Chapter 27
Nematoda
Subprotocol 1
Female Reproductive Organs of Root Knot Nematodes
(Tylenchida)
Hirumi et al. (1968) cultured reproductive organs of the root knot nematode Meloidogyne incognita acrita, in which intra-uteral development
of embryos is taking place.
Materials
- Dissecting microscope
- Maximov slide
- Forceps
- Needles
- Sharp knives
- Culture flasks
PROTOCOL
Equipment
- 1,000 p.p.m. Hyamine 2389 (Rohm & Haas)
Solutions
- Earl's balanced salt solution
- 1,000 p.p.m. HgCl z
- Stock salt solution (120 mg NaHzP04·HzO, 300 mg MgClz·6H z O, 400 mg
MgS0 4 ·7H z O, 300 mg KCI, 100 mg CaCl z ·2H z O, 70 mg glucose, 40 mg
sucrose,40 mg fructose, 1,000 mg lactalbumin hydrolysate, and 0.5 ml
of 0.5% phenol red solution in 100 ml distilled water)
- Culture medium, consisting of 20 ml TC-199 medium, 20 ml stock salt
solution, and 6 ml FBS with 100 IV/ml penicillin and 100 f..Lg/ml streptomycin
- Root knot formed on tomato root by M. incognita acrita
Animals
Procedure
1. Collect infested tomato roots, and dip them in 1,000 p.p.m. HgCl z for Sterilization
15 min.
2. Wash the roots three times with sterilized triple-distilled water.
3. Excise matured female nematodes gently from the galls.
4. Surface sterilize the female nematodes with 1,000 p.p.m. Hyamine 2389
for 10 min.