Nematode Pests of Horticultural Crops and their Management (1+1)

Exercise 8

Practical - 8

Exercise: Preparation of Temporary and Permanent Mounts. 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. Materials required: Nematode suspension, desiccators, Calcium chloride (anhydrous), cavity blocks, ethyl alcohol, formalin, lactophenol, glycerol, glass wool, glass slides, cover slips, Whatmann filter paper/ blotting paper, glyceel/nail enamel. 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. 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. 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. 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) 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). 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) 3. Mounting Procedure (Fig. 14) Take a drop of anhydrous glycerin in the centre of a glass slide. Glycerin can be made anhydrous by keeping 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. 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. 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. Fig. 8.1 Permanent slide 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.