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Temperature: 25°C

M9 buffer
KH₂PO₄, 3.0 g
Na₂HPO₄, 6.0 g
NaCl, 5.0 g
MgSO₄ (1M), 1.0 ml
Distilled H₂O, 1.0 L
Dissolve ingredients in 1 L of distilled water. Distribute 200 to 500 ml aliquots into appropriate sized bottles and autoclave for 15 minutes.

Harvest and Preservation

1. To harvest the Nematocida culture, add 5 ml of M9 buffer to an infected NGM plate and transfer the suspension to a 15 ml centrifuge tube.
2. Centrifuge at 200 x g for 5 min. Remove the supernatant, resuspend the pellet in 5 ml of M9 buffer, and repeat the centrifugation step.
3. Repeat step 2 a minimum of five times in order to wash the infected worms.
4. After the last wash, resupend the pellet in 1 ml of M9 buffer and transfer the suspension to a 2 ml microcentrifuge tube. Add Silicon carbide beads (BioSpec Products, Inc.) to the tube and vortex for 1 minute. Repeat the procedure 4-5 times.  Filter the worm extract through a Whatman filter paper number 1 to remove eggs and any remaining intact worms.
5. Perform a spore count of the worm extract and adjust the concentration to ≥ 3 x 10⁷ spores/ml.
   NOTE: If the concentration of spores is too low, harvest infected worms from additional NGM plates to yield the desired concentration.
6. Mix the extract with an equal volume of M9 buffer containing 30% glycerol. The final concentration of the extract will be ≥ 1.5 x 10⁷ spores/ml and 15% glycerol.
7. Dispense 70 ml aliquots into 1.0-2.0 ml sterile plastic screw-capped cryovials.
8. 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^o 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.)
9. Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator.

Troemel ER, et al. Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans. PLoS Biol. 6: 2736-2752, 2008. PubMed: 19071962

Nematocida parisii ERTm1 genome sequencing project Available from: NCBI BioProject