^.f'oV'i^^B CORNELL UNIVERSITY. THE THE GIFT OF y<i'it"(..,r' The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/cletails/cu31924104225275 STUDIES IN LABORATORY WORK STUDIES LABORATORY WORK BY C. W. ^ANIELS, M.B.Camb., M.R.C.S.Eng- Director of the London School of Tropical Medicine; Lecturer on Tropical Diseases at the London Hospital ; formerly Director of the Institute for Medical Research, Federated Malay States ; Member of the Royal Society Malaria Com- mission in India and Africa, and in the Medical Service of the Colonies of Fiji and British Guiana AND A. T. STANTON, M.D.Tor., M.R.C.S.Eng., D.pi\^Tr,Camb. Demonstrator London School of Tropical M^icine - ' •' ^ ^. 1 wt , ; i ■■^Y, J^" \s^ K/ '■,_ % l!V. SECOND EDITION Thoroughly revised, with many new and additional illustrations PH ILA.de LP HI A P. BLAKISTON'S SON, & CO. IOI2 WALNUT STREET 1907 [All Rights Reserved] J) No-.^dnr JOHN BALE, SONS AND DANIELSSON, LTD. GREAT TITCHFIELD STREET, W PREFACE TO SECOND EDITION. Rapid advance has been made in all branches of tropical medicine since the first edition was published. In this edition these advances are considered, and especially information as to the known carriers of disease has been added, including ticks, biting flies and fleas. The subjects are now so large that a certain amount of systematic classification has become necessary, and tables are therefore included showing, in brief, the zoological relationship of parasites and of their carriers. A'sub-division of the chapters has been made, as it is hoped from time to time, by publication of the further advances made, to supplement the information given in each chapter. The general idea remains the same, the book is intended for the lonely worker in his private laboratory. We are indebted to many friends for advice and assistance, particularly in the revision of proofs, and especially to Dr. W. H. B. Newham, and A. W. Balch, Surgeon, U.S. Navy. C. W. D. A. T. S. September, 1907. PREFACE TO FIRST EDITION. The object of this work is to assist practitioners in the Tropics in the application of simple laboratory methods to the practice of medicine. The writer has had personal experience in several countries of the peculiar difficulties that a student desirous of advancing the. knowledge of tropical medicine, or of practising it con- scientiously, will meet, and the plan of study advocated is the outcome of this .experience. The subjects include an outline of animal parasitology and the development of the best known of these parasites. The part played by insects in spreading disease is so important that it is necessary to have a sound working knowledge of the more important known carriers of disease. Chapter vii. has been kindly written by Mr. F. V. Theobald for this book, so as to enable the student to differentiate the more important genera of the Diptera. No exhaustive study of any one subject has been made, but it is hoped that suffi- cient information is given to enable the practitioner to com- mence the effective study from the laboratory point of view of the more important problems. Simple methods are selected as far as possible, and those recommended are in the main those adopted by the writer for teaching purposes at the London School of Tropical Medicine, and can be relied on as applicable to the circumstances. Few references are given, as the practitioner in the Tropics has rarely access to a library. I am much indebted to Dr. G. C. Low, Medical Superin- tendent at the London School of Tropical Medicine, for valuable assistance, and the revision of the proofs has also been kindly undertaken by him. CONTENTS. The Laboratory... Material Chapter I. Chapter II. Chapter III. Blood ... ... ... ... ... ... ... 40 Chapter IV. Animal Parasites found in the Blood ... ... ... 69 Chapter V. Parasites found in the Blood of Animals ... ... ... 98 Chapter VI. , Parasites found in Blood Plasma ... ... ... ... 106 Chapter VII. Parasites other than Protozoa found in Human Blood ... 117 Chapter VIII. Certain Properties of Blood Plasma and Blood Serum ... 134 Chapter IX. Arthropoda — Insecta ... ... ... ... ... 148 Chapter X. Diptera ... ... ... ... ... ... 153 Chapter XI. Mosquitoes ... ... ... ... ... ... 194 X. CONTENTS PAGE Chapter XII. Dissection of Mosquitoes... ... ... ••• ••■ 229 Chapter XIII. Demonstration of Development of Parasites in Mosquitoes ... 243 Chapter XIV. Eggs, Larvae and Pupse of Mosquitoes ... ... ••• 252 Chapter XV. Fleas, Lice, and Bed-bugs ... ... ... -.- 273 Chapter XVI. Arachnoidea — Ticks, Mites, Porocephalus. Crustacea — Cyclops 286 Chapter XVII. Pigment Deposits and Degenerations in Tissues ... ... 301 Chapter XVIII. Parasites in the Tissues ... ... ... ... •■• 31' Chapter XIX. Fasces ... ... ... ... ... ■■■ ... 318 Chapter XX. Intestinal Parasites ... ... ... ... ... 332 Chapter XXI. Urine ... ... ... ... ... ... ... 357 Chapter XXII. Bacteriology ... ... ... ... ... ... 365 Chapter XXIII. Measurements ... ... ... ... ... ... 415 Chapter XXIV. Statistics ... ... ... ... ... ... 429 Appendix. Tables ... ... ... ... ... ... ... 455 Instruments and Reagents ... ... ... ... 457 Index... ... ... ... ... ... ... 459 LIST OF ILLUSTRATIONS. I — Automatic Bunsen Burner for Methylated Spirit ... 3 2 — " Primus " Paraffin Lamp ... ... ... ... 4 3 — A useful Microscope for tropical work... ... ... 5 4 — Micrometer Eye pieces ... ... ... ... 14 5 — Micrometer Eye pieces ... ... ... ... 14 6 — Micrometer Eye pieces ... ... ... ... 14 7 — Koch's Steam Sterihser ... ... ... ... 17 8— Hot Air Steriliser ... ... ... ... ... 18 9 — Hearson's Incubator, working with Petroleum Lamp ... ig 10 — Hot air Oven for paraffin ... ... ... ... 31 II — Paraffin Bath ... ... ... ... ... 31 12 — Block for moulding paraffin ... ... ... ... 31 13 — Cathcart's Microtome, with spray bellows ... ... 34 14 — Swift's Freezing Microtome ... ... ... ... 35 15 — Parts of Swift's Microtome ... ... ... ... 35 16 — Cambridge Rocking Microtome, new pattern for cutting flat sections, with large articulating apparatus and one razor ... ... ... ... ... 2>1 17 — Diagram to illustrate the making of a wet blood film ... 42 18 — Braddon's method of making blood films ... ... 43 19 — Crenated, vacuolated and buckled corpuscles ... ... 45 20 — Method of making dry films with two slides ... ... 49 21 — Method of making dry films with needle ... ... 49 22 — Method of making dry film with gutta percha ... ... 50 23 — Method of making dry film with two cover glasses ... 50 24 — Leucocytes ... ... ... ... ... 54 25 — Myelocytes ... ... ... -... ... 59 26 — Wide necked stoppered bottle for staining and fixing blood films ... ... ... ... ... 72 27 — Schematic view of the asexual and sexual phases of the malarial parasite ... ... ... ... 75 28 — Parasites in capillaries ... ... ... ... 84 29 — Phases in the asexual and sexual development of the quartan parasite ... ... ... ... 86 30 — Phases in the asexual and sexual development of the benign tertian parasite ... ... ... ... 87 xii. LIST OF ILLUSTRATIONS FIG. PAGte 31 — Phases in the asexual and sexual development of the malignant malarial parasite ... ... ... 89 32 — Proteosoma and Halteridium ... ... ... 99 33 — Development of Piroplasma ... ... ... ... loi 34a — Drepanidium ... .•■ ... ... ••• 103 34i5 — Hasmogregarina balfouri ... ... ... ... 103 35 — Development of H. balfouri (Plate) ... ... ... 104 36 — A Trypanosoma dividing ... ... ... ... no 37 — Y ornis oi Spirochceia obermeiri ... ... ... 112 38 — Trypanosome in various stages ... ... ... 113 39 — Plate of Leishman Donovan bodies ... ... ... 114 40 — Trypanosomes and Leishman-Donovan bodies ... 115 41 — Method of making a film for the examination for filarise... 119 42 — Glass rack for staining a number of slides ... ... 120 43 — Cobb's formula for measuring filariae ... ... ... 125 44 — Head of Filaria bancrofti ... ... ... ... 126 45 — Head of Filaria oszardi ... ... ... ... 126 46 — Tail of Filaria bancrofti ... ... ... ... 127 47 — TaW oi Filaria ozsardi ... ... ... ... 127 48 — Head of Filaria demarguai ... ... ... ... 130 49 — Head of Filaria Persians ... ... ... ... 130 50 — Tail of Filaria demarquai ... ... ... ... 131 51 — Tail of Filaria Persians ... ... ... ... 131 52 — Some of the important Spectra ... ... ... 137 53 — Wright's Tubes ... ... ... ... ... 141 54— Mixing tube ... ... ... ... ... 142 55 — Wright's tube with rubber teat ... ... ... 144 56 — Antennae ... ... ... ... ... ... 155 57— Mouth of an £w/zj ... ... ... ... 157 58 — Wing of Tipula ... ... ... ... ... 158 59 — Base of wing, calyptrate diptera ... ... ... 159 60 — Puparium of a " Screw-worm " ... ... ... 159 61 — Wing of a Cecidomyia ... ... ... ... 162 62 — Wing of Anopheles maculipennis ... ... ... 163 63 — Wing of a Culex ... ... ... ... ... 163 64 — Wing of Chironomus ... ... ... ... 164 65 — A Ceratopogon ... ... ... ... ... jg^ 66 — Wing of C"^n?/o/of<7« (after Leonard!) ... ... 165 67 — Phlebotomus ... ... ... ... ... 165 68 — Wmg of Simulium... ... ... ... ... 167 69 — Head of Tabanus ... ... ... ... ... jgg 70 — Wmg of a. Tabanus... ... ... ... ... jgg 71 — Tabanus bovinus ... ... ... ... ... 170 72 — Hcematopota pluvialis ... ... ... ... j 70 73 — Head of Hcematopota ... ... ... ... jyj LIST OF ILLUSTRATIONS Xlll. FIG. PAGE 74 — "^'vn^ oi HcBmatopota pluvialis ... ... ... 171 75 — Chrysops Distinctipennis ... ... ... ... 172 76 — Leptis scolopacea ... ... ... ... ... 173 77 — Wing of Empis ... ... ... ... ... 174 78 — Dermatobia noxialis ... ... ... ... 161 79 — Dermatobia noxialis ... ... ... ... i6i 80 — Stomoxys ... ... ... ... ... ... 179 81 — Wvag oi Stomoxys calcitrans ... ... ... 180 82 — Cross-section of proboscis of ^/owziJ^yj ... ... 180 83 — Dissections of the abdomen of 5/(?»?ox)/i' ... ... 182 84 — Transverse section of the proboscis of Glossifia palpalis 184 85 — Glossina morsitans ... ... ... ... ... 184 86 — Lucilia ccesar ... ... ... ... ... 187 87 — Yi&a.A. oi Lucilia ccesar ... ... ... ... 187 88 — Chrysomyia macellaria ... ... ... ... 188 89 — Auchmerom.yia luteola ... ... ... ... 188 90 — Homalomyia canicularis ... ... ... ... 1 90 91 — 'Lax-vdi oi Homalomyia ... ... ... ... 191 92 — Wmg oi Hydroicea ciliata ... ... ... ... 191 93 — Hippobosca equina ... ... ... ... ... 192 94 — Melophagus ovinus ... ... ... ... 192 95 — Pinning Mosquitoes on discs ... ... ... 196 96 — Examination of scales on Mosquito ... ... ... 197 97 — Types of scales, head ornamentation, forms of clypeus ... 198 98 — Anatomy ... ... ... ... ... ... 