Nematode Paste in Horticultural Crops and their Management

Practical - 7

Exercise: Preparation of Fixatives and their Recipes/Compositions.
Objectives:
To prepare different types of fixatives and solutions required for preparing temporary and permanaent whole mounts of the neamatodes.
Definition: Fixatives are the chemicals used to preserve the nematode specimens without any postmartum changes and also help in optical differentiation of the tissue.
Types of fixatives: Following are some of the common fixatives.
1. Formalin 4%

Formalin (= 37% formaldehyde) 10.8 ml
Distilled water 89.2 ml
The body structure of some species becomes grainy when using formalin as a fixative. This is probably due to the formation of ‘free formic acid’, and can be prevented by adding a bit of calcium carbonate to the stock solution (Baker 1945).

2. F.A. 4:1 or F.A. 4:10

Formalin (= 37% formaldehyde) 10.8 ml
Glacial acetic acid 1 or 10 ml
Distilled water Add to obtain a total volume of 100 ml
Acetic acid neutralizes the shrinking effect caused by formalin. The disadvantage of this mixture (especially 4:10) is that nematodes become brown and the posterior part of the spear fades.

3. F.P. 4:1

Formalin (= 37% formaldehyde) 10.8 ml
Propionic acid 1 ml
Distilled water 88.2 ml
Propionic acid neutralizes the shrinking effect caused by formalin, and slightly enhances the contrast. F.P. is useful for year-long fixation, although Netscher and Seinhorst (1969) prefer fixing in formalin 4%, after killing with hot F.P. 4:1 (or F.A. 4:1).

4. F.G. 4:1

Formalin (= 37% formaldehyde) 8.5 ml
Glycerine 2 ml
Distilled water 89.5 ml
If other components of the liquid evaporate (e.g. if the jar was not properly closed), nematodes kept in this fixative stay in the glycerine, preventing them from drying out.

5. T.A.F.

Formalin (= 37% formaldehyde) 7.6 ml
Tri-ethylamine 2 ml
Distilled water 90.4 ml
Fixing in T.A.F. (Courtney et al. 1955) gives good results as specimens remain more or less life like, but Hooper et al. (1983) detected degeneration of the cuticle of some nematodes after 20 years of storage. Therefore, Southey (1986) does not recommend T.A.F. as a long-term fixative.

6. F.A.A. (Ditlevsen’s)

Formalin (= 37% formaldehyde) 6.5 ml
Ethanol 96% 20 ml
Glacial acetic acid 1 ml
Distilled water 39.5 ml
Because of the alcohol, F.A.A. has a dehydrating, thus shrinking, effect. This may be useful, among others, for making annulation or natural indentations more visible (Southey 1986).

7. Bouin’s Fixative

Formalin (= 37% formaldehyde) 26.5 ml
Saturated aqueous Picric acid, 75 ml
Glacial acetic acid 5 ml]

Nematodes are picked and placed in any of the above mentioned fixatives heated to 50^oC.

Double strength (DS) of all above mentioned fixatives can be prepared by reducing the quantity of water to just half of the above mentioned quantities. DSFs are used for fixing nematode suspension. The suspension is poured in equal volume of double strength boiling fixative for this purpose.

Infested plant material is fixed in 5-10% formaldehyde after thorough washing with clean water. However, plants loose their natural colour , when fixed in formaldehyde. If natural green colur of plants is to be retained, they should be fixed in aqueous Copper sulphate solution (CuSO₄ 14.0g mixed in 2.5 l water) bubbled with Sulpher Dioxide gas (SO₂) until solution turns clear.