30 GNATS OE MOSQUITOES — CHAPTER II history of the genus Cidex can be followed out with great ease, but this is by no means equally the case with Anopheles, the species of which are very difficult to maintain in captivity, and I have not yet succeeded in rearing any batch from egg to imago. Very often the females refuse to deposit their eggs, or when they do so the larvae rapidly die, and the experiences of numerous correspondents have been similar. It is difficult to say why this is so, as I have used vessels holding much more water than many natural collections I have met with containing Anopheles' larvae, have had them partly filled with mud from genuine Anopheles' pools, and tried in every way to as nearly as possible imitate natural conditions. At any rate, it is useless to attempt to experiment in test tubes and such-like small receptacles, and I am inclined to think that the best plan will be to have made a frame of wire gauze large enough to cover a natural pool. In the north-western provinces of India, at least, the scale of such an experiment need be neither unwieldy nor costly, as here, perhaps, the commonest natural haunt of Anopheles' larvae are certain small masonry tanks, one or more of which are to be found in the garden of almost every bungalow, and these are often no more than two or three feet square.
For rearing perfect insects from a comparatively limited number of larvae taken from some natural source, a very handy little apparatus may be improvised from one of the ordinary prune bottles fitted with a screwed metal top. The greater part of the middle of the cap is cut out, taking care to leave at one point of the circumference a tongue-like projection to serve as a support for food. A single loop of wire is soldered on to the sides of the cap, of sufficient height to serve as a support for the bag of gauze or " leno," which when bound with string to the rim of the cap completes the apparatus. All appliances for observing captive Anopheles should be well fitted, as they will discover and creep through the smallest interstice, and much of the ordinary Mosquito netting sold is quite valueless as a protection against them.
The study of the habitual resting position of living
COLLECTING, PKESERVING AND APPLIANCES 31 insects is an interesting one, and has attracted considerable attention lately. With reference to the habitual attitude of Anopheles as contrasted with Culex, and with the view of more accurately studying this point, I have devised an arrangement for photographing living, resting Mosquitoes in profile. The appliance consists of a frame of thin wood about lin. deep, and 4jin. x 3jin. in dimensions. This frame is converted into a box with glass top and bottom by means of a couple of the glasses of waste quarter-plate negatives, kept in position by adjustable brass clips. In each of the two vertical sides, is an oval opening, closed by a small piece of gauze glued on to the inside of the frame. When a living Mosquito is introduced into the box it generally settles on the gauze, and if the box be then placed in front of a long extension camera so that the edge of the vertical side of the frame crosses the field of view, one can generally obtain a satisfactory picture.
10. — Box WITH Glass Front and Back for Photographinc LIVING Mosquitoes in Profile.
Mosquitoes make excellent sitters, and I have obtained negatives showing most minute detail, although the exposure has to be some ten to fifteen seconds, as they are uneasy and restless in too strong a light. It is useless trying to get too great magnification, as with any lens of less than 4^in. focal length, placed so as to enlarge about two and a half times, it is impossible to get all the parts simultaneously in focus. For most purposes a sheet of white paper is the best background, but it is quite possible to get
32 GNATS OR MOSQUITOES — CHAPTER II negatives showing much detail by reflected light, in which case a dark background should be substituted. With the wet collodion process and subsequent enlargement, it would, I believe, be possible to produce useful records of the markings, not only of Mosquitoes, but of other insects.
For certain experiments it may be necessary to transport living Mosquitoes to a distance, and for this purpose Dr. L. Sambon contrived a method, described as below in the Brit. Med. Journal, September 29, 1900, p. 949, from which also are copied the subjoined figures. Fig. 11.— Case for Mosquitoes. Fig. 12. -Ventilated Box for forwarding Mosquitoes.
"When the insects had fed. Dr. L. Sambon, who had gone to Eome on Experiment No. 2, placed them in small cylindrical cages made of Mosquito netting stretched on a wire frame (fig. 11). Four such cylinders were packed in a well-ventilated box (fig. 12) and forwarded to the London School of Tropical Medicine through the British Embassy in Eome. The box was 9in. in depth and 8|in. on the sides. The wire openings were Sin. square on each side. By the courtesy of the Postmaster-General they came forward by the Indian mail, so that they arrived in London some forty-eight hours after leaving Kome. A good many of the Mosquitoes died on the journey or soon after arrival ; but a fair proportion survived and appeared to be healthy and vigorous."
