AN INTRODUCTION TO METHODS OF STUDYING THE MORBID HISTOLOGY OF DISEASE-CARRYING INSECTS.

243 AN INTRODUCTION TO METHODS OF STUDYING THE MORBID HISTOLOGY OF DISEASE-CARRYING INSECTS. By CAPTAIN A. E. HAMERTON, D.S.O. Royal Army Medical Oorps. THE great technical improvements in modern histological research have simplified the investigation of the' stages of development, and have enabled us to follow out the life-history of some of the pathogenic protozoa which are dependent upon an invertebrate host for the fulfilment of their biological destiny. Much remains to be discovered in this comparatively new field of medical and veterinary research, and there are some of us who have opportunities for pursuing such investigations in the well-equipped laboratories which are nowadays provided in most large military stations abroad. It is proposed, by way of an introduction to methods of studying the morbid histology of arthropoda, to briefly outline some broad principles of the technique required. Fixing.-The fixing of the tissue elements is of primary importance for all classes of objects, whether it be desired to cut them into sections or to treat them in any other special way. The term "fixing" implies not only the rapid killing of the element, so that it may not have time to change the form it had during life, but also the hardening of it to such a degree as to enable it to retain its original shape and size when submitted to the action of the reagents with which it may subsequently be treated. It follows, therefore, that the parts of blood-sucking insects required for histology should be immersed in the fixing fluid immediately the insects are killed. A strip of the chitinous wall of the abdomen, of the thorax, and of the head, if a large fly, should first be snipped off, or the parts to be examined may be dissected out in the fixative, in order to permit of rapid fixing and hardening of the cells of the internal organs. A useful fixing fluid for this purpose is picro-acetic acid, as suggested in THE JOURNAL OF THE ROYAL ARMY MEDICAL CORPS for July, 1908. Viscera of insects or pieces of tissue several millimetres in thickness require one to six hours immersion, according to size. The insect and its dissected parts should then be transferred to 60 per cent. alcohol, in which they may be kept indefinitely until it is convenient to embed them. Picric acid should always be

244 The Morbid Histology of Disease-carrying Insects washed out with alcohol. The extraction is more rapid with warm alcohol to which a little lithium carbonate has been added. Where it is possible to proceed at once to embedding the material, it is better to treat the object with one of the following solutions :- (1) Saturated solution of corrosive sublimate to which 2 per cent. of glacial acetic acid has been added; or better:- (2) Absolute alcohol Glacial acetic acid.. Chloroform.. Corrosive sublimate.... 1 vol... 1".. 1".. to saturation. Objects should be removed from the fixing bath and washed in 70 per cent. alcohol as soon as they appear to have become opaque throughout; the time required varies from a few seconds for the salivary gland of a fly to a few minutes for larger objects. Another excellent fixative for all purposes is Flemming's solution, which can be obtained from Griibler ready made up, or may be prepared as follows:- Chromic acid, 1 per cent... Osmic acid, 2 per cent. Glacial acetic acid.... 15 vols. 4 " 1 " Flemming's fluid may be allowed to act for from one to many hours or days. Some workers state that objects may be left in this fluid for several weeks or months without detriment. After fixing in Flemming's solution, it is necessary to wash the solution out of the object very thoroughly by leaving it for several hours in running water. Dehydrating.-The tissue having been fixed and washed, it is now necessary to dehydrate it. This should be done by passing through strengths of alcohol in consecutive order, commencing with a 10 per cent. solution and increasing 10 per cent. until finally a bath of absolute alcohol is reached. For small objects a minute or two in each alcohol bath suffices. They should, however, be left in the absolute alcohol bath for some hours in order to harden. Embedding.-Put into a test tube sufficient cedar-wood oil to cover the object, then gently pour a little absolute alcohol into the tube, so as to have a thin layer of alcohol floating upon the top of the cedar-wood oil. Take the already dehydrated piece of tissue, and put carefully on to the layer of alcohol in the tube, and leave it until it has sunk to the bottom of the cedar-wood oil. Let the object remain in the oil for, say, half an hour, then transfer it rapidly into paraffin kept at melting point in a watch-glass. The

