in order to eliminate undisrupted C. elegans eggs, larvae, and other debris. (Filter becomes saturated after passing ~100 µl packed nematodes; use additional filters as necessary.)
- Adjust the concentration of the filtrate containing Nematocida spores to 2.0 - 4.0 x 107 spores/ml with fresh buffer solution (i.e., ATCC medium 1323, M9 buffer, or a PBS solution).
- Prepare a 30% (v/v) solution of sterile glycerol in fresh buffer solution.
- Combine the filtrate and glycerol stock solutions in equal volumes to yield a final concentration of 1.0 - 2.0 x 107 spores/ml and 15% glycerol.
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Dispense in 0.5 ml aliquots to 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
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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.)
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Store in either the vapor or liquid phase of a nitrogen refrigerator.
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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 1 minute. Do not agitate the ampule. Do not leave ampule in water bath after thawed.
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When completely thawed, dilute the spore preparation by addition of 0.25 to 0.5 ml of a balanced saline buffer solution such as ATCC medium 1323, M9 buffer, or a PBS solution.
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Infect C. elegans nematodes by adding the dilute spore preparation onto an agar plate containing an established C. elegans culture. Seal the plate with parafilm and incubate upright at 25°C. Follow the protocol for maintenance in-vivo.
For further information on cultivation/preservation of Nematocida, reference the following:
Estes, KA, et al., 2011. PLoS Pathogens 7(9): e1002227.
Troemel, ER, et al., 2008. PLoS Biology 6: 2736–2752.
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