Culture System: in-vivo cultivation, Caenorhabditis elegans (Nematoda)
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:
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.
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.
Dispense in 0.5 ml aliquots to 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
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.)
Store in either the vapor or liquid phase of a nitrogen refrigerator.
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.
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.
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.