W^ith respect to instrumental outfit, a good hand lens
COLLECTING, PRESERVING AND APPLIANCES 33 magnifying about 10 — 15 diameters is indispensable ; and for dissecting one must have a simple microscope. Nothing elaborate is required, and indeed a perfectly practical dissecting stand can be improvised from a cigar-box and a scrap of mirror, with a bent wire to hold the lens ; but, in choosing an instrument, be careful to select one with broad, gently-sloping wings on which to rest the hands. It is a mistake to fit it with expensive lenses, as all such, work very close to the object in proportion to their power, and room to manipulate is of more importance than excellence of definition. Two simple plano-convex lenses of lin. and ^in. focus, are all that is required for the actual dissecting, but one modern achromatic lens of high power may be added, not for working under, but for the purpose of examining the preparation at intervals.
As regards the compound microscope, the practitioner in the tropics requires something that he can conveniently take to the bedside of the patient for the diagnosis of malaria.
As myself the designer of a folding microscope, I can with the better grace say, that none of them appear to me sufficiently rigid for use with high powers ; and therefore prefer a portable, but not a folding stand, and for the last year have been using that catalogued by Leitz. Messrs. Smith and Beck also showed me a somewhat similar and equally excellent instrument. Both these stands have sufficient weight and rigidity for use with the highest powers. For diagnostic purposes the 3mm. Zeiss dry apochromatic is a most useful lens, but for critical work on the parasite the highest powers of the best makers are required, though there is no need to spend additional money on lenses of exceptional angular aperture.
I give these details, which may seem somewhat out of place here, as one is often asked what should be recommended as the most convenient outfit for the tropical medical practitioner.
Entire larvae are best mounted as microscopic objects in 4 per cent, formol solution, to which a very little glycerine has been added, but I should doubt if such mounts would 3
34 GNATS OR MOSQUITOES — CHAPTEH II be very permanent, especially in hot climates, so that it will be well to preserve a few slides with Farrant's solution. The slide should be first prepared by forming on it a shallow cell of Hollis' liquid glue, of such size that the edge of the cover rests on the middle of the cell-wall, leaving an edge of the glue outside it. This should be allowed to set, but not to dry hard before using, so that the edge of the cover can be imbedded in the semi- solid material. When all superfluous preservative has been drawn off with blottingpaper, a ring of the glue is run round the edges of the cover and the preparation set aside to dry. The sectionising of perfect insects is even more difficult than that of larvae, as unless the razor be exceptionally keen, it carries the dense chitinous covering before it instead of cutting, and so crushes the internal parts.
There are, however, many structures alike in the larva and adult insect which can be demonstrated in no other way than by the method of serial sections, and as already mentioned, there are especial difficulties in applying this plan to animals with a chitinous integument. Thin as it is in most of the species with which we have to deal, it yet is apt to resist anything but the sharpest of razors, and what is even worse, is well-nigh impervious to the entry of preservative and other fluids, so that I have found it quite impracticable to adopt the plan of staining eii masse either in the larva or adult insect, and this must hence be done after the sections have been fixed on the slide. After some experimentation I find that the following method may be relied upon to yield satisfactory and wellpreserved preparations. For many of its details I am indebted to suggestions from Mr. Allen, the Director of the Marine Biological Laboratory at Plymouth, who has recently been working on certain copepods, which are not altogether dissimilar organisms, as far as consistence is concerned.