A. E. Hamerton 245 melting point of the paraffin should be the lowest possible in the climate. A paraffin melting at 50 C. if the temperature of the laboratory is between 15 C. and 17 C. is a useful standard; for higher temperatures use a harder paraffin, and for low temperatures paraffin with a low melting point is required. After about half an hour it is advisable to transfer the object into another watch-glass containing fresh clean paraffin. Minute pieces of tissues like the viscera of insects should be left in the paraffin bath for one to three hours according to size. When the object is thoroughly soaked, a paraffin block should be prepared as follows: Take a clean Petrie dish, pour into it enough glycerine to form a.thin layer on the bottom and heat slightly in the spirit-flame. Now pour over the layer of glycerine a thin layer of the melted paraffin. When the layer of paraffin is just about to solidify pour over it quickly the remainder of the paraffin containing the pieces of tissue and quickly separate and arrange the pieces of tissue with a hot needle. The Petrie dish should now be floated in cold water to produce rapid solidification of the paraffin. On immersing the Petrie dish in the water, the cake of paraffin will float up out of the dish. Blocks of paraffin with the pieces of tissue embedded therein can then be cut out of the cake, trimmed as required, and mounted on the object-carrier of the microtome. The preparation of sections of such chitinous structures as the hard proboscis of Stomoxys or the mouth parts of other biting insects requires special consideration. Chitinous structures may be fixed in either picro-acetic acid or, better still, in either of the corrosive sublimate solutions given above. After washing out the fixative the chitin should be softened by boiling gently for about an hour in a 10 per cent. solution of caustic potash. The alkali should then be thoroughly washed out in running water and the object dehydrated and embedded as described above. In studying the anatomy of the mouth parts of biting insects by the method of cutting serial sections it is necessary to endeavour to preserve the integrity of these brittle structures and to keep the component parts of the piercing organs in their natural position. In order to obtain this difficult ideal the following method, though tedious, gives good results: Take four small test tubes. In tube I. make a saturated solution of gum acacia in ether. In tube Il. make a thick syrupy solution of celloidin in ether. Now mix the contents of tubes I. and n. in equal quantities. This makes the stock solution in tube Ill. When proceeding to cut sections take a.nother tube, IV., and put into it 2 cc. of ether and a few drops.. 17

246 The Morbid Histology (If Disease-carrying Insects of absolute alcohol. Then with a camel-hair brush moistened in water take a drop or two of the stock solution from tube Ill. and mix it well with the contents of tube IV. With the mixture so obtained paint the surface of the paraffin block after each cutting of a section. The ether evaporates, leaving over the object to be cut a thin film of adhesive protective material. When sections so treated have been mounted and fixed on the slide the celloidin must be dissolved off by washing the slide over with ether before passing it into xylol. For details of another method of dealing with chitinous structures by immersing in increasing strengths of celloidin solution before embedding, I would refer the reader to Stephens and Christopher's book, "The Practical Study of Malaria" (third edition). Cutting.-When cutting thin sections (8 to 10 p,) the following points must be attended to in order to avoid crumpling and rolling; First and foremost the paraffin must not be too hard. If, after cutting, the paraffin is found to be too hard, it may be softened by placing near the embedded object a couple of Bunsen or spirit lamp flames, so arranged as to ensure equal warming of the two sides of the block. If the paraffin is too soft the block may be cooled by a lump of ice or alcohol spray. Secondly, the knife should be set square and the block trimmed to a four-sided prism. If ribbons are to be cut the block must be oriented, with one of its sides parallel to the knife edge and the opposite side parallel to this one. Ribbons of sections will often cut perfectly flat, even when the same mass will only give rolled and crumpled sections when cut disconnectedly. Mounting.-The following method of mounting minute sections is convenient. Make up Meyer's albumen solution as follows ;- White of new-laid egg Pure glycerine Sodium salicylate.. " 25 cc. " 25 cc. 0'5 gramme For use put about two drops of this solution into a watch-glass full of water and mix. Now transfer a few drops of this albumen solution from the watch-glass on to a slide by means of a camel- 'hair brush. Using the brush again, take up a short ribbon of sections and float it, shiny surface downwards, upon the fluid on the slide. Drain off the excess of albumen solution, and arrange the ribbon in a straight line and let it dry thoroughly on the slide, leaving it overnight if possible, remembering to protect it from,dust. When the sections are dried on the slide, the latter should

A. E. Hamerton 247 be gently warmed until the paraffin is just melting. The slide should now be flushed with xylol to remove the excess of paraffin, and then immersed in xylol for a few minutes. The next step is to put the slide for a few minutes into 90 per cent. alcohol to remove the xylol. Now pass the slide rapidly through alcohol baths from 80 per cent. to 70, 60, 50, 40, 30, 20, and 10 per cent., finally flushing the slide with distilled water. Unstained sections so fixed on the slide can now be transferred for staining to any aqueous stain which may be selected. Staining.-Tissues like the organs of mosquitoes, bugs or flies, which have been fixed in picro-acetic acid, may, after removal of the acid by washing in alcohol, be stained in bulk by transferring them from the alcohol to Grenacher's alcoholic borax carmine stain, as sold ready made up by Griibler or prepared according to the following formula :- Make a concentrated solution of carmine in borax solution (3 per cent. carmine to 4- per cent. borax solution in water) by boiling for half an hour; dilute it with an equal volume of 90 per cent.. alcohol, allow it to stand for a time, and then filter and keep for a week to ripen. Preparations should remain in this stain until thoroughly penetrated, that is, for about half an hour in the case of delicate insect tissues, or several hours for coarser tissues. From the stain they should be transferred direct to 70 per cent. alcohol, which has been acidulated with four drops of hydrochloric acid to each 100 cc. of alcohol solution. They are left in the acid alcohol for a few minutes to allow of differentiation, and are then passed into absolute alcohol and cedar-wood oil, when they may be embedded in the usual way. Tissues may also be stained in bulk in Delafield's hffimatoxylin. This stain gives good results when applied to unstained sections mounted on the slide. Delafield's hffimatoxylin can be obtained ready made up from Griibler and Co., Leipzig, or it may be prepared as follows: To 400 cc. of saturated solution of ammoniaalum (ammonia-alum dissolves in about eleven parts of water) add 4 grammes of hffimatoxylin crystals dissolved in 25 cc. of strong alcohol. Leave it exposed to the light and air in an un stoppered bottle for three to four days, filter, and add 100 cc. of glycerine and 100 cc. of methylic alcohol. Allow the solution to stand until the colour is sufficiently dark, then filter and keep in a tightly stoppered bottle. This solution stains best when a. few months old; it will keep for years. It is a powerful stain and must be diluted before use with equal parts of distilled water, to

