4. Infective juveniles are best heat killed at 60
C in hot Ringer’s
solution.
Note:
(a) Live nematodes are distinguished from dead nematode using
staining methods, namely Mendola Blue, Nile Blue A, New
Blue R.
(b) Nematode structure can be examined by staining methods
acid fuchsin, gold chloride, picric acid-iodine.
(c) In order to preserve nematodes, it must be killed by gentle
heat. Live nematodes placed directly in fixative become
distorted.
(d) To kill the nematodes, place the vial containing them in a
water bath at 57
C for 10 min or in an oven for 15 min.
Fixatives
Materials Required
l
TAF fixative [165].
– Formalin: 7 ml.
– Triethanolamine: 2 ml.
– Distilled water: 91 ml.
The above solution is stable. Fix nematodes in TAF. Yet if the
fixative evaporates, the hygroscopic triethanolamine prevents specimens from drying.
Procedure
1. Prepare double-strength TAF fixative for 100 ml.
2. Heat-kill nematodes in Ringer solution.
3. Check they are all dead and stretched or relaxed.
4. Place the nematodes immediately in fixatives after heat
relaxing.
5. Add an equal volume of triethanolamine-formalin (TAF) fixative (2 ml) and heat the fixative (65–70
C).
6. Mix and then rest the vial/beaker separately for 1 day, so that
the fixative infiltrates into all tissues and reacts with them.
7. Translocate the TAF-fixed worms into a cavity block for further
study using a stereo microscope.
Note: To avoid post fixation denaturation and deterioration,
nematodes are generally relocated to glycerin using a dehydration
technique. This will also make the specimen more transparent and
perfect for study with high-powered light microscopes. Stains are
usually not used.
Morphological Characterization
71
C in hot Ringer’s
solution.
Note:
(a) Live nematodes are distinguished from dead nematode using
staining methods, namely Mendola Blue, Nile Blue A, New
Blue R.
(b) Nematode structure can be examined by staining methods
acid fuchsin, gold chloride, picric acid-iodine.
(c) In order to preserve nematodes, it must be killed by gentle
heat. Live nematodes placed directly in fixative become
distorted.
(d) To kill the nematodes, place the vial containing them in a
water bath at 57
C for 10 min or in an oven for 15 min.
Fixatives
Materials Required
l
TAF fixative [165].
– Formalin: 7 ml.
– Triethanolamine: 2 ml.
– Distilled water: 91 ml.
The above solution is stable. Fix nematodes in TAF. Yet if the
fixative evaporates, the hygroscopic triethanolamine prevents specimens from drying.
Procedure
1. Prepare double-strength TAF fixative for 100 ml.
2. Heat-kill nematodes in Ringer solution.
3. Check they are all dead and stretched or relaxed.
4. Place the nematodes immediately in fixatives after heat
relaxing.
5. Add an equal volume of triethanolamine-formalin (TAF) fixative (2 ml) and heat the fixative (65–70
C).
6. Mix and then rest the vial/beaker separately for 1 day, so that
the fixative infiltrates into all tissues and reacts with them.
7. Translocate the TAF-fixed worms into a cavity block for further
study using a stereo microscope.
Note: To avoid post fixation denaturation and deterioration,
nematodes are generally relocated to glycerin using a dehydration
technique. This will also make the specimen more transparent and
perfect for study with high-powered light microscopes. Stains are
usually not used.
Morphological Characterization
71