The larva or adult insect, as the case may be, is killed by immersion in a solution consisting of two parts of alcohol (90 per cent.) to one part of aqueous solution of perchloride of mercury (1 per mille.), in a test tube, which
COLLECTING. PRESERVING AND APPLIANCES 35 is then gently boiled for a minute or two so as to expel the air contained within the trachea. As the fluid cools it is necessarily drawn through the stigmata into the body of the insect, and is thus at once carried to all its tissues. It is left in this fluid for a few hours, and is then placed first in 90 per cent., and finally in absolute alcohol. To imbed it, it is first placed for at least twenty-four hours in oil of turpentine and is then imbedded in the usual manner in paraffin. As the various structures are very loosely connected it is very important to choose a specimen of paraffin with a melting point suitable to the temperature of the air of the place in which one happens to be working, for the least curling of the sections is fatal to the production of really satisfactory sections, so that it is well to try a sample of the paraffin in the microtome before employing it for imbedding. In Europe, a paraffin with a melting point of about 105" is not at all too soft for the ordinary temperature of the laboratory, but in the tropics I have found samples melting from 115° to 125° most generally useful ; the former for the cold and the latter for the hot weather. The specimens should be kept for at least six hours in the bath of melted paraffin, and are then, with due attention to orientation, placed in the microtome, which, it is needless to say, should be one of a type constructed to produce ribbands of serial sections, the ordinary pathological instrument being quite useless for this purpose. As the sections are to be stained on the slide, albumen, and not creasote-shellac, must be used for fixation. A single drop of Mayer's albumen mixture (equal parts white of egg and glycerin, with 1 per cent, salicylate of soda, well beaten up with an egg whisk and filtered) is added to a watch-glass of water and the sHde is prepared by brushing over it a liberal allowance of this very dilute albumen, so that the sections rest on a thin layer of fluid. AVhen as many of the series as the slide will accommodate have been arranged in position, the slide is placed on the warm plate of the imbedding apparatus and warmed just sufficiently to flatten the paraffin and no more. It is then placed aside to dry as far as the presence of the glycerin
36 GNATS OR MOSQUITOES— CHAPTER II in the mixture will allow ; they are then placed on the warm plate and the paraffin melted for a moment, after which they are successively passed through batbs of turpentine, absolute alcohol, and 40 per cent, spirit, and are then ready for staining.
For this purpose I find no dye better than Manson's methylen blue (borax 5 per cent., methylen blue 2 per cent., aqueous solution) ; this is allowed to act for several minutes and then washed off with water, after which it is well to give a ground staining of watery solution of eosine or fuchsine. After staining the slides are passed successively through baths of 90 per cent, spirit, absolute alcohol, and turpentine ; and finally mounted in balsam. Gentian violet also gives good, and Ehrlich's hsematoxylin fair, staining, but I have not been able to get any result with boraxcarmine. Working in England I *have not been able to test this plan on infected Mosquitoes, but the perfect way in which the most delicate tissue elements are preserved, and the fact that it is so well suited to the use of Manson's stain, makes it a hopeful method for demonstrating the parasites in the salivary glands. Great care must be taken to lose none of the series of sections, as the salivary glands are so small that they may easily be missed unless the series be fairly complete.
As the majority of workers at tropical medicine must necessarily conduct their investigations in places where there is no gas supply, such as is required for the working of the ordinary imbedding apparatus and other appliances involving the employment of self-regulating appliances for maintaining a constant temperature, it may not be out of place to describe a simple piece of apparatus for the purpose which I have used for many years in India. It consists of a sheet of copper about 15in. long by Sin. broad and at least xV^- thick. This is supported in a horizontal position on two wooden feet sufficiently high to admit of the chimney of a small paraffin lamp being placed under one end.
In addition to the ordinary copper capsules for containing the melted paraffin for imbedding, a special long narrow one
COLLECTING, PRESERVING AND APPLIANCES 37 is required. This is filled with a sample of the paraffin which is selected for use, and is then placed near the middle of the copper plate, not across, but parallel with the length of the plate. If the lamp be now lighted and placed under one of the projecting ends of the plate, its heat is conducted by the copper to the narrow tray, and it will be found that a greater or less proportion of its length will become melted. At the point where the melted and solid portions meet it is clear that the paraffin is just at its melting point, and opposite this point are placed the small capsules of paraffin in which the structures for imbedding are to be placed. The long narrow tray, in fact, acts as a thermometer, and if the plate be allowed to reach, so to say, a settled condition before placing the capsules of tissues on it, it will be found that the heat of the lamp is quite uniform enough to render little or no close supervision necessary, and that in a still atmosphere it may often be left for hours without touching. I do not, of course, propose such an appliance as a substitute for the self-regalating one where gas is available, but where, as in most tropical countries, there is no gas supply, it will be found to be something more than an inefficient makeshift, and with a small amount of occasional attention will yield as good results as can be desired.
With the exception of the wings, which are best mounted dry, the cover being merely secured by a perforated label, parts of Mosquitoes are best mounted in balsam, after passing through absolute alcohol and clove oil.
The subject of the methods of determining the relation of Mosquitoes to blood parasites is rather outside the scope of a book like the present, but a few words on the subject may not be out of place. To infect a Mosquito all that is necessary is to introduce a few of the species under investigation under a Mosquito netting, beneath which the patient harbouring the haematozoa is to sleep. The insects should be females, and to avoid the possible fallacy of their having been previously infected, it is better, if possible, to employ for the purpose only insects that have been reared in captivity from larvae. Although a certain number of confirmatory experiments of the infection of the vertebrate host