Nematocida sp. 1

1.   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 it is thawed.

2.   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 Page's Balanced Saline, M9 buffer, or a Phosphate Buffered Saline (PBS; ATCC cat. 30-2200).

3.   Infect C. elegans nematodes by adding the dilute spore preparation onto an agar plate containing an established C. elegans culture (stage L1 or L4/young adults).  Seal the plate with parafilm and incubate upright at 25°C.  The worms become infected by simply ingesting the spores.  Follow the protocol for maintenance in-vivo.

NG agar
Agar (Difco 214010)                 17.0 g
NaCl                                        3.0 g
Bacto peptone (Difco 211677)    2.5 g
Cholesterol (5 mg/mL)              1.0 mL
CaCl₂ (1 M)                              1.0 mL
MgSO₄ (1 M)                            1.0 mL
KH₂PO₄ (1 M)                          25.0 mL

Mix the first three reagents in 800 mL of distilled water and autoclave. After the mixture is cool, add the last four reagents aseptically and adjust the volume of the medium to 1 L with sterile distilled water. See www.atcc.org for ATCC medium formulations. For further information on cultivation of C. elegans nematades, reference the following:
Brenner, S, 1974. Genetics 77: 71–94.

MAINTENANCE OF NEMATOCIDA CULTURE IN-VIVO:
Nematocida infection typically results in 50% host worms showing symptoms of infection at 2-3 d, and 50% host worm mortality at 4-5d. The infection may be propagated by transfer of a few (<10) infected host worms to a fresh plate culture of uninfected nematodes. Observe worms daily for symptoms of infection by preparing wet mounts for phase microscopy at approximately 600x (DIC imaging recommended). Nematocida infection manifests as distinct, granule-free regions within the C. elegans intestinal cells.

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:  

1. 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. 
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.  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.  (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.  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.) 
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.  Do not agitate the ampule.  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.  Seal the plate with parafilm and incubate upright at 25°C.  Follow the protocol for maintenance in-vivo.

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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