| Nematode Extraction Procedures |
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Some Procedures for Collecting and Preparing Nematodes For Study.
Zafar A. Handoo and Donna Ellington
(Note: These procedures may be similar to or modified from those used by other workers.)
Use of Sieves for Extraction of Nematodes From Soil
1. Materials: 1 bucket, 20 mesh sieve, 60 mesh sieve, 325 mesh sieve, and stand to hold sieves.
2. Procedure:
Stack the 3 sieves (be sure to wash them, they work better wet!), one on top of the other from top to bottom, 20 mesh, 60 mesh and 325 mesh. Place on stand. Run water through the sieves to be sure they are wet and the water runs freely through them.
Place a soil sample (approximately 250 ml) into bucket. Stir with hand and break up clumps of soil while adding tap water to bucket (approx. 3/4 full).
When solution is uniform, wait 30 seconds for some of the heavy sediment to settle, lift bucket gently and pour through the stacked sieves (20, 60, 325) at one time (leaving the settled sediment in the bottom of the bucket).
Discard the 20 mesh sieve. Then collect cysts and large eel shaped forms from the 60 mesh sieve by backwashing into a 250 ml beaker. Collect the other eel-shaped forms by the same method and backwash the 325 mesh sieve into a separate 250 ml beaker.
After the beakers have settled for about ? an hour, decant the clear water from the top and put the settled sediment at the bottom of the beaker into a watchglass for further examination.
To save nematodes for further study kill and fix extracted nematodes by adding one of the following fixatives to the material, let settle down again, decant and place the rest in a vial.
Fixative Solutions (solutions work better when hot):
Looss's Fluid (a small amount of shrinkage of specimens may occur)
70% ethanol - 9 parts
glycerol - 1 part
3% formaldehyde fixative (more desirable)
formalin (40% formaldehyde) - 8 parts
distilled water - 92 parts
3% formaldehyde-2% glycerine fixative (more desirable)
formalin (40% formaldehyde) - 8 parts
glycerol - 2 parts
distilled water - 90 parts
Note: These chemicals listed above are hazadous and should be handled properly!
3. Types of nematodes to collect from various sieves
20 mesh - Discard
60 mesh - Cysts, white females, sometimes large males & large eel-shaped forms
325 mesh - Larvae & eel-shaped forms
Collecting Cyst-Forms from Greenhouse and Field Samples
1. Materials: 1 bucket, 20 mesh sieve, 60 mesh sieve, 325 mesh sieve (if collecting eel-shaped forms) and stand for sieves.
2. Procedure:
Place a sample (approximately 250 ml) into bucket. Stir with hand and break up clumps of soil while adding tap water to bucket.
When solution is uniform, wait 15 seconds for some of the heavy sediment to settle, lift bucket gently and pour through the stacked sieves (20, 60, 325) at one time (leaving the settled sediment in the bottom of the bucket), The cysts should not be settled in this short period of time.
Gently wash down the 3 stacked screens as one and then discard the 20 mesh sieve which should only hold debris. Then collect cysts from the 60 mesh sieve by backwashing into a beaker. Collect the eel-shaped forms from the 325 mesh sieve by the same method and wash into a separate beaker to go through the funnels.
After the beaker holding the cysts has settled a few minutes decant the clear water from the top and pour small samples from the bottom of the beaker into watchglasses and search for cysts under the dissecting scope.
3. Types of nematodes to collect from various sieves
20 mesh - Discard
60 mesh - Cysts, white females & sometimes large males
100 mesh - Root-knot males, Dolichodorus, Hoplolaimus & other large forms
325 mesh - Larvae & eel shaped forms
Cyst Cone Mounts (For Heterodera spp.)
1. Cut cyst in 3% formaldehyde.
2. Trim cyst cone portion (decide size so it can stand up well) in lactophenol or glycerine.
3. Transfer to water to remove above solutions.
4. Transfer to clove oil to remove water, air bubbles, and clean dirt off surface of cone. This clears cone.
5. Put a very tiny drop of clove oil on slide, put cone in this.
6. Put Euparal on round 12 mm coverslip and drop on top of cone in clove oil. Press down gently. If hard melt Euparal. (Heat slide so Euparal melts. Air bubbles will eventually come out and disappear.)
7. Check every day for the first week to make sure the cone is still standing. Get coverslip to lay on the vulva cone. If bubbles are inside the cone, turn the slide over and they will eventually squeeze out. Use round 12 mm coverglass.
Note: These chemicals listed above are hazardous and should be handled properly!
Modified Seinhorst (1959) Method of Taking Nematodes to Glycerin.
1. Make up two solutions to be used in the process and label.
Solution #1
glycerol - 1 part
95% ethanol - 20 parts
distilled water - 79 parts
Solution #2
glycerol - 5 parts
95% ethanol - 95 parts
2. Nemas should be hand picked with clean tools! Place nematodes in small deep dish (a Stender prep dish can be used which holds about 10 ml of solution) containing Solution #1 (about 75% of the volume of the dish). Both the dish and the solution must be extremely clean as any small particles will adhere to the specimens.
3. Place open small dish into larger closed glass vessel, eg. a desiccator, or staining dish, containing about 1/10 of its volume of 95% ethanol and leave the dish in this saturated atmosphere for at least 12 hours in an oven at 35 - 40C. This removes almost all the water and leaves the nematodes in a mixture of glycerol and ethanol.
4. Decrease the volume in small dish by about ? and add Solution #2 to the dish after the 12 hours.
5. Place small dish in covered petri dish and replace it in the oven.
6. Several hours later repeat step #5 (we've seen less collapsing of certain nematodes using this method).
7. Keep in covered petri dish in oven and add Solution #2 every day for a week. Then leave in the oven until the alcohol evaporates and the nematodes in the dish are in pure glycerol and can be mounted.
Note: These chemicals listed above are hazardous and should be handled properly!
