Nematocida sp. 1
PRA-372 ™
- Product category
-
Protists
- Product type
-
Parasitic protozoan
- Strain designation
- ERTm6
- Type strain
- No
- Isolation source
- Wild-caught Caenorhabditis briggsae
- Geographical isolation
- Key Largo
- Product format
- Frozen
Documentation
ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.
ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
Detailed product information
Characteristics
- Comments
-
Reference strain for genome sequencing project(Nematocida sp. 1 ERTm6 genome sequencing project Available from: NCBI BioProject)
Handling information
- Host
-
in vivo cultivation, Caenorhabditis elegans (Nematoda)
- Handling procedure
-
Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen. If liquid nitrogen storage facilities are not available, frozen ampoules may be stored at or below -70°C for approximately one week. Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C). Storage of frozen material at this temperature will result in the death of the culture.
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.
- Culture maintenance
-
MAINTENANCE OF HOST C. ELEGANS CULTURE:
C. elegans nematodes may be maintained at 25°C on agar plates of ATCC medium 997, NG, or similar media containing a lawn of E. coli bacteria. If desired, a uracil-requiring strain of E. coli (such as strain OP50-1, available from the Caenorhabditis Genetics Center, University of Minnesota) may be used on a growth medium containing limited uracil, thereby precluding bacterial overgrowth which may obscure view of the nematodes.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 mLMix 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 - Reagents for cryopreservation
-
M9 buffer
KH2PO4 3.0 g
Na2HPO4 6.0 g
NaCl 5.0 g
MgSO4 (1M) 1.0 ml
Distilled H2O 1.0 LAutoclave 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:
- Cryopreservation
-
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.
History
- Depositors
- ER Troemel
- Chain of custody
- ER Troemel <-- MA Felix
- Type of isolate
- Animal
Legal disclaimers
- Intended use
- This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
- Warranty
-
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.
- Disclaimers
-
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.
While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.
This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.
Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.
Permits & Restrictions
Import Permit for the State of Hawaii
If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.
Frequently Asked Questions
References
Curated Citations
Nematocida sp. 1 ERTm6 genome sequencing project Available from: NCBI BioProject
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