In certain countries, e.g., Fiji, Samoa, Philippines, West Africa, larvæ, apparently those of Filaria bancrofti, show no periodicity. In Fiji the usual intermediate host is Stegomyia pseudoscutellaris, a day-biting mosquito, so that possibly, as Bahr suggests, the mikrofilariæ have partly adapted themselves to the habits of their intermediate host, as the nocturnal mikrofilariæ are adapted for transmission by a nocturnal feeding mosquito, e.g., Culex fatigans, but how this could come about is a mystery. It is not certain in all cases whether the non-periodic mikrofilariæ really belong to Filaria bancrofti; some may be L. loa larvæ, or possibly unknown larvæ. An exact morphological description of these larvæ is therefore always necessary.
Preservation of Living Larvæ.—Blood from the vein (or finger puncture) is shaken up with twenty times its volume of sterile 0·9 per cent. salt solution, and kept in an ice cupboard (Fülleborn).
Concentration of Larvæ.—(a) The above mixture is hæmolysed with water and then sufficient salt solution added to make up to 0·9 per cent. The solution is allowed to stand or can be centrifugalized. (b) The blood is mixed with sodium citrate and centrifugalized; the larvæ are found in the leucocytic layer (Bahr). (c) Allow blood to clot in a small tube; the larvæ appear on the surface of the clot and are so got in pure serum. A drop of blood may also be allowed to clot on the slide; the larvæ are found in the clear areas of serum. (d) Hæmolyse blood with water or acetic acid. Centrifugalize, make smears from, or examine the sediment.
Removal of Red Corpuscles.—The blood film is allowed to stand for some minutes in a moist atmosphere. The staining solution is sucked through with blotting paper: the larvæ stick to the slide, while the corpuscles are washed out.
Morphology of Larvæ.—Wet staining: Azur II one part, 0·9 per cent., salt solution 3,000, or very dilute Giemsa or ripened methylene blue or neutral red solutions. Place a drop on the slide and add a drop of blood to this. The larvæ remain alive for one or more days; it sometimes takes twenty-four hours to stain some particular structure. Differentiation by drawing through weak eosin solution is often useful. This method is the best for finest details. The excretory pore, anal pore, excretory cell, and chief “genital” cell stain first, then the matrix cells and finally the column of nuclei.
Wet fixation and staining: The blood is spread on a large cover-glass—floated on the surface of 70 per cent. alcohol heated to about 70° C. Wash in water, (1) overstain with 1 in 1,000 azur II solution, warming slightly; (2) differentiate with (a) absolute alcohol (containing, if necessary, a trace of HCl), or (b) with absolute alcohol 96 per cent. ninety parts, anilin oil ten parts; (3) clear in origanum, bergamot or cajeput oil; (4) mount in balsam. Or stain with hæmatoxylin, e.g., Mayer’s glycerine alumhæmatein, heating till slightly steaming. Differentiate with acid (2 per cent. HCl) alcohol if overstained. Clear and mount as above.
Dry fixation and staining: (1) With azur II as above, or (2) with hæmatein (warm). Examine the dried films in the usual way without a cover-glass. The azur stains the excretory and genital cells clearly.
Thick films: (1) The blood is smeared out fairly thickly over an area as big as a sixpence.
(2) Dry quickly to prevent shrinking, using carefully a spirit lamp in a moist climate.