200 99 — Types of metathorax (Theobald) ... ... ... 201 100 — Neuration of wing (Theobald) ... ... ... 203 loi — Various forms of wing-scales (Theobald) ... ... 204 102 — Mosquitoes ... ... ... ... ... 208 103 — Culex (male and female) and Anopheles (male and female) 209 104 — Head and mouth-parts of Mosquito ... ... ... 229 105 — Maxilte and mandibles of Mosquito ... ... ... 230 106 — Tip of Proboscis of Mosquito ... ... ... 231 107 — Cross-section of proboscis of Mosquito ... ... 232 108 — Method for dissecting Mosquitoes ... ... ... 234 109 — Method for dissecting Mosquitoes ... ... .... 235 no — Method for the dissection of the salivary glands ... 237 (Method for the dissection of the salivary glands ... 238 '" I Stomach of the Mosquito, showing zygotes ... ... 246 112 — Eggs of Mosquitoes ... ... ... ... 254 113 — Method for catching larvae ... ... ... ... 256 114 — Method for catching larvae ... ... ... ... 256 115 — Breeding grounds of Mosquitoes ... ... ... 266 116 — Breeding grounds of Mosquitoes ... ... ... 266 117 — Mosquito box ... ... ... ... ... 270 118 — Folding mosquito cage ... ... ... ... 271 XIV. LIST OF ILLUSTRATIONS 119 — Mosquito house 120 — Mouth-parts of a Flea (after Wagner) 121 — External anatomy of flea 122 — Types of Fleas 1 2 3 — Pediculus vestimenti 124 — Phthirius inguinalis 125 — Cimex lectularius ... 126 — Legs of Ticks 127 — Ixodina (female) 128 — Ixodina (males) 129 — Mouth-parts of Ornithodoros 130 — Mouth-parts of /rarfgj 131 — Mouth-parts of Rhipicephalus \2)'2. — Ornithodoros savignyi 1 3 3 — Demodex follicularum. 134 — Stages of Cyclops ... 135 — Wire gauze strainer 136 — Eggs of some of the Intestinal Worms 137 — Anatomy of a segment of a Tapeworm 138 — Genital pores of some of the Tapeworms 139 — Diagrammatic view of suckers of Trematodes 140 — Anatomy of a Fluke 141 — Oxyuris vermicularis (male and female) 142 — Trichocephalus dispar (male and female) 143 — Male and female Ankylostomes 144 — Head and tail of male A. duodenale; head male N. americanus- 145 — Scheme of development of Amoeba 146 — Cercomonas hominis 147 — Balantidium colt 148 — Erlenmeyer's Flask 149 — Petri's Dish 150 — Cornet's Forceps 151 — Durham's tubes 152 — Centrifuge 153 — Thoma's Hasmocytometer, by Zeiss 154 — Oliver's Tintometer 15s — Cowers' Hsemoglobinometer 156 — Von Fleischl's Hasmometer ... and tail of PAGE 272 274 276 278 282 283 284 288 289 291 294 295 295 297 298 300 327 329 335 339 340 345 345 347 348 353 355 355 369 373 381 391 395 418 425 426 427 Six Statistical Charts 444, 445: 446, 447, 448, 451 Coloured Plates at End. Plate I. 27 figures Plate II. 19 figures Plate III. 21 figures Plate IV. 25 figures Studies in Laboratory Work. CHAPTER I. The Laboratory. — In few places in the Tropics is there any institution that corresponds to the British idea of a laboratory. Tap-water, gas and electric light usually have to be dispensed with and substitutes employed. The isolated worker has to arrange and make his own laboratory, either in the house or attached to a hospital. A separate building will rarely be available. The first essential is a good light, and if, as is usual, work is done by daylight, the light must come neither from east nor west. A north or south aspect should be chosen, according to whether the worker is north or south of the line, so as to avoid direct sunlight. A corner of a verandah can be made into a good laboratory, by placing blinds or jalousies on two sides, and leaving only the one side, that facing north or south, open. The side from which the light is received should be closed in with a window if possible, to prevent the entrance of rain and dust. Another important consideration is wind, and with the wind the amount of dust. If there is a glass window this is of less importance, but if working on an open verandah, a portion of the verandah sheltered from the prevailing wind must be selected, even if this choice involves the sacrifice of the most favourable light. If a room with a north or south aspect is not available 2 THE LABORATORY any other aspect will suffice, provided that there is a deep, low verandah outside the window. On the wall of the laboratory should be fixed a number of plain wooden shelves. One of the lower of these, at a convenient height, should be strong and broad enough to receive heavy weights. On this shelf may be kept mosquito cages, maturing larvae and other objects awaiting immediate examination or requiring constant attention. It is convenient to have two tables — one on which to work with the microscope, and also for papers, notebooks and any book actually in use, another on which staining processes, dissections and the rougher and more messy work can be done. Individual habits of neatness and arrangement make a difference. Though much excellent work has been done by untidy workers, there is no doubt that in the limited space available on the narrow veran- dahs usual in many parts of the Tropics, work is easier and more comfortable if the habit of tidiness be culti- vated. Persons who are exceptionally neat and method- ical in their habits will probably find one long table more convenient than the two recommended here. For work with the microscope a firm steady table is required, and this should be placed a few feet from the window. The second table, which should be also strong, must be placed in a good light, and it is better to have the light falling from the left-hand side of the table. For the other side of the laboratory jalousies are most convenient, as they let in plenty of air and can be turned so as to regulate the amount of air and to stop the entrance of rain. A cheaper arrangement is to use reeds,, as natives in the most parts of the world are good workers with reeds. Sufficient air will pass through to keep the room cool. Native mats can be used, but must be nailed on to a framework, otherwise they will get blown about by the wind. Water must be kept in bulk, as tap-water is rarely available. A small tank — an empty thoroughly cleaned. THE LABORATORY kerosine tin will serve — should be kept filled with water. This should be filtered, and a glass syphon tube with a rubber tube and clamp attached can be used to draw Fig. I.— Automatic Bunsbn Burner for Methylated Spirit. it off. The tank must be kept covered with a well- fitting lid, and a basin or other receptacle should be placed underneath to receive the waste and washings of stains, &c. 4 LABORATORY APPARATUS Distilled water must be kept in bulk in a well-stop- pered bottle, from which a sufficient amount is taken as required into a wash bottle for immediate use. An excellent substitute for the ordinary gas Bunsen burner is the spirit Bunsen (fig. i). The "Primus" Kerosine Smokeless Burner will be found very useful for heating vessels on a larger scale (fig. 2). Fig. 2. — "Primus" Paraffin Lamp. An incubator is an enormous advantage and for accurate bacteriological work is essential. The temperature in most tropical places ranges from 75° upwards, and organisms grow better at " room " temperature than in England. In many places the nocturnal and diurnal variations are small, and in such the need for an incubator is not so great. In others there is a great difference between the day and night temperature, and in these the need is great.* A cold incubator is useless unless ice can be obtained. * With practice and the exercise of some ingenuity a workable incubator can be made by placing one tin inside a larger one ; (or a chemist's water-oven may be employed). The space between THE MICROSCOPE 5 Cultures should be kept in a dark cupboard, which must be as dry as possible. Above the long broad shelf running along the wall two or three rows of narrow shelves should be fitted up on which stains in use can be kept. These are better kept exposed than in cupboards. The main stock can, of course, be kept out of sight. For night work a good lamp is required. The lamp must be low, and the flame not more than six inches from the table.* Equipment : A good microscope with a sub-stage con- denser, iris diaphragm and mechanical stage is essential. An oil immersion ^Sg-inch objective, a low power, say f-inch, and a fairly high power, say ^-inch, will be required. For many purposes a ^-inch is a very useful lens. It is well to have two eye-pieces. The choice of suitable microscopes is a large one, and the differences between those of different makers are not very great, the points of difference being such that it is difficult to say which is the best. In the choice much depends on the conditions under which the work has to be conducted. If much travelling has to be done it is advisable to have a microscope that is easily carried and can be set up for use at a moment's notice. Of these travelling or portable microscopes there are several different forms all fulfilling the main requirements — light- ness, compactness, and usefulness. The folding micro- scopes made by some makers, though compact and easily packed, are heavy and therefore inconvenient to carry. If most of the work can be done at a fixed station the two is filled with water. A small kerosine lamp placed beneath the tins will heat the water, and by varying the height of the lamp a sufficiently equable temperature can be maintained. * For the best definition the narrow edge of the flame should be used as the source of the illumination and focussed accurately on the object. 5 THE MICROSCOPE one of the ordinary forms of microscope is the most convenient to work with. If the expense ^^ "° ^^^^^ it is well to have two stands, one portable and one tor stationary work. The objectives and eye-pieces can be used for either, and therefore the additional expense is not very great. THE MICROSCOPE 7 Parts of a Microscope.