571 After staining in boiling lactophenol-cotton blue, roots are washed in water to remove excess stain. They are then placed in an acid solution comprising 1 g zinc chloride per 1.7 ml of 12N hydrochloric acid (Holloway & Baker, 1968) for destaining. Time of destaining varies with temperature, depth of staining, root thickness, and the purpose for which the acid treatment is required. For example, to count nematodes directly the roots are left longer in the acid solution than if the aim is merely to soften the roots to allow the nematodes to be dissected out. In the acid solution, the plant tissues, the cortex first, soften, the cells separate and the stain is removed simultaneously. With time, all the cells of the root destain, and finally break down. Nematodes eventually destain, swell and 'burst'; but are more resistant and hence take longer for each stage of the process. The optimum temperature for differentiation of Heterodera avenae in cereal roots is between 25 and 30° C. At temperatures above 30°, the nematodes de-stain and 'burst' after about 15 min. exposure to the acid solution, but this process takes several hours at 20°. At 30°, nematodes in young thin roots can be counted directly from roots while still in the acid solution, 10-20 min after immersion. Overstained roots and older, thicker roots take proportionately longer to de-stain. The process can be hastened by gently squashing the roots before immersion in the acid solution. HOLLOWAY, P. J. & BAKER, E. A. (1968). Isolation of cuticles with zinc chloride-hydrochloric acid solution. Pl. Physiol. 43, 1878-1879. ERRATUM In the paper by M. R. Siddiqi: Systematics of the genus Trichodorus, Nematologica 19: 259-278: 1. P. 259, line 19 : Diptherophora should be Diphtherophora. 2. P. 275, line 22: tanssaniensis should be tansaniensis. 3. P. 278, lines 35 and 43: Stationary should be Stationery.
Nematologica – Brill
Published: Jan 1, 1973
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