Handling, processing and mounting plant parasitic nematodes

Handling, Processing and Mounting Plant Parasitic Nematodes

Killing and Fixing

Nematodes are best killed by gentle heat (i.e. 55-60deg.C) as this does not disrupt the body contents and specimens killed by this means assume a characteristic 'relaxed' shape which can help in their identification. Nematodes can either be killed first and then fixed, or killed and fixed at the same time. The method chosen is usually down to personal preference.

Treating complete nematode suspensions is generally the most convenient method. After extraction, the nematode suspension is concentrated into a small volume of water (< 20 ml) by decanting, siphoning or centrifuging. This can be heated to a temperature of 55-60deg.C directly over a flame, a hot-plate or in an oven; or by partially immersing container with nematode suspension in a large volume of water at approximately 80-90deg.C (off the boil) for a few minutes. Whichever technique is used the nematodes must not be boiled or over heated. After killing in this way the sample is left to cool and then the whole nematode suspension is fixed by adding an equal volume of 'double strength' fixative. Alternatively, individual nematode specimens are picked out of the suspension and transferred into the cold fixatives at the normal strength shown below.

Fixing and killing of nematodes in suspension can be done using formaldehyde or FA 4:1. Double strength fixative is heated to 80-90deg.C (just off the boil) and poured into an equal volume of nematode suspension.

Single, or small numbers of nematodes, can be killed and fixed in a drop of water on a glass slide in the same way as above, but overheating (and boiling!) is a real danger and should be avoided if you want to remain on speaking terms with the taxonomist.

The main fixatives used are:

                                       Normal strength    Double strength
(1) TAF

40% Formaldehyde (formalin) 7 ml 7 ml Triethanolamine 2 ml 2 ml Distilled water 91 ml 45 ml (2) FA 4:1

40% Formaldehyde (formalin) 10 ml 10 ml Glacial acetic acid 1 ml 1 ml Distilled water 89 ml 45 ml (3) Formaldehyde (formalin) 2% (5%) 4% (10%)

Nematodes in fixative should be left for 12 hours or overnight before processing further.

Correct fixation is essential for accurate identification of nematodes and care should be taken to see that the process is properly carried out.

NEVER put live nematodes into cold fixative.

Handling

Nematodes are picked out of the suspension under the low magnification of a stereoscopic microscope. The handling needles or implements that can be used are many and varied, e.g. slivers of bamboo or quills sharpened to a fine point under the microscope; eyelashes or cactus spines mounted on needles with water resistant glue, or a dentist's probe. The microscopically fine point of the needle is positioned under the nematode which is then gradually worked upwards to the water surface before then being removed by draping over the end of the point. The water suspension should be shallow and re-focusing of the microscope is necessary as the nematode is worked upwards. The technique requires some practice before nematodes can be removed quickly.

Processing for Mounting

Semi-permanent mounts

  1. Lactophenol

    Transfer fixed nematodes to a drop of stained or unstained lactophenol on a glass microscope slide which has been heated to 60deg.C (fuming). This process is not recommended becuse of the hazardous fumes from the lactophenol. If it is employed, care should be taken either to use a fume cupboard or to ensure adequate ventilation.
    Lactophenol formula:
    Phenol crystals 1 part [CAUTION! Hazardous chemical]
    Lactic acid 1 part
    Glycerine 2 parts
    Distilled water 1 part
    Allow to cool before transferring nematodes to a drop of unstained lactophenol on a slide ready for mounting.

  2. Lacto-glycerol

    This method is basically the same as above but with the dangerous chemical, phenol, being omitted. Nematodes can be stained during the processing to semi-permanent mounts by adding a suitable stain to the lactophenol or lacto-glycerol.
    Lacto-glycerol formula:
    Equal parts by volume of lactic acid, glycerol and distilled water.
    Stains:
    0.0025-0.01% Cotton blue (blue)
    Acid fuchsin (red) [CAUTION! Hazardous Chemical]
    Picrofuchsin (yellow) [CAUTION! Hazardous Chemical]

    In normal circumstances, lacto-glycerol should be used in preference to lactophenol.

Permanent Mounts

  1. Seinhorst Glycerol-ethanol evaporation method
    Transfer nematodes directly from the fixative to a watch glass (cavity glass block, etc.) containing 0.5 ml of the following solution:
    96% Ethanol: 20 ml
    Glycerol: 1 ml
    Distilled water: 79 ml
    Place in a closed desiccator containing 96% ethanol. Leave for 12 hours in an oven at 35-40deg.C. This removes most of the water. The watch glass with nematodes is then topped up with a solution of:
    96% Ethanol: 95 ml
    Glycerol: 5ml
    Place in a partly closed Petri dish in an oven at 40deg.C until all the alcohol has evaporated (approximately 3 hours). The nematodes are then in pure glycerol and are transferred to a drop of glycerol on a glass slide ready for mounting.
  2. Simple Evaporation method.
    Transfer nematodes directly from the fixative to a glass cavity block (etc.) containing the following solution:
    95% Ethanol: 70 ml
    Glycerol: 5 ml
    Distilled water: 25 ml
    Partially cover with a glass plate or microscope slide for the first day, and gradually remove the cover over the next two days until the solution is fully exposed and nematodes are in pure glycerol. (The final stage may need to be done in an oven or desiccator in humid climates.) Transfer nematodes to a drop of glycerol on a glass slide ready for mounting.
  3. Slow glycerol evaporation method
    Transfer nematodes directly from fixative to a few mls. of 2-5% glycerol (glycerol and distilled water) in a watch glass, etc., and leave exposed in a desiccator or oven set to about 35-40deg.C for a week or longer. If the processing is done in an oven, the watch glass should be partially covered at first so as to slow down the evaporation process. When all the water has been removed the nematodes can be transferred to a drop of anhydrous glycerol on a glass slide ready for mounting.
    NB. In all the above glycerol methods the glycerol used in the final stage of mounting should be free of water (and air bubbles). This can be done by keeping the glycerol for mounting in a desiccator.

