Preparing Carrot Discs for Nematode Culture

Rev. 05/19/2005

Adapted from:

Dr. John J. Chitambar

California Department of Food and Agriculture

April 2003

This technique is appropriate for Ditylenchus spp., Pratylenchus spp. and Radopholus spp.

1. Place carrots in lukewarm soapy water and scrub thoroughly with nylon brush

2. Rinse carrots in cold clean water.

3. Prepare a 10% household bleach solution to volume of distilled water. Pour solution into a clean tub. Add carrots. Ascertain that carrots are completely submerged in bleach solution. Leave carrots in solution for 30 minutes. Drain.

4. Place carrots on paper towels on trays and cover with paper towels. Transfer carrots on tray to a laminar airflow chamber. Remaining operations are to be done in chamber.

5. In the airflow chamber, sterilize a knife by dipping it in 95% alcohol and passing it over a flame. Cut off the crown end of the carrot. Cutting operations may be done over layer of paper towels on the chamber counter. Hold carrot at the tapered end and using an alcohol-dipped and flamed vegetable peeler, peel the carrot downwards in slightly overlapping longitudinal strips. Sterilize peeler after every two strips. Peeled portion should be of a diameter small enough to fit into a wide mouth flint glass bottle.

6. Using an alcohol-dipped and flamed sharp knife, cut the peeled carrot portion into 3-4 cm long, and not less than 2 cm wide cylindrical pieces. Sterilize the knife after every cut. Cutting may be done over a sterile disposable petri plate so that cut pieces fall into the plate.

7. Transfer cut carrot cylinders to autoclaved wide-mouth flint glass bottles (or jars) using a set of sterilized forceps. Sterilize the forceps after every cylinder. Put at least four cylinders per bottle.

8. Tighten bottle cap, label bottle and store in dark at 24-25 C.

After 1-2 weeks, or as soon as white specks of callus are visible on the carrots, inoculate with nematodes.

Nematodes do not need to be surface sterilized, but should be rinsed in a few changes of sterile water and inoculated in a few milliliters of the same.

Additional Notes from Dr. Chitambar:

I continue to have much success using sterilized carrot callus discs, either in 1% water agar plates or in sterile flint-glass wide mouth bottles. I stopped using water agar as I was obtaining very heavy numbers on discs alone.

The D. dipsaci I use were originally obtained from field collections of cutivated (and stored) garlic cloves, and naturally growing bush lupine shrubs along the Northern California coast. Also, I have had success culturing D. dipsaci from Narcissus bulbs from Northern California.

I have started cultures with as few as 5 individuals, but recommend using an inoculum of at least 50-100 for starters. The nematodes do not need to be surface sterilized. Subsequent and timely subculturing will soon clean up the nematodes naturally.

The carrots are obtained from our local grocery store, and I have never worried about the variety. I am careful in selecting carrots that are not blemished, cracked or too thick and woody, but are more or less cylindrical rather than tapering.

I take considerable pains in preparing the discs under near to sterile conditions in a laminar flow hood. I peel the carrots twice in overlapping longitudinal strips before giving the peeler (and knife) an alcohol dip and flame "bath".

The cultures seem to do better in glass bottles than plastic or other materials. The cultures can be stored in the dark at a room temperature of 24-25 C.

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