— The base or stand is a stage fixed either to a tripod or to a vertical column rigidly attached to a solid and heavy footplate. The tripod is to be preferred, as from the wide spread of the legs greater stability is secured, and the level is less affected by irregularities in the table on wrhich the micro- scope is placed. In the folding and portable microscope the legs of the tripod are jointed at or near their junction with the stage, and can be folded back so as to economise space in packing. The stage itself is a fixed plate firmly attached to the upright carrying the optical parts of the instrument, viz., the mirror and sub-stage condenser below the stage, and the tube, eye-piece and objective above. To this solid plate is fixed the mechanical stage, of which there are two main types : — (i) Those in which a lighter stage carrying the . object to be examined is attached above the fixed stage. This can be moved by a rack and pinion in two directions at right angles to each other. (2) Those in which the slide is seized by catches and moved over the solid stage. Some mechanical stages have in addition to the rectan- gular motions a circular one in the same plane. This motion is not required. Of the two types, preference should be given to the first, as it can be used for objects of all sizes and shapes, not simply, as with the second, for objects mounted on the regulation slides. With care it does not get out of order any more readily than that of the second type. The microscope tube is attached to the upright in such a manner that it can be moved up and down parallel to the upright, but allows no lateral movement in any direction. The length of the tube is important, as with the higher objectives the best definition is obtained with a certain known length of tube. This distance varies with the objectives of different makers. To provide for 8 THE MICROSCOPE this variation there is a second or draw-tube inside the outer tube, which can be drawn out so as to lengthen the tube to the required extent. The length of tube required for an objective should be ascertained, and the draw-tube should be, and usually is, marked so that the correspond- ing length can be obtained. In the portable and folding microscope the outer tube is so short that it is always necessary to use the draw- tube. The adjustments by which the object is focussed are of two kinds : — (i) The coarse adjustment, by which the tube is moved by a rack and pinion and brought approxi- mately into focus. The range of the coarse adjust- ment is great, but the movement is too coarse to focus easily and correctly with higher powers. (2) The fine adjustment, which may be a differen- tial screw or of the lever pattern. The range of this adjustment is small, but very delicate movement is obtained. Illuminating Apparatus. — Good illumination is abso lutely necessary for useful work with high powers. The parts of the microscope providing for this illumination and modifying it are the mirror, the sub-stage condensor and the iris diaphragm where, as is most usually the case, the object is to be examined by transmitted light. For opaque objects which can only be usefully examined with low powers illumination comes from above the stage. The Mirror is attached below the condensor. It has two surfaces, one concave and the other plane. The plane mirror is that employed for work with higher powers. Too small a mirror should not be used. The Sub-stage Condenser. — This is placed between the mirror and the stage, and collects the rays of light received from the mirror into a cone of large aperture, which can be focussed on to the plane of the object. It must be centred so that the optical axis corresponds with that of the objective, and must be movable so that THE MICROSCOPE 9 it can be moved up or down in this axis. The move- ment is better performed by a rack and pinion, but in most of the portable microscopes this has to be done by hand. The Lenses. — To the tube are fixed at each end the two systems of lenses used for the magnification of the object. The lower system of lenses, which is screwed on to the lower end of the tube, is the objective and forms a real image of the object, which is further magni- fied by the system of lenses at the upper end of the tube — the eye-piece. To save time, annoyance, and wear of screws a nose- piece is fitted to the lower end of the tube, to which can be screwed the three objectives in use instead of screwing them directly to the lower end of the tube. These are the essentials of a microscope for the work here contemplated. It can be purchased complete for about £^o from several well-known makers. The price varies a little, but the reader is strongly advised to pay little attention to slight differences of price in the selec- tion of an instrument that suits him. Much more expen- sive instruments can be purchased, but at about the above-mentioned price an instrument can be obtained suitable for the work contemplated. The portable microscopes with the same objectives are about £'i, or ;^4 less. No microscope should be bought without spending some time in careful examination and testing of the lenses and adjustments. The points to which special attention should be paid are : (i) The rigidity of the stand. This rigidity must be constant both with the tube vertical and inclined. (2) All the adjustments and screw movements must be tested to see that they work smoothly and evenly, and that every movement of the milled head results in effective movement of the screw and of the part of the instrument which it is intended to move; lO THE MICROSCOPE With the mechanical stage it is further necessary to satisfy one's self that the movement imparted to the stage is all in one plane, otherwise as the object is moved it will also move out of focus. This can be ascertained by examining an object, such as a uniform blood film, under various powers and determining how far the object remains in focus. When using a ^^-inch objective, even with the best stages, some focussing will be necessary, but it should be slight, and the object should be very little out of focus with considerable movements of the mechanical stage. A slide and film of uniform thickness must be used for this test, and the result of the examina- tion should be confirmed by using a series of slides. The nose-piece should centralise the objective cor- rectly, otherwise an object that is in the centre of the field with a low power may not be in the field with a higher power. This is tested by centralising with the highest power some object that is visible with the lowest power, and seeing how near the centre of the field this object is when viewed with the other objectives. The order should also be reversed if the test appears to be satisfactory. In testing the objectives the points to be most closely investigated are : — (i) Definition. Unless the object is sharply and clearly defined the magnification is wasted. (2) Flatness of field. Many lenses give good and sharp definition at the centre of the field, whilst objects a little removed from the centre are blurred, and those at the periphery are out of focus. In using such lenses, if any other part of the field is brought into focus the objects in the centre of the field will be out of focus. With such a lens the field is not flat. It is perhaps too much to hope that the periphery of the field will be in sharp focus at the same time as the centre, but at any rate for blood work the greater part of the field must be flat, other- wise objects such as malaria parasites may easily be overlooked. ILLUMINATION II (3) Chromatic aberration must be entirely corrected and no particoloured fringe seen round the edge of the field. (4) Magnification. As a test object a well-stained, evenly spread blood film is as good an object as any, and as the object is a familiar one the degree of magnification can be readily estimated. Both eye- pieces should be used in turn. In the use of the microscope great attention must be paid to the illumination. The light in the Tropics is not good, as it so often has to be derived from blue sky. The mirror should be turned so as to receive the light from a white cloud when possible. When very sharp definition is required the narrow edge of a flat flame should be used as the source of illumination. In using the low power this should be focussed on the object, and by means of the centring screws of the condenser brought right across the field of vision. A higher power, say ^, should then be used, and again the condenser should be centred so that the image of the flame stretches across the field. Finally, this pro- cess is repeated with the oil immersion lens. The condenser must be raised until the image of the flame is as sharply defined as the object under examina- tion. In this way very sharp definition is obtained, but the greater part of the field is not illuminated. For the finer work the condenser as well as the objectives must be apochromatic. In using a low power the condensor should be low so as to be out of focus, or if the stand permits it, swung out so as not to be between the mirror and the object. With a |-inch objective it should be higher, and with the ^^^inch oil immersion objective close to the under- surface of the slide. The brightest and most uniform light that can be obtained with the iris diaphragm open is the best. If it is desired to reduce the light, it should be done by closing the diaphragm, not by altering the position of the con- denser or of the mirror. 12 USE OF MICROSCOPE Both the mirror and condenser should be kept clean. It is well to have a spare mirror, as these silvered mirrors sometimes deteriorate rapidly in the Tropics. In focussing it is well to bring the objective nearer to the object than is necessary, and then, using the coarse adjustment, whilst looking down the microscope to withdraw the objective from the object till it is seen more or less distinctly. For exact focussing the fine adjustment should be used, but not till the object is nearly in focus. The range of the fine adjustment is small, and if used over too extensive a range there is risk of straining it. When working with the oil immersion lens it is well to place the oil on the object and screw down the tube till the objective touches the oil. In doing this the drop of oil should be viewed from the side, and it will then be easy to see when the objective touches the oil. After- wards very slowly focus on the object. Before using an oil immersion lens the field should be examined with a low power to make certain that there is something visible in the field. In a fresh blood film, for instance, if a part be selected in which there are no corpuscles there may be nothing to focus on, and in such a case there is risk of screwing the objective down on the cover- glass. If black specks are visible in the field it is well to rotate the eye-piece ; if these rotate with the eye-piece there are particles of dirt in some part of the eye-piece. Dirt on the objective shows as a general haziness ; such haziness may also be due to a cloudy or dirty cover-glass or a badly prepared specimen. All glass, and particularly, the softer and more highly i-efractile glass of which lenses are made, is liable in a hot, moist climate to deteriorate and become cloudy