Practical assignment on preparation nematode mounting

1. Topic:- Preparation of nematode mounting
By
PRAVEEN KUMAR
BSc Ag 1st year 2nd sem
BTC CARS BILASPUR CHHATTISGARH
IGKV RAIPUR CHHATTISGARH
PLANT PATHOLOGY

2. Objectives:-
• Temporary mounts of nematodes are prepared to study those
structures which become obscure after fixation but may form
important identification characters.
• Semi-permanent mounts are prepared to study those
structures which do not remain clear for a very long period
after fixation.
• Permanent mounts are prepared to preserve the slides for
detailed morphological studies and to maintain the
specimens for a long time for future investigations.

3. Materials required:
1. Nematode suspension
2. desiccators,
3. Calcium chloride (anhydrous),
4. cavity blocks,
5. ethyl alcohol,
6. formalin,
7. lactophenol,
8. glycerol,
9. glass wool,
10. glass slides,
11. Cover slips,
12. Whatmann filter paper/ blotting paper, glyceel/nail enamel.

4. Procedure:-
■ Procedure involves steps like killing, fixing, clearing and mounting of
nematodes.
a. Killing:-
■ A temperature of 50oC is optimum for killing the nematodes.
■ For killing a few individual specimens, place the nematodes in a drop
of water on a glass slide and warm the slide slightly while moving it
regularly on the burner.
■ Be cautious that the drop does not dry. Add one more drop of water.
■ This method of killing is use for making temporary mounts.

5. b. Killing and Fixing
■ Take measured quantity of live nematode suspension in a test
tube
■ Add 8% boiling formaldehyde solution/ FA4:1/TAF as fixative in
equal volume to it so that the temperature of suspension is not
more than 50oC.
■ Leave this fixed suspension in specimen vial and cork it. Let it
stand for minimum 24 hours.
■ If not required for use immediately, fixed nematodes can be
preserved for any period of time without further deterioration.
■ This method is generally used for making semi-permanent and
permanent mounts.

6. Temporary mounts:-
Procedure:-
■ Place a drop of water on the centre of slide.
■ Pick the freshly killed nematode specimens and place it in this
water drop.
■ Place a coverslip in such a way that no air bubble remains.
■ Such specimens can be observed under microscope only for a
few minutes until water dries up.

7. 2. Clearing
■ Internal structural of nematodes specially, the reproductive
organs are obscured due to the presence of food material in the
intestine.To make the contents of intestine transparent, clearing
is done by either of the following methods.
■ (A) Rapid Lactophenol Method (For semi-permanent mounts)
■ (B) Glycerol- ethanol (Seinhorst’s) method (for permanent
mounts)

8. a) Rapid Lactophenol Method (For
semi-permanent mounts)
■ Procedure:-
■ Take a drop of lactophenol on a slide.Lactophenol can be
prepared by mixing lactic acid, phenol, glycerin and water in a
ratio of 1:1:2:1.
■ Warm it to 55-60oC for 1-2minute.
■ Transfer the desired nematode to a drop of warm
lactophenol.
■ The nematode will become transparent and is ready for
mounting in lactophenol (semi-permanent mount).

9. b) Glycerol- ethanol (Seinhorst’s)
method (for permanent mounts)
Procedure:-
■ Take Seinhorst’s solution – I (96% ethanol-20ml, glycerine-1ml,
distilled water-79 ml) in a cavity block and transfer the desired
nematodes into it.
■ Cover the cavity block partly and put it in a glass vessel containing
96% alcohol.
■ Place the glass vessels in an oven 40oC for 12hours.
■ Refill the cavity block with Seinhorst’s solution- II (Glycerin-5 parts,
96% alcohol-95 parts).
■ Cover the cavity block partly and leave it in oven at 40oC for 4 hours
■ The nematodes are left in the pure glycerin and are ready for
mounting in glycerin (permanent mount)

10. 3. Mounting
■ Take a drop of anhydrous glycerin in the centre of a glass slide.
Glycerin can be made anhydrous by keep it in an open vial in a
desiccators having anhydrous calcium chloride.
■ Pick 2-3 cleared nematodes and transfer them on to the slide.
■ Arrange them parallel (under stereomicroscope) ensuring that
nematodes are resting on the surface of the slide.
■ Arrange them parallel (under stereomicroscope) ensuring that
nematodes are resting on the surface of the slide
■ Pick three glass wool pieces (diameter equal to nematode) and
arrange in the drop on the slide radially.
■ Take a cover slip and put it over the drop of lacto phenol/glycerin
and tap it gently.

11. ■ Remove the excess of lacto phenol/glycerin with the help of a blotting
paper.
■ Seal it with glyceel/nail polish first at three places and after a few
minutes ring it completely with the nail polish. After 5 minutes each, give
two coating of nail polish again.
■ Label the slide for nematode species, host, locality and date.

12. Precautions
■ While killing temperature should not exceed 50oC as high
temperature distorts various body structures permanently.
■ Glycerine drop should be placed in the centre and needs to be
just enough to cover the cover slip completely. A big drop
may slide the specimens out of cover slip area.
■ The thickness of the glass wool should be almost same or
slightly more than the thickness of the nematode.
■ The cover glass should be placed slowly in a sliding phase so
that no air bubble remains.

13. References:-
■ http://ecoursesonline.iasri.res.in/mod/paghtmiew.php?id=11697
■ http://cabweb.org/Identification/Iipprep2.htm

14. Thank you