248 The Mm-bid Histology of Disease-carrying Inseots which one drop of glacial acetic acid has been added. As the stain is in aqueous solution, objects must not be transferred directly into it from strong alcohol; they must first be passed through the grades of diminishing strengths of alcohol as given previously. Tissues in bulk may be stained overnight in this solution. Tissues in section should be examined at intervals under the low power of the microscope to guard against over-staining. About three hours immersion in the stain is usually sufficient. For accurate work in cytology and histology staining is most satisfactorily done after cutting and fixing the sections on to the slide. It is advisable to prepare half a dozen or more slides of the tissue to be examined, and to stain by several different methods. An aqueous solution of Leishman's stains gives good results with some tissues. I would refer the reader to the British Medical Journal of June 20, 1908, for a good method of fixing and staining by the Giemsa stain. It is generally admitted that the most reliable of all known stains is iron-hrematoxylin applied according to Heidenhain's method. This is a very powerful stain of a certain optical quality peculiarly suited to the employment of high powers. It will take effect on any material, is absolutely permanent, and it permits of the use of a large variety of counter-stains. As this is a standard stain, the technique of its application will be given in detail. The sections having been fixed on the slides as above described and the paraffin washed out with xylol, the slides are resting in 90 per cent. alcohol. It is now necessary to transfer the specimens into 80 per cent. alcohol, containing iodine and potassium iodide in the following proportions :- Make up a concentrated solution of potassium iodide in water, and of iodine in alcohol. Mix them together, and add to the mixture some 80 per cent. alcohol until the mixture becomes a dark brown colour. Leave the slides in this solution for five to ten minutes, and then pass them into 30 per cent. alcohol, and through decreasing strengths of alcohol to distilled water. The specimens should now be immersed for about an hour in the following solution of iron-alum (eisenoxydammon, Griibler), 3 per cent. dissolved in distilled water. The slides are then washed with water and stained in an aqueous solution of hrematoxylin prepared as follows: Hrematoxylin (pure), t per cent., dissolved in distilled water to which a few drops of concentrated solution of lithium carbonate may be added by way of improving the stain. This solution keeps indefinitely and improves with age. The

A. E. Hamerton 249 sections should be left in the hrematoxylin for half an hour to two hours. They are then washed with water and again treated with iron-alum solution, which slowly washes out the stain. The progress of the differentiation ought to be controlled by frequent microscopic examination under the low power, the sections being removed from time to time out of the iron-alum, rinsed with water and examined. When a proper differentiation has been obtained, i.e., the nuclei appearing black and other parts of the cell fairly clear or smoky, the preparations should be washed for at least a quarter of an hour in running tap-water. In order to make a pretty preparation with a good contrast of colour, it is advisable to counter-stain in one of the following solutions: Saffranin 0 (Griibler), 1 gramme dissolved in 10 cc. of absolute alcohol and added to 90 cc. of aniline oil water. After differentiation and washing in tap-water, the preparations may be transferred directly into this stain, and left therein until the tissue has become sufficiently coloured. They must then be rapidly dehydrated with alcohol, cleared in xylol and mounted. Orange G (Griibler), a little of the stain is put into 80 per cent. alcohol to form a saturated solution. The slides should be passed through increasing strengths of alcohols to the 80 per cent. solution containing the stain. They may be left in the latter for about ten minutes. Eosin or acid fuchsin prepared in the same way may be used as counter-stains. For further information I would refer the reader to the two books named in the footnote. l The preparation of microscopic specimens, like the preparation of photographs, admits of considerable modification and improvement according to the experience and fancy of the worker. Every microtomist having once mastered the theoretical and practical rudiments of his craft, will soon know how to select fixatives and stains suitable to his material. Although we cannot all expect to unfold great truths which would point out the way for the advance of practical preventative medicine, we cannot fail to derive interest and pleasure from contemplating the exceeding beauty and wonderful complexity of such minute organs as the proventriculus of a fly or the sucking apparatus of a mosquito. In conclusion, I desire to express my thanks to Professor E. A. Minchin, M.A., F.Z.S., of London University, for his great kindness in demonstrating to me many of the methods described above. 1 "The Microtomist's Vade-mecum," Lee, Sixth Edition; and "Practical Study of Malaria," Third Edition, Christopher and Stephens.