or white, resembling very fine ground glass. When lenses become affected in this way they require regrinding. Some lenses spoil more quickly than others, and in purchasing objec- tives it should be stated that they are required for work DETERIORATION 1 3 in the Tropics. Various less serious conditions are some- times mistaken for this change in the glass. The cement may run so that it partly covers the inner aspect of the objective. In other cases water condenses between two lenses and causes a want of definition similar to that due to frosting of the glass. Either of these conditions may be detected by unscrewing the lenses and examining the surface with a watchmaker's glass or hand-lens. These conditions, when discovered, are easily remedied. It is well to use only lenses that can be unscrewed, and from time to time to unscrew and clean the surface of the lenses carefully. They will keep longer if this is done, but must not be expected to last as long as they do in England. Lenses not in use are best kept in a perfectly dry stoppered bottle. There is no objection to having some dehydrating agent, such as well-dried calcium chloride, in a separate compartment in the same bottle. A camera lucida or drawing camera is a great conve- nience, and so useful for measurements that some form of this instrument should be used. That of Leitz is a cheap and simple form, the use of which it is easy to learn. For measurements a micrometer slide ruled to ^^^ of a millimetre is a useful accessory ; failing it any of the standard ruled scales, such as the counting chamber of a Thoma-Zeiss' or Gowers' haemocytometer, can be used as a substitute. A micrometer scale to be placed in the eye-piece in focus with the front lens is useful for some measure- ments, but can be dispensed with if measurements are made with a camera lucida. A more useful form of eye- piece micrometer is ruled in squares. Once they are standardised these can be used for blood counts, and the ruled scales used for the counting chamber of a haemo- cytometer, &c., dispensed with. For many purposes it is convenient to subdivide the field, and this can be more readily done with a micrometer eye-piece ruled in squares than in any other way. H MICROMETER EYE-PIECE These eye-piece scales are simply placed in the eye- piece and rest on the diaphragm between the two lenses. It will usually be necessary to move the diaphragm Fig. 4. /^^^ ""^^ / ^ V ; Fig / -: -.:: • 5- -V s, ::::-: v :::::::::::: \::\'-:::\':V---- \ 7 . . _ 7 .i^ Fig. 6. slightly in order to bring the scale sharply into focus, but this is easily accomplished. These eye-pieces require standardisation for the value DISSECTING MICROSCOPE 1 5 of the squares or scale. The micro-millimetre scale is used as the object, and for each objective the number of micro-millimetres in a division of the scale noted. This can be done once for all and the records preserved. There is no object in having the divisions of a scale or the squares of an accurately known size. As seen in the eye-piece they are magnified. A warm stage is not so much needed in the Tropics as in England, but is a convenience. The simplest form is a copper plate perforated with a hole the size of a shilling. From the plate a copper tongue extends in front for about six inches. The under-surface of the plate is covered with cloth and is placed on the stage so that the aperture corresponds to the central aperture in the stage. The object is placed on the slide on the copper plate and examined, and by heating the tip of the tongue of copper projecting from the plate by means of a spirit lamp, the heat will be conducted to the plate and the slide kept warm. By heating the tongue nearer to the plate a higher temperature will be obtained, and by lowering the spirit lamp, or moving it further off, a lower temperature. With a little practice there is no difficulty in maintaining a fairly uniform temperature which can be estimated by touch. More elaborate warm stages are to be procured in which the temperature is kept steady by the circula- tion of hot water. A dissecting microscope, is useful but not essential ; it consists of a single compound lens fixed on a vertical carrier which can be raised or lowered by a rack and pinion ; the stage is of glass and there are wooden movable hand-rests at each side. For illumination there is a plane reflector, and as an alternative on the other side of the mirror a plaster of Paris disc. For most of the purposes for which the dissecting microscope is used a watchmaker's glass does equally well, and for some purposes it is better, as both hands are free, and no stage is required. A good large hand-lens on a handle is useful for observing the habits of mosquito larvae. 1 6 COVER GLASSES AND SLIDES Reagents, stains, slides and cover-glasses are required. The slides should not be of the best quality ; the thin, white slides deteriorate in the Tropics more rapidly than the coarser glasses. No. 2 quality is to be preferred. They require thorough cleaning, and a stock cleaned and ready for use, requiring only to be wiped, should be kept in hand. They are best cleaned by placing in a saturated solution of carbonate of soda which is just brought to the boil. Afterwards they are well washed in running water, wiped with a soft linen rag, and kept in spirit in a stoppered or well-corked, wide-mouthed bottle. Before use the slide must be taken out of the spirit and well rubbed with a soft, clean linen rag. Cover-glasses are best sent out in oil or covered with oil, as even in the course of a voyage lasting only two weeks they may become frosted. The whole mass of cover-glasses, say half an ounce, is placed in oil of cloves, and the cover-glasses are separated so that the oil pene- trates between them. They are then taken .out of the oil, wrapped in cotton-wool and can be replaced in their boxes. Treated in this way they will keep for months even in the worst climates. They also keep well if sent out in spirit, but this is not recommended, as if the spirit evaporates completely the glasses deteriorate very quickly. Cover-glasses treated with oil are not very easy to clean, as the oil will have hardened and dried to a large extent. A good deal of the oil can be removed by plac- ing the cover-glasses in i per cent, lysol solution or in xylol, and separating them or stirring them up. This saves the spirit which is necessary to more completely remove the oil. They must not be left more than an hour in the lysol, and then should be placed and kept in spirit, which will gradually remove nearly all the oil. A small stock should be further prepared so as to be ready for immediate use. This may be done by first just bringing them up to the boiling point in a saturated solution of carbonate of soda, washing well, preferably COVER GLASSES 17 in running water, and transferring them to strong sul- phuric acid ; in this they should be left over night, then again well washed in water and finally transferred to a wide-necked, well-stoppered bottle half filled with spirit. For use they should be taken out with forceps and well rubbed with a soft linen rag. Fig. 7. Such cover-glasses should be free from both grease and grit, and are then fit to use for making fresh fluid blood films or for other preparations. As alternatives to the treatment with carbonate of soda and sulphuric apid, some prefer strong nitric acid and others bichromate of potash (2 parts), sulphuric acid (3 parts) and water (25 parts) ; others, again, sulphuric acid alone. In any of these solutions the cover-glass can be kept indefinitely and washed in water immediately before use. Cover-glasses should be of the best quality, and for blood work the thinnest (No. i) should be used. A i8 STERILISERS smaller stock of thicker cover-glasses should be kept for the examination of faeces and making " squash " prepara- tions. These thicker cover-glasses do not deteriorate so rapidly. For bacteriological work some form of steam steriliser is necessary to sterilise vessels, media, &c. With this all requisite sterilisation for ordinary work can be done, but a hot air steriliser is an advantage for the quicker and easier sterilisation of vessels, Petri dishes and some instruments. Fig. 8. — Hot Air Steriliser. A steam steriliser (fig. 7) is simply a tall metal vessel covered with a lid with a vent for the escape of steam, and containing water at the bottom. As it is not well to iriimerse the objects to be sterilised in the water, there is a perforated false bottom above the level of the water on which such objects rest. The whole vessel to pre- vent loss of heat is covered with some non-conducting material. There is no great difficulty in improvising such a steriliser, but those sold are more sightly and convenient. STERILISERS 19 They can be heated by a paraffin lamp, and the " Primus " is one of the best. At a pinch a wood or other fire may be used. The hot air steriHser (fig. 8) is a metal case enclosed in a second larger one, the two being separated by an Fig. 9.— Hearson's Incdbator, working with Petroleum Lamp. air space. The double case can be dispensed with, but the heating is then less uniform. A temperature of about 160° C. is required. Incubators are needed where it is importaht that growth 20 INCUBATOR should take place at a uniform temperature, and are essential when it is desired to describe accurately the character of the growth of an organism, or to compare one growth with another, or with the description of another. Where gas cannot be obtained a form of incubator which can be used with kerosine must be employed (fig- 9). At " room temperature " in the Tropics most organisms grow well, and much useful work can be done without an incubator. If there is no incubator a dark cupboard must be used, as light has a deleterious effect on most bacteria. This cupboard shotild be fixed in a dry place, where the tem- perature is as uniform as possible. 