(3) Place films downwards in water for a few minutes.
(4) Fix in alcohol.
(5) Stain with azur II, 1 in 1,000. Differentiate as above. Examine as a dry film. This method suffices for showing the excretory cell and the G1 cell; or
(6) Stain with hæmatein (slightly steaming), especially for the column of nuclei and the sheath. The fixation in alcohol in this case may be omitted.
(7) The removal of the hæmoglobin and the fixation may be combined by using Ruge’s mixture (formalin 2 per cent., containing 1 per cent. acetic acid) or acetic alcohol (glacial acetic 1, alcohol 3).[303]
Structure of Larvæ.—(1) Subcuticular cells: By vital staining, at intervals underneath the cuticle are seen a series of spindle-shaped cells—the subcuticular matrix cells of Rodenwaldt, the muscle cells of Fülleborn. There are thirty or forty or more of these.
(2) Nerve ring: Appears as a break in the nuclear column about 20 per cent. of total length from the head.
(3) Excretory system: Consists of a lateral spherical hollow excretory pore which shows a radial striation. Connected with the pore is an excretory cell which appears to be canalized. Excretory pore, 29·6 per cent. of length from head. Excretory cell, 30·6 per cent. of length from head.
(4) “Genital” cells and anal pore: Consists of a pore opening ventrally on a very fine papilla with which are connected four other cells in series, the chief “genital” cell (G1) being some distance from the three others, which lie close to the pore. G1, 70·6 per cent., anal pore, 82·4 per cent. of length from head.