Mounting

Arrange nematodes in the centre of the drop of mounting fluid, separated from each other and resting on the glass slide ie. not floating in the mountant. Place 3 or 4 pieces of fine glass fibre (or pieces of No. 0 coverslip) radially around the nematodes to act as supports for the coverslip and to prevent nematodes from being flattened. Warm a No. 0 or 1 coverslip and gently lower it onto the drop. There should be no excess fluid seeping from under the coverslip. The coverslip is then sealed by ringing with glyceel, nail varnish, or candle wax (from the long wick of a candle which has just been extinguished). Two coats are necessary to give a complete seal.

The coverslips can be bedded onto a ring of wax prior to sealing which serves both to give a better and more permanent mount, and contain the mounting fluid. A heated glass or metal tube (e.g. cork borer or copper pipe) is pressed into a block of paraffin wax (m.p. 55-60deg.C) and then onto the centre of a slide thus transferring the wax which sets as a ring. A drop of glycerol is then placed inside the ring. The coverslip is rested on the wax ring and the slide gently heated on a hot plate, flame or in an oven causing the wax to just melt, thus allowing the coverslip to contact the glycerol and rest on the supports. Do not overheat the wax or leave too long on a hot plate as the worms may start to cook! The coverslip is then sealed as above after the wax has set.

Nematodes in Plant Tissues

Fixing

Nematodes in plant material can be preserved in 2% formaldehyde (5% formalin). Immersing the plant material in hot fixative will produce better nematode specimens.

Staining

Wash roots (or other plant material) to remove soil, etc. and immerse in a boiling solution of lacto-glycerol containing 0.1% cotton blue or acid fuchsin stain for approximately 3-5 minutes. (NB. This is not the ideal method to obtain nematodes for identification!)
Lacto-glycerol:
Lactic Acid 1 part
Glycerol 1 part
Distilled water 1 part

The time required in the boiling solution will depend on the thickness of roots, very thick roots need to be cut longitudinally prior to staining.

Allow to cool in the stain and then wash under running water to remove excess stain. Place in a clear glycerol solution (equal parts glycerol and water) and leave for 24 hours or longer. The stain is removed from most of the plant tissues leaving the nematodes deeply stained. If plant material is to be kept in this solution or mounted, a drop of formalin should be added for preservation.

Mounting

Slide mounts of infested material treated in the above manner can be made after removal of water by the evaporation methods mentioned previously. The plant material can also be permanently mounted in 'Euparol' after passing through an ethanol series or in Canada Balsam by passing through a graded ethanol series to absolute and clearing in clove oil.

Common symbols and abbreviations used in nematode morphometrics

L = Total body length.
a = Total body length divided by maximum body width.
b = Total body length divided by oesophageal length.
b' = Total body length divided by distance from anterior end of body to posterior end of oesophageal glands.
c = Total body length divided by tail length.
c' = Tail length divided by anal body width.
V = Position of vulva from anterior end expressed as percentage of body length. Superior figures refer to the extent of anterior and/or posterior gonad or uterine sac, also as percentage of body length.
V' = Position of vulva from anterior end expressed as percentage of distance from head to anus.
T = Distance between cloaca and anteriormost part of testis expressed as percentage of body length.
R = Total number of body annules.
R ex = Number of annules from anterior end to excretory pore.
R V = Number of annules from posterior end to vulva.
R Van = Number of annules from vulva to anus.
R an = Number of annules from posterior end to anus.
m = Length of conical part of spear as percentage of whole spear length.
MB = Distance of median bulb from anterior end expressed as a percentage of total oesophageal length.
Caudal ratio A = Length of hyaline tail divided by its proximal width.
Caudal ratio B = Length of hyaline tail divided by its width at a point 5 um from its terminus.
um = One thousandth of a millimetre.

References

  • Alves, L.M. and Bergeson, G.B. (1967). A quick destaining procedure for showing contrast between nematodes and root tissue. Pl. Dis. Reptr. 51: 511-514.
  • Baker, A.D. (1953). Rapid method for mounting nematodes in glycerine. Can. Entomol. 85: 77-78.
  • Davies, K.A. (1973). A rapid method for assessing nematodes in roots. Nematologica 19: 570-571.
  • Esser, R.P. (1973). A four minute lactophenol fixation method for nematodes. Pl. Dis. Reptr. 57: 1045-1046.
  • Esser, R.P. (1974). Two permanent mounting methods compared after six years. Proc. helm. Soc. Wash. 41: 10-13.
  • Haverkate, F. (1972). Specific staining of living plant nematodes with 4- dimethylaminobenzenediazocyanide. Nematologica 18: 414-416.
  • Janowski, G.J. Cleveland, D.J. and Georgi, J.R. (1972). A glycol-methacrylate embedding technique for nematode whole mounts. Cornell Vet 62(2): 333-336.
  • Lamberti, F. and Sher, S.A. (1969). A comparison of preparation techniques in taxonomic studies on Longidorus africanus Merry. J. Nematology 1: 193-200.
  • Seinhorst, J.W. (1962). On the killing, fixation and transferring to glycerine of nematodes. Nematologica 8: 29-32.
  • Seinhorst, J.W. (1973). How small is a small drop of water? Nematologica 19: 121.
  • *Southey, J.F. (1986). Laboratory Methods for Work with Plant and Soil Nematodes. Reference Book 402. Ministry of Agriculture, Fisheries and Food, HMSO. 202 pp.