21 CHAPTER II. Post-mortem Examinations. Post-mortem examinations in the Tropics present certain differences from these examinations in temperate climates. Post-mortem, changes are more rapid, so that it is essential that the examination should be made as soon as possible after death. This is not only on account of the rapidity with which putrefactive changes occur, but also because many of the animal parasites die, and some, such as the sporozoa, disintegrate and cease to stain well even before putrefactive changes set in. A large proportion of tropical diseases are those affect- ing the abdominal viscera, and to study the exact relation- ship of the parts it is often advisable to remove the thoracic and abdominal viscera en masse. The abdomen should be freely opened and room gained by subcuta- neous division of the muscles attached to the pubes below, and sternum and ribs above. The attachments of the diaphragm to the sternum and costal margins must next be divided with the knife close to the chest wall, and the parietal peritoneum stripped off the abdominal wall with the hand as far as possible. The trachea and vessels going to the neck are then to be freely divided by passing the hand and knife in front of the lungs and cutting transversely above the root of the lungs, whilst with the hand the thoracic viscera are grasped firmly at the root of the lungs and steady traction exercised. When the division is complete, the lungs and heart will be easily pulled downwards through the lower opening of the thorax. With the knife the posterior 22 POST-MORTEM EXAMINATIONS attachments of the diaphragm are divided from above, and steady traction, aided by a few touches with the knife, will strip the peritoneum off the remainder of the wall of the abdomen, and all the abdominal viscera with the aorta and kidneys will be completely separated except at their pelvic attachments. These can be divided, or better, the peritoneum stripped off the pelvis at each side, and the urethra and rectum divided as near the perineum as possible. The mass of organs can be now examined from every aspect and the relations of the different parts readily observed.. By this method the root of the mesentery, the posterior mediastinum, and other parts which are usually over- looked can be displayed, and in these regions para- sites are sometimes found. Certain special observations are worthy of attention : — (i) The weights and relative weights of the organs vary considerably from European standards, both in health and in disease. With the lungs, in recording the weight, it is essential to note also the time that has elapsed between death and the examination, as the weights of these organs increase a few hours after death, probably by aspiration of fluid. If the examination is made two or three hours after death the lungs will be barely half the weight taken as the standard in Europe, whereas, if the examination is made later the weights may be much the same.* (2) Variation in weight of the organs with age differs in different races, and the curves obtained for the organs are in many cases different from those recorded in Europe. For example, the brain weight in Europe attains its maximum between 45 and 50, whilst in the negro the maximum is reached between 20 and 25.* (3) Abnormalities are common, and some of them occur with unusual frequency in certain races, such as Meckel's diverticulum in the Chinese, and deeply PUTREFRACTIVE CHANGES 23 fissured lungs in the negro races. Diseases also aifect organs differently according to race, and of this the age incidence of splenic enlargement in negro and other races living under the same conditions is a striking example. While in childhood all are equally affected by malarial disease, in adult life the spleen in the negro tends to subside, but in other races, Indian, Chinese and aboriginals, it remains increased in size, and is commonly iound post mortem to be three or four times the weight of the European normal.* (4) Abnormal appearances, such as congestion, ecchymosis, &c., are more common in the Tropics, and are observed under different conditions. On the one hand, as the examinations are made much earlier the appearances resemble more closely those in the living subject ; and on the other hand, as putrefactive changes occur so easily, particularly in the vicinity of the intestine, patchy,, irregular post- mortem staining is common, and is frequently mis- taken for disease. (5) Certain special putrefactive changes may be a source of error. As a result of putrefaction some of the organs, and particularly the spleen, often appear of a slaty colour, which may be mistaken for malarial pigmentation. Section of the organ will show that the discoloration of early putrefaction only extends for a short distance into its substance. The substance of the spleen in section sometimes appears very dark, but this colour can be dis- tinguished from that of acute malaria by noticing that the dark colour changes to bright , red after ex- posure to air. The only satisfactory test of malarial pigmentation of an organ is by examination of a portion of the tissue with the microscope. It is not necessary to cut sections, a small portion of the * Vz'de tables in Appendix. 24 EXAMINATION FOR ENTOZOA organ can be pulped between two slides and ex- amined at once for pigment. A diffluent spleen is often described, but is not met with in post-mortem examinations made early. The spleen, even in the most acute cases of malaria, though enlarged and black, is firm, and wedges with acute angles can be cut. These angles retain their sharpness even when exposed to a jet of water. Such a spleen is easily pulped, and if allowed to decompose speedily becomes " diffluent." Organs, shortly after death, are firm and hard from rigor mortis of the tissue elements. When this passes off the organs become flaccid and softer. Many of the early putrefactive organisms form gas, and consequently emphysematous changes are pro- duced; such emphysema of the liver and other organs is common. In the intestines small emphysematous patches form in the submucosa, and present a peculiar and rather deceptive appearance. Gaseous distension of the whole intestine is very com- mon, and the stretched walls appear unusually thin and are often described as atrophied. Examination for Entozoa. — In the examination of intestines in the Tropics it is not advisable to wash out the intestine before opening it. The intestine should be opened and examined for entozoa first and subse- quently washed to see the condition of the mucosa. If the intestines are washed out first, the entozoa will be carried away, and may escape notice. Worms and intestinal parasites die as a rule within some six to twelve hours after the death of the host, and some, such as the ankylostomes, . lose their hold on the intestinal walls even earlier. Museum Preparations. — In the older methods for the fixation and hardening of macroscopical specimens, alcohol, and, later, formalin were employed. These methods preserved the form and relations of organs well, but were practically valueless for preserving the natural colours. MUSEUM PREPARATIONS 25 Modern methods in use result in the preservation of the natural colours of the specimens and harden them at the same time. The basis of the methods is the use of a first bath of formalin, which has the power of changing oxyhsemoglobin into acid haematin, of a second bath of spirit, which converts the acid haematin into alkali haematin, which in colour is very similar to oxyhsemoglobin, so that the tissues regain their natural colours ; finally the specimens are preserved in a solution containing potassium acetate, glycerine and water. Kaiserling's original method, which gives as uniformly satisfactory results as any of its modifications, is as follows : — (i) The organs are fixed in the formol mixture until they are just hardened — thirty-six to forty-eight hours or even longer. The best results are obtained by fixing in the dark. Formalin... ... ... 200 cc. Water ... ... ... 1,000 cc. Nitrate of potassium ... 15 grammes. Acetate of potassium ... 30 grammes. As a substitute 10 per cent, formalin may be used. Weak formalin must not be used, as this dissolves out the haemoglobin. A section of a solid organ such as the spleen or liver can be safely placed in a vessel containing this fluid, but in dealing with softer tissues it is advisable to wrap the specimen loosely in cotton-wool before immersion, and to give it plenty of room. In dealing with an entire organ it is best to inject it with the fluid, as the organ then retains its shape better, and the haemoglobin in its interior is more completely converted, and does not afterwards leak out and colour the mounting medium. (2) After fixation the specimen is placed in the second bath, which consists of spirit. It was origin- ally recommended to employ increasing strengths of spirit, but equally good results are obtained by 26 MICROSCOPIC PREPARATIONS placing the specimen at once into 90 per cent, spirit. The length of time necessary for this bath must be determined by the appearance of the specimen ; it should be removed when the original colour fully returns, which is usually in twenty-four to thirty-six hours. (3) Mount in jars containing : — Glycerine 400 cc. Acetate of potassium ... 200 grammes. Water 2,000 cc. A few crystals of thymol or a trace of formalin may be added to prevent the growth of moulds. Preparation of Tissues for Microscopic Examination. Parts of an organ or tissues kept for microscopical examination may be examined fresh or preserved and hardened. From the examination of the fresh specimen much information may be gained. Smears of the fluids that exude from the cut surface may be made, small portions of the tissue may be squashed between a slide and cover- glass, or the material may be frozen and sections made. In the last case treatment in a strong solution of gum arable is desirable. The details of the methods useful for the determination of parasites is considered with the description of these parasites. It is from the fresh specimens that cultures must be made. For this pur- pose the spleen and lymphatic glands are the most suitable places. Blood should be withdrawn by syringe from the unopened heart for culture-making and for inoculation of animals. Fixation and Hardening. — Although formalin is com- monly recommended as a fixing fluid when ordinary tissue changes are to be demonstrated, it will be found in tropical practice that alcohol is the most generally useful reagent, as protozoal and bacterial organisms stain FIXATION AND HARDENING 27 best after fixation by this method. It has the disadvan- tage of causing great shrinking of thetissues from rapid dehydration. To fix, the tissue should be first placed in 80 per cent, alcohol and in two to four hours transferred to 95 per cent, alcohol. The tissue must be in small cubes not more than half an inch in their greatest length, and placed in at least ten times their volume of alcohol in a closed glass vessel. If a piece of tissue is simply put into a bottle and spirit poured