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NEMA 505 NEMATOLOGICAL TECHNIQUES                                                                                 (2L+2P) I

Objective
  Understanding the principles, theoretical aspects and developing skills in nematological techniques.

Theory

UNIT I
  Principles and use of light, scanning and transmission electron microscopes, and other laboratory equipments.

UNIT II
  Methods of survey, surveillance and sampling for nematodes; Extraction of active and sedentary nematodes from soil and plant tissues; Nematode population estimation.

UNIT III
  Principles and techniques of killing, anaesthetizing, fixing, clearing and mounting (temporary or permanent) of nematodes; preparation of perineal patterns, vulval cones of cyst nematodes, en- face views and body section of nematodes; Sketching and measurement of nematodes using camera Lucida/drawing tube and image analyzer; Microphotography of nematodes.

UNIT IV
  Methods for raising of pure culture of plant and insect parasitic and microbivorous nematodes. Experimental techniques for studying pathogenicity. Evaluation of nematicides. Screening of crop varieties for locating sources of resistance. Staining nematodes in plant tissues; microtomy for histopathological studies; collection of plant root exudates and their bioassay; preparation of plant materials for exhibition.

UNIT V
  Spectrophotometry, electrophoresis, PCR, Chromatography, Centrifugation

Practicals
  Baermann’s funnel technique; Cobb’s sieving and decanting technique; Oostenbrink elutriation technique ; Cotton wool filter technique, Two flask Technique; Estimation of nematode population in soil; Picking tools and nematode handling; Root incubation method; Centrifugation floatation technique ; Root knot nematode- Eggmass collection and their hatching, Extraction of root knot with NaOCl; Inoculation of nematodes in pots and maintenance of nematode culture; Observation on and extraction of different stages of root knot nematodes from roots; Lactophenol method for staining of nematodes in plants; Byrd’s method for staining of nematodes in plants; Microscope Handling and taking photos through microscope; Cyst extraction with Fenwick can; Cyst extraction through Sieve; Cyst population estimation; Drawing and measuring nematodes; Temporary mount preparation; Permanent mount preparation special preparation of nematodes - perineal patterns, vulval cones, en-face and body sections; collection of root exudates, preparation of exhibits of nematode diseased plant material, in vitro culturing techniques of nematodes- callous culture, excised root and carrot disc techniques. Protein extraction and estimation, DNA extraction and estimation, PAGE, Agarose gel electrophoresis, DNA amplification using PCR.

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