Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Wild-caught Caenorhabditis briggsae, Cape Verde islands

Reference strain for genome sequencing project

Culture System: in-vivo cultivation, Caenorhabditis elegans (Nematoda)

M9 buffer
KH₂PO₄ xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 3.0 g
Na₂HPO₄ xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 6.0 g
NaCl xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 5.0 g
MgSO₄ (1M) xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.0 ml
Distilled H₂O xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0 1.0 L

1. Harvest infected nematodes when most worms are filled with spores.xa0 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.xa0
2. 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.
3. Reduce volume of supernatant to ~1 ml, resuspend pelleted worms and transfer to a 2-ml microcentrifuge tube.xa0 Add ~500 µl silicon carbide beads (BioSpec Products, Inc. cat. 11079110sc) to the tube and vortex for 1 min, repeating four to five times.
4. 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.xa0 (Filter becomes saturated after passing ~100 µl packed nematodes; use additional filters as necessary.)
5. 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).
6. Prepare a 30% (v/v) solution of sterile glycerol in fresh buffer solution.
7. 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.
8. Dispense in 0.5 ml aliquots to 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
9. Place the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If the freezing unit can compensate for the heat of fusion, maintain rate atxa0xa0xa0xa0xa0xa0xa0 -1°C/min through the heat of fusion.xa0 At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.)xa0
10. Store in either the vapor or liquid phase of a nitrogen refrigerator.
11. 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.xa0 Do not agitate the ampule.xa0 Do not leave ampule in water bath after thawed.
12. 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.
13. Infect C. elegans nematodes by adding the dilute spore preparation onto an agar plate containing an established C. elegans culture.xa0 Seal the plate with parafilm and incubate upright at 25°C.xa0 Follow the protocol for maintenance in-vivo.

ER Troemel <-- MA Felix