on it, the blood coagulates at the edges where in contact with the glass, and the fluid does not penetrate between the glass and tissue. This is avoided by placing some cotton- wool or small pieces of crumpled paper at the bottom of the bottle. In certain cases, especially where it is desirable that the blood should be retained in the vessels, a larger piece of tissue can be taken, and after partial fixation subdivided into pieces of the right size. This is par- ticularly to be recommended when the object is the examination of the tissue for malaria parasites or filaria in situ. At the end of six hours the alcohol should be changed, and again changed in twelve hours. By this time in a warm climate the specimen will be sufficiently fixed, and longer immersion in absolute alcohol will render the specimen too brittle. In colder weather, average temperature under 70° F., it can be left some hours longer in the alcohol. The specimens when fixed can be kept in methylated spirit till required. If greater accuracy is required the specimens can be kept in 60 per cent, absolute alcohol. This will keep the specimens, and stronger alcohol at tropical temperatures soon overhardens them. FORMOL Alcohol. — For more rapid fixation of tissues in which examination for malaria parasites is not required, alcohol and formalin give excellent results. This solu- tion is made by the addition of formalin in the pro- portion of 2 to 10 per cent, to the absolute alcohol. It 28 FIXATION AND HARDENING penetrates rapidly and causes less shrinking than alcohol alone, but the tissues should not be left in this solution for more than twelve hours or they will be overhardened. They are then fit for further processes or can be kept in spirit. Muller's Fluid. — Pot. bichromate 2*5 parts, sodium sulphate i part, and water 100 parts, is very extensively used and gives good results, but is slow in its action. The fragments of the tissue are placed in abundance of the fluid, which should be changed in a few hours, and again daily for a week, after that once a week will be sufficient. Some tissues will be sufficiently fixed in two or three weeks, but others, as the parts of the central nervous system, may, even in a warm tropical climate, require many weeks. When fixation is complete the specimens should be washed for twenty-four hours in abundance of water, which is frequently changed, pre- ferably in running water, and then kept in methylated spirit. Animal parasites do not stain well in tissues which have been fixed in bichromate solutions. Orth's Fluid. — Muller-formol is made by adding 10 per cent, of formalin to Muller's fluid. This must be added immediately before use. It is a rapid fixative, and at blood heat only three or four hours are required for thin pieces of tissue. Two days are usually sufficient at room temperature. When hardened the tissues may be cut directly on the freezing microtome or passed through increasing strengths of alcohol and then imbedded. Zenker's Fluid. — For the examination of skin, which is readily overhardened, Zenker's fluid gives good results. This is composed of 5 parts of corrosive sublimate, 2'5 parts of potassium bichromate, i part of sodium sulphate, 5 parts glacial acetic acid, and 100 parts of water. The slices of tissue to be examined must be very thin, not more than a tenth of an inch in thickness. The time required for fixation is twelve to twenty-four hours, IMBEDDING 29 according to the thickness of the specimen and the temperature. After the tissues are fixed they must be thoroughly washed in water, which is frequently changed, for at least twelve hours, and should then be placed in spirit to which a little tincture of iodine has been added, to remove any mercury deposited in the tissues. If the colour of iodine disappears from the fluid more iodine is to be added until the colour no longer disappears. Or the specimens can be kept in spirit and the cleaning with iodine done after the sections are cut. The specimen can then be kept in spirit till required for use. OsMic Acid Mixtures. — Two other useful fixatives are Flemming's solution : — Chromic acid i per cent., aqueous solution 15 cc. Osmic sohttion 2 per cent., aqueous solution 4 cc. Glacial acetic acid i cc. and Hermann's solution, in which i per cent, solution of platinum chloride is substituted for the i per cent, solu- tion of chromic acid in Flemming's solution. These solutions must be freshly made up before use, and as the penetrating power of the fixative is low, the specimens must be very thin, not more than one-twelfth of an inch in thickness. Fixation takes from one to two days, and the speci- mens require to be thoroughly washed, preferably in running water, for one day, and then placed in 80 per cent, alcohol. Boiling Method. — For the rapid fixation of tissues they may be placed in water that has been heated just to the boiling point. If small pieces of tissue are used a few minutes will suffice to coagulate the albumins. This method is also useful for the examination of renal casts and for the contents of cysts. Imbedding. — The tissues having now been fixed, hardened and completely dehydrated, are assumed to be in absolute alcohol. Sections of hardened tissues can be cut with a razor by hand, or with a microtome 30 IMBEDDING knife after fastening the specimen in the microtome clamp. Fair sections of firm tissues can sometimes be obtained in this way or by means of the freezing micro- tome. With these methods, however, portions of the tissue are Hkely to fall out of the sections, and it is desir- able to have the tissues imbedded in some material with which they become permeated and which preserves the component parts in their relative positions and surrounds them with a protective coating. They can then be cut into thin sections on a microtome. The two substances in common use for imbedding purposes are paraffin and celloidin. Paraffin imbedding is the most useful if very thin sections are desired. For certain purposes celloidin is indispensable, as when it is desired to keep any loose bodies in situ in a tissue, there being no necessity to remove the celloidin before mounting in Canada balsam. Hard tissues, and tissues which easily become brittle, such as muscle and skin, are cut with considerable diffi- culty by the paraffin method. (i) Paraffin Imbedding. — The general principle is to pass the specimen through alcohol till it is thoroughly dehydrated, then to place it in a fluid in which paraffin is soluble, which will dissolve out the alcohol, and then to replace this fluid by first a weak solution of paraffin, then a strong solution of paraffin, and finally melted paraffin wax. Excess of paraffin is poured round the tissue and it is allowed to cool ; when the paraffin solidifies not only is the piece of tissue enclosed in a solid block of paraffin wax but the tissues will be permeated with the wax. There are many modifications, some of which are rendered necessary for special tissues. For general work with specimens taken from strong spirit : — (i) Place the specimen in absolute alcohol for twenty-four hours. If the specimen has been re- moved from weaker spirit or from water, before IMBEDDING 31 placing in the absolute alcohol it should be placed in methylated spirit for forty-eight hours. (2) Remove from spirit, drain off excess of spirit for a few minutes and place in aniline oil. One day. (3) Place in xylol. One day. (4) Place in paraffin and xylol, pqual parts. One day. Fig. 10. Fig. II. Fig. 12. (5) Place in melted paraffin wax for one day. The paraffin wax can be kept melted in a drying oven (fig. 10) at the required temperature, or a paraffin embedding bath can be used for this purpose (fig 11). As a considerable amount of spirit is required for 32 RAPID PARAFFIN METHOD the spirit lamp to maintain the required temperature, it is well to imbed as many specimens as possible at the same time. The imbedded specimens keep well. (6) Imbed and cool quickly. The imbedding may be done by filling small paper boxes with melted paraffin and placing the pieces of tissue in this melted paraffin. The box is then placed in a dish of cold water on which it floats and is rapidly cooled, so that the paraffin sets without crystallising. Or L-shaped pieces of metal are placed in contact on a smooth slab, as in the diagram (fig. 12), and the space between filled with the melted paraffin and the specimens placed in as before. Modifications. — The paraffin used in England melts at too low a temperature for satisfactory work in the Tropics. It is well therefore to keep two varieties of paraffin, one melting at 48° C. and the other at 60° C, and to use 3. mixture of them. Such a mixture with a melting point about 54° C. is usually sufficient, but in the warmest weather either a larger • admixture of the paraffin at the higher melting point will be required, or the pure paraffin melting at 60° C. Rapid Paraffin Imbedding Method. — For the rapid examination of small objects the process of imbedding may be shortened by the use of acetone, which hardens the tissues and at the same time prepares it for immersion in paraffin. (i) Fix small pieces of tissue in 10 per cent, for- malin for one-half to four hours. (2) Place in pure acetone for one-half to one and a half hours. (3) Transfer directly to fluid paraffin (52° to 56° C), and place in the oven for one-half to one and a half hours. The acetone evaporates and the paraffin permeates the tissues. (4) Prepare the paraffin block. Cut and stain as usual. The acetone may be used again by placing fired copper sulphate at the bottom of the vessel. CELLOIDIN IMBEDDING 33 (2) CELLOIDIN Imbedding. — Commercial celloidin (Schering) is a purified gun-cotton. It is sold in granules, in shavings, or in flat slabs. The latter require to be cut into small squares before using. To imbed in celloidin the general principle is the same as that for paraffin, but the agents employed and the methods differ. (i) The specimen is kept in absolute alcohol, after being in weaker spirit, for twenty-four hours. (2) It is then soaked in a mixture of equal parts of ether and absolute alcohol for twenty-four hours. (3) Place in a weak solution of celloidin (3 per cent.) in alcohol and ether for twenty-four hours or more ; two days is usually ample. (4) It is then to be transferred to a thicker celloidin solution, 6 per cent, celloidin dissolved in alcohol and ether, and kept in this for at least one day, and better for several days. (5) The specimen is then placed on a small block of wood, on which a few drops of the thick celloidin have been placed. Leave exposed to the air for a few minutes and pour a little thick celloidin solution over the specimen. Expose to air for a few minutes and place in 60 per cent, alcohol, which will harden the celloidin. In cutting celloidin specimens the knife must be oblique and must be moistened with spirit. Section - Cutting. — For this purpose a microtome is required, and the greater number of those available may be used for either celloidin or paraffin. For cutting frozen sections a special instrument is necessary. Cathcart's microtome (fig. 13) is the simplest efficient freezing microtome, but in the Tropics its use is neces- sarily limited, as freezing with ether is not practicable in most tropical countries. Imbedded sections can be cut with this microtome, but must not be frozen. The carrier is heated and the paraffin block pressed against it. The paraffin will be 34 SECTION-CUTTING melted and will then adhere to the zinc plate ; but better sections can be obtained with other microtomes. For freezing in the Tropics a mixture such as ice and salt is the best. Swift's microtome consists of a circular wooden box (figs. 14, 15) (a) from the centre of which rises a metal tube surmounted by a horizontal zinc plate raised above Fig. 13.