Practical 4

Practical - 4

Exercise: Extraction of Nematode from Plant Material.
Objectives:
  • To study the nematode fauna present in the plants and their quantitative estimation.
Material required:
  • Infested plant material, scissors, Petri-plates, wire gauge, facial tissue paper, warring blender, acid fuchsin, lactophenol, etc.
i. Direct examination technique
Procedure:
  • Wash the infested plant material thoroughly and chop it into small pieces and place it in a Petri-plate filled with water.
  • The migratory forms semiendo/endoparasitic nematodes come out of the chopped material into water by 24 hours and can be observed directly under stereomicroscope.
  • Alternatively the chopped material can be processed by modified Baermann’s technique.
ii. Waring blender technique
Procedure:
  • Wash the roots under tap water so that adhering soil particles are removed.
  • Chop the roots into small pieces, and put them in a blender jar. Add about 100 ml water. Cover the jar with lid.
  • Operate the blender just for 10-15 seconds.
  • Take out this material from the blender and place it on the modified Baermann’s funnel assembly as described earlier.
iii. Acid fuchsin staining technique (for semi-endoparasites and endoparasites)
Procedure:
  • Wash the roots gently under the tap water to remove the soil particles.
  • Place them on a bloting paper so that excess water is removed.
  • Chop the roots in to small pieces of about 1-2 cm.
  • Take a weighed quantity of chopped roots (0.5 to 1 g) and wrap them in a piece of muslin.
  • Boil them in 0.1% acid fuchsinlactophenol solution for 1-3 min depending upon the hardness of the root.
  • Remove the excess stain under running tap water.
  • Leave the stained root bits in plain lactophenol (lactic acid 1 part+ glycerine 2 parts + phenol 1 part + water 1 part) overnight to distain the roots.
  • Examine the roots under a stereomicroscope. The nematodes appear red in more or less transparent root tissues.
  • The stained but dead nematodes can be dissected out of the roots under a stereomicroscope.
Precautions:
  • Plant material should be chopped into small pieces for easy and efficient extraction.
  • Excess stain should be washed off and destaining should be for appropriate period.
  • Roots should not be boiled excessively.

Last modified: Friday, 22 June 2012, 6:13 AM