Permanent Mounts of Nematodes

 Rev 10/30/2019  

I. Seinhorst's Method (Nematologica 8:29-32) 

1. Kill nematodes by pouring in an equal volume of hot (80C) 1.0 acetic acid to create 0.5%. Alternatively, kill in hot FA 4:1 fixative. 
2. Fix for 48 hours in FA 4:1 fixative. 
3. Rinse specimens in distilled water. 
4. Place in Seinhorst I solution (ethanol-glycerin mix) in a BPI watchglass. 
5. Add 1-2 drops of saturated aqueous picric acid to provide a yellow stain and prevent clearing of stylet. 
6. Place this open watchglass in a larger container (desiccator) surrounded by 95% ethanol and incubate at 35-40C for 12 hours. 
7. Add Seinhorst II solution (ethanol-glycerin mix) and store in a partly-closed container (covered petri dish) at 40C. 
8. Transfer to pure glycerin. 

II. Thorne's Method (Principles of Nematology, 1961) 

1. Place specimens in a drop of water on a ringed slide. 
2. Kill nematodes with gentle heat. 
3. Transfer nematodes to BPI watchglass filled with FAA fixative. 
4. Store in fixative at room temperature for 48 hours. 
5. Transfer nematodes to BPI watchglass of 1.25% glycerin in 95% ethanol. 
6. Store in desiccator over CaCO₃ at 35C for 4 weeks for slow transfer to pure glycerin. 

FAA:

80 parts distilled water

60 parts 95% ethanol

2.4 parts formalin

1.6 parts acetic acid 

III. A.D. Baker's Rapid Method (Can. Ent. 85:32-38) 

1. Kill with gentle heat. 
2. Fix 
3. Transfer to warm (55C) lactophenol. 
4. Series transfer to glycerin (all done at 50-55C, on a hot plate. for about 10 min in each step): 

Method works well with heteroderid females and other large nematodes. 

Transfer series:  

Lactophenol:

20 parts liquid phenol

20 parts lactic acid

40 parts glycerin

20 parts distilled water. 

Greater Detail on Preparation of Nematode Mounts for Microscopic Observation 

Three basic steps in preparation of nematode mounts:

1. Killing and fixing nematodes
2. Processing nematodes to glycerine
3. Mounting nematodes

Step I. Killing and fixing nematodes.

1. Collect live nematode specimens in distilled or deionized water in a small beaker or watch glass.
2. Concentrate the nematodes in a minimal volume of water and add equal volume of hot (90 C) fixative solution, buffered formalin (Humason, 1972), to it. Nematodes may be killed with heat before adding fixative although adding hot fixative directly is also effective. Buffered formalin provides very good fixation.

Leave the specimens in the fixative for 1-2 days. Nematodes may be stored in buffered formalin indefinitely, it does not clear characters.

Buffered formalin solution is prepared as follows:

Formalin (ca 40% formaldehyde) ---------- 10.0 ml

Water ------------------------------------- 90.0 ml

Sodium acid phosphate --------------------- 0.4 g

Anhydrous disodium phosphate ------------- 0.65 g

Step II. Processing specimens to glycerin. (Seinhorst, 1959).

1. 1. Prepare the following two solutions and keep them at room temperature.

   Seinhorst I solution: 20 parts 95% ethanol 1 part glycerin 79 parts water

   Seinhorst II solution: 95 parts 95% ethanol 5 parts glycerin

2. Place fixed nematodes in a BPI dish. Draw-off excessive fixative and concentrate the nematodes in a small volume. Add ca 6-8 ml of Seinhorst solution 1 to the nematode suspension. (A very small quantity of rose bengal, acid fuchsin or aqueous picric acid may be added to the solution to stain the nematodes. This is optional.)
3. Place the open BPI dish in a larger closed glass container (desiccator) with 95% ethanol at the bottom and place in oven at 35-40C for at least 12 hours. This removes most of the water in the BPI dish. (Do not close or allow ethanol from the glass container to over-flow into the BPI dish.)
4. Remove dishes from oven and draw-off excess Seinhorst solution 1 from the BPI dish, using a pipette under a dissecting microscope to avoid loss of specimens.
5. Add Seinhorst solution 2 to the BPI dish, place it in a partially covered petri-dish and return to oven at 40 C.
6. Several hours (at least 3 hours) later, draw-off excess solution from the BPI dish and repeat step 5. Keep the dishes in oven until all the alcohol has evaporated (at least 3 hours) and nematodes are in pure glycerin.

Step III. Mounting nematodes.

A. Temporary mounts

1. Place a small drop of the fixative in the center of a clean glass slide.
2. Using a 'nematode pick' under a dissecting microscope, pick up the desired specimens and place them in the fixative on the center of the slide.
3. Place the slide under the dissecting microscope, arrange the nematodes in the center of the slide and bottom of the drop.
4. Place glass wool (about 5 mm in length) or glass microbeads in a triangular position near the edge of the drop.
5. Place a cover glass (18 mm wide) gently over the drop using a forceps or supporting it with a needle. Draw off excess fixative carefully using filter paper.
6. Apply Zut, Glyceel or nail polish with a small brush to the edge of the cover glass, to seal it. After the sealant has dried, the slides can be observed under a compound microscope.

B. Permanent mounts

1. Fix a clean cover glass (25 mm wide) in the center of a Cobb aluminum slide by supporting with appropriate sized white cardboard pieces.
2. Place a small drop of anhydrous glycerin in the center of the cover glass in the aluminum slide.
3. Pick up nematodes from the fixative as in step A 2, and place them in the drop of glycerin.
4. Arrange the nematodes in the center of the slide and place glass wool as in steps A 3-4.
5. Carefully place a cover glass (18 mm wide) over the drop and seal the edges of the cover glass as in steps A 5-6.
6. After the sealant has dried, a second coat of sealant may be added. Allow to dry, label the slides on the white cardboard, and examine under a compound microscope Excess glycerin on the slide is difficult to remove and can cause smudges, which interferes with the sealing process.
7. Store the slides in a flat position to avoid settling of nematodes towards the edge of the cover glass. Use of aluminum slides enables viewing of the nematodes from both sides of the slide.

Other methods for fixing, processing and mounting nematodes (see Zuckerman et al., 1985; Southey 1986) may be followed as per individual preferences.

References:

1. Humason, G. L. 1972. Animal tissue techniques. San Francisco: W. H. Freeman and Company.
2. Seinhorst, J. W. 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4:67-69.
3. Zuckerman, B. M., W. F. Mai, and M. B. Harrison. 1985. Plant nematology laboratory manual. Amherst: The University of Massachussets Agricultural Experiment Station.
4. Southey, J. F. 1986. Laboratory methods for work with plant and soil nematodes. Technical bulletin 2. London: Her Majesty's Stationery Office.

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