— Microtome, Cathcart's, with Spray Bellows. the level of the top of the box. The box is covered with a glass plate perforated in the centre with a hole large enough to allow the tube and zinc plate to pass through. When arranged for use the box is filled with a mixture of well-crushed ice and salt. The lid is placed on and the zinc plate projects above its level. The FREEZING MICROTOME 35 substance to be frozen is placed on the zinc plate and well-covered with a strong solution of gum. If the air temperature is not too high the specimen freezes with the surrounding gum. In many tropical countries this does not suffice unless the specimen is also surrounded by a cold atmosphere. This is produced by placing a second metal box (fig. 15) Fig. 14. Fig. 15. (B) on the top of the glass plate. This metal box has a central tube rising from the bottom and open below, and this tube must be wide enough to enclose the zinc plate and specimen. If the metal box is also filled with the freezing mixture the air in the central tube will be cold and the specimen surrounded by this cold air freezes readily. When frozen the upper metal box can be removed and sections cut. 36 FREEZING MICROTOME In this instrument the specimen remains fixed, and the thickness of the section is regulated by alterations in the level of the razor. This is arranged by having the razor blade fixed on a tripod ; the length of the legs of this tripod can be regulated by turning the milled heads of the screws. The feet of the tripod are tipped with bone so as to slide evenly over the glass. For use the blade of the razor must be wetted with water, and the tripod carrying it is so arranged that one leg is anterior. The two posterior screws are turned till the edge of the razor is horizontal or parallel with the sur- face of the glass. Any alteration in the screw of the anterior leg will then raise or lower the edge of the blade. The sections are cut by gliding the tripod over the plate till the edge of the razor touches the specimen and cuts through it. A slightly oblique motion is the best, and the tripod must be pressed firmly on the glass plate and the movements must be rapid, more like a thrust. The knack of making the correct movement is soon acquired. When a section is cut the tripod is drawn back, slightly tilting it to avoid touching the specimen, the anterior screw turned to an amount regulated by the thickness of the desired section and again thrust forward. This process should be repeated until there are several sections on the upper surface of the blade of the razor, and these can be removed with a camels' hair brush to a vessel con- taining water which has been recently boiled, so as to be free from air. The sections will float and can be floated on to a slide and either examined directly or stained. Sections of fresh, unfixed tissues can be cut and examined unstained, or, to show structure better, they can be stained. They should be soaked in gum before freezing. Well-fixed specimens can also be frozen and cut. It is necessary before freezing to thoroughly remove the last traces of alcohol by washing in water, and to soak in an aqueous solution of gum arable for some hours. PARAFFIN SECTIONS 37 Where ice cannot be obtained further hardening and imbedding is necessary. Paraffin Sections.— Of the simpler and cheaper forms of microtome suitable for cutting paraffin sections, the Cambridge Rocker is the most convenient. Full directions for the use of this instrument are sent with the microtome, but the chief points to observe are : — Fig. 16. — Cambridge Rocking Microtome, new pattern for cutting flat sections, with large articulating apparatus and one razor. (i) That the razor must be rigidly clamped. (2) That the paraffin block must be firmly fixed on the metal carrier. This is done by heating the carrier and applying the paraffin block firmly to it, and keeping it in position till the carrier is cold. (3) That the thickness of section must be graduated in accordance with the nature of the tissue, its brittle- ness, and object of the section. If the specimen is too hard or brittle it is useless to expect thin sections. Sections showing large parasites, such as filaria, in tissues like the lung, should not be too thin. For the details of nerve structure, and for sections showing bacilli, or the 38 CELLOIDIN SECTIONS parasites of malaria, the thinnest possible sections are the best. Celloidin Sections. — For cutting celloidin sections a slide microtome, such as that of Jung, is best, but quite good sections may be obtained with the Cambridge Rocker. A form of this instrument should be selected in which the razor can be placed obliquely. These sections are generally cut in alcohol. The sur- face of the celloidin block should be moistened with alcohol by means of a camel's hair brush. Place the knife so that it forms a slight angle with the block. Transfer the cut section to 70 per cent, alcohol with a brush or with the finger. From paraffin blocks sections 5 to 7 /^ in thickness may be obtained ; from celloidin blocks sections 10 to 15 jj, are generally cut. Fixation of Paraffin Sections on Slide. — For ordinary work the sections, when cut, are placed on the surface of some warm water about 44° C, or rather more if if paraf&n of a higher melting point is used, and floated on to a slide. The water is allowed to drain off, and the slide is then placed in the hot incubator for twelve to fifteen hours. The section will then be fixed to the slide. Occasionally it will be found that the sections after removal of the paraffin fall off. In such a case the other sections may be very gently warmed over a flame till the paraffin begins to appear more translucent. Another method is to employ a mixture of albumin and glycerine to fix the section. This mixture is pre- pared by beating up the white of an egg, filtering, and adding an equal quantity of glycerine. A crystal of carbolic acid or thymol may be added to prevent the growth Of moulds. A thin film of the mixture is placed on a slide, and on this the dry paraffin section is arranged and flattened gently. As before the slides are placed in the hot incubator. Paraffin sections can be rapidly fixed by floating them out on a drop of water placed on the slide. The slide is FIXATION AND TREATMENT OF SECTIONS 39 warmed till the section flattens out, and the water is then drained off and the section firmly pressed to the slide with a piece of blotting paper. This should leave the dried section closely applied to the dried slide. The slide is now again heated until the paraffin begins to appear translucent, and again firmly pressed to the slide. This method should not be used for delicate tissues such as the brain, as the firm pressure required is harmful. Treatment of the Sections. — To remove paraffin from the sections so that aqueous and other stains can be used, the slide carrying the section should be placed in xylol and agitated in it for two or three minutes. This dissolves the paraffin. To remove the xylol place in strong spirit or absolute alcohol and again agitate, so that fresh surfaces of spirit are brought in contact with the section. As a precaution it is well to rinse in fresh spirit. The slide can then be placed in water to remove the spirit, and stained as is considered advisable. After staining, dehydrate in alcohol, clear in oil of cloves, wash with xylol if aniline stains are used, and mount in xylol Canada balsam. If the alcohol used is strong enough the oil of cloves need not be used. 40 CHAPTER III. Blood. Examination of the blood is of, such importance in tropical work that in all cases of difficulty and doubt recourse to this method of diagnosis is essential. A thorough knowledge of normal blood is a necessary pre- liminary. The abnormal forms of cells met with in various diseases must be readily recognised. Last, but not least, the various methods used for the finding and recognition of parasites must be known. Many methods of examining blood have been employed and most of them are good. Fallacies and mistakes have occurred with all, and the sources and causes of these errors and the recognition of them have to be studied. These are dealt with under each method described. Blood is composed of a nearly colourless fluid, the plasma, in which are floating cellular elements, the red and white blood corpuscles and blood-plates. The more solid elements, the blood corpuscles, will be considered first. They vary in number, in their relative proportions and in their characters. Other cells, not normally present in the blood, are found under certain conditions in that fluid. Most of these cells are normally present in the tissues of the healthy body, though not normally in the blood. Parasites occur in the red corpuscles and in the plasma, and are sometimes found, in a more or less disorganised condition, in white corpuscles or phagocytes, which have devoured them. None have been observed in the blood- plates. BLOOD FILMS 41 The two main methods employed for the examination of blood are : — (i) In the fresh and fluid condition. (2) As films which are allowed to dry, and fixed and stained in various ways. These two methods are of general application. For special purposes, so as to reveal abnormal bodies scantily present in the blood, thick films can be made and the hemoglobin removed. In this way a quantity of blood that would not be sufficiently transparent if treated by the ordinary methods can be rapidly examined. Examination of Fresh Blood. — This method is the only one by which vital changes can be observed. Of the normal blood elements, the amoeboid and phagocytic properties of the leucocytes can thus be observed. Living organisms abnormally present, such as filarise, tryanosomes, and the parasites of malaria, can be watched, and such developmental and degenerative changes as occur in shed blood observed. No description or observation of new parasites is com- plete without an examination of these parasites in the living conditions. It is noteworthy that most of the important mistakes made even by experienced observers in the description of bodies met with in blood have been due to neglect of the examination of fresh fluid blood. The essential point in the preparation of fresh fluid blood films is that a great part of the film should be so thin that the blood corpuscles are lying flat and separate from each other. (i) The simplest method of making such a film is to take a small drop of blood on the centre of a cover-glass and drop it on the slide (fig. 17). In a well-made film by this method three zones are apparent. The edge of the film is thick and irregular. Here the corpuscles are in rolls or masses and it is too thick for the examination of the individual red corpuscles (fig. 17, a). Internally to this is an area with a slightly opaque or ground-glass appearance. Here the red corpuscles will 42 FRESH FILMS be found lying flat and not to any great extent over- lapping each other. This is the part of the film best suited for examination of the red corpuscles and the parasites contained in them (fig. 17, c). The centre of the film is clear and transparent, and here few corpuscles are found, as this part is composed almost entirely of the plasma (fig. 17, b). 00x6 o b Fig. 17. To get good films by this method there are certain points to be observed : — (i) The slide and cover-glass must be free from grease, otherwise the blood will not spread out {vide cleaning slides and cover-glasses). (2) They must be freed from grit ; this is best done by rubbing well, immediately before use, with a soft linen rag. (3) The drop of blood must be so small that, when spread out, it does not extend to the edges of the cover-glass. If the blood is too abundant it floats up the cover-glass, and sufficient space is left between the slide and cover-glass to allow of the formation of rouleaux. (2) Another method is that of Braddon. Here the FRESH FILMS 43 cover-glass, freed from grease and grit, is placed on a slide similarly cleaned. The cover-glass is so placed that its edge corresponds with one edge of the slide. Pressure is exercised on the centre of the cover-glass, or it can be fixed with Cornet forceps. Vasehne is then ZIZ Fig. i8. applied to the slide at the edges of the cover-glass, leaving the side applied to the edge of the slide and a small space at the edge opposite to this free (fig. i8). These slides can be prepared in the house or laboratory and are then ready for use. If the edge of the slide be applied to a drop of blood, the blood will run up by 44 EXAMINATION OF FILMS capillary attraction and spread itself in the space between the cover-glass and slide in a film thin enough for examination. (3) A third method is to make the film between two cover-glasses, the lower one being much the longer. The blood spreads more readily between two cover-glasses than between a slide and cover-glass, and the free edge of the lower glass can be clamped on to the slide (Herder's method). Beautiful films are obtained, and cover-glasses can be carried in larger numbers than slides on account of the smaller weight, but the greater fragility of cover-glasses is a serious objection to the general adop- tion of this method. All of the above methods give good results. The first has decided advantages in that the blood elements are all present and, to some extent, distributed evenly through- out the best part of the film. The second is very con- venient for class work, as there is no delay at the bedside, and a large number of preparations can be made quickly. It is useful with nervous patients, as no preparation is necessary at the bedside. The more adhesive elements of the blood, the blood-plates and leucocytes, are crowded together near the edge where the blood has entered. In the thinner part of the field, which is farther from the edge, these elements have been " filtered out " and few solid elements but the red corpuscles are left. In the freshly drawn blood the elements normally present are : — (i) Red corpuscles or erythrocytes. (2) White corpuscles or leucocytes. (3) Blood-plates or platelets, Hayem's haemato- blasts. (4) Plasma. In the red corpuscles the points to note are the colour, the size and the shape, and variations in these. In many of the corpuscles, particularly if pressure has been applied to the cover-glass, clear, transparent spaces. CRENATION 45 vacuoles, which may be either circular, oval, or even slit-shaped, will be found (fig. 19 c). These must be recognised for what they are, and not confounded with unpigmented parasites nor with the natural deficiency in colour seen towards the centre of the corpuscles. From both of these the vacuoles can be distinguished by the sharpness of their outline. An oscillatory or vibratory motion of the hzemoglobin edge of the vacuole is highly characteristic, but must not be mistaken for amoeboid movement. Fig. 19.— a, b, Crenated corpuscles ; c, vacuolated corpuscle ; d, e, buckled corpuscles. Crenation. — If the blood corpuscles be watched for some time they will be seen to become distorted and projections are thrown out, either as a few blunt pro- cesses or as sharper projections (fig. 19 a, b)., This change is known as crenation, and the projection may be feebly motile and portions may break off and be discharged into the plasma. These crenations are readily recognised when they occur at the edge of the corpuscles. When they occur on the flat surface they produce an irregularity in the colouring and, as they are not flat, cause refraction, and so produce an appearance of dark spots surrounded 46 LEUCOCYTES by a lighter ring, or a light spot surrounded by a dark ring, according to the focussing. Some of the red corpuscles, particularly if pressure has been used in making the film, are bent on themselves or "buckled" (fig. 19, d, e). Such corpuscles may assume very varied shapes, and, as the haemoglobin is readily expressed from any part of the corpuscle, compression irregularities in colour are usual. All these appearances can be easily distinguished from parasites by careful focussing. Occasionally, however, one meets with red blood' cells within which are bodies closely simulating ring forms. These may be motionless, or have a slight oscillatory movement at the edge, but true amoeboid movement is not seen. In shape they may be rounded or oval, and not uncommonly appear to possess a central dark dot, which is probably due to refraction. , Similar bodies described by Cropper and since noted by many observers in working with fresh blood have a marked rotary movement. They are found in other animals besides man, and in man and animals in non-tropical countries. They have never been stained in dried preparations and their precise nature has not been determined, but they are most frequently found in cases where there is evidence of degenerative changes in the blood. Leucocytes are distinguished by their size and the absence of colour. As seen in the fresh blood they are usually granular, the granules being best seen on closing the iris diaphragm of the microscope. Variation in the granules will be noted, and the coarse, highly refracting granules of the eosinophile leucocytes are quite charac- teristic. These granules are often mistaken for pigment by beginners. Letting in more light, which brings out pigment granules more strongly and shows these normal granules to be translucent, will remove this difficulty. The characters of the nuclei and of the granules are best studied in stained specimens. The amoeboid movements and phagocytic properties are best seen in these fresh living fluid films. STAINING OF FRESH FILMS 47 The Blood Platelets are the most difficult objects to see, as they are colourless, non-granular and differ little in refractive index from the plasma. The size and arrangement in groups, points that vary in different specimens of blood, should be noted. The irregular serrated margins they acquire in a short time, from the formation of filaments of fibrin, are character- istic of these bodies. These elements are more readily seen in stained or over-stained specimens. Staining of Fresh Films. — Many methods of staining blood, whilst still in a fluid condition, by admixture with stains have been employed. The usual practice is to place a drop of sufficiently dilute stain on the slide, then take a minute drop of blood on the cover-glass and drop this on the drop of stain, so that the blood and stain spread out together. A certain admixture takes place at the edge of the drop of blood, and in a little time the stain diffuses further into the blood. Various solutions of stain have been used. Braddon's is perhaps as good as any.* In this as well as in other aqueous stains, the water causes a liberation of the haemoglobin, and the dissolved haemoglobin precipitates the stain, or debris is stained by the stain. If this process takes place in the serum little confusion is caused, but if, as frequently happens, it takes place as the stain penetrates the red corpuscles it causes the formation of a complicated arrangement of stain in the interior of the red corpuscles, which has been mistaken for parasitic growth. To avoid this error, a strong salt solution is used by some, so that the red corpuscles are not destroyed. Others, for the same reason, use ascitic fluid. Malaria parasites are well stained by this method, and * Braddon's solution is composed of i per cent. pot. citrate, ^ to 3 per cent, methylene blue ; water to 100 parts. 48 DRIED FILMS it has the advantage of requiring no fixation, and conse- quently is rapid. Dried Films. — These can be made in many ways, most of which after a little practice give excellent results. In all methods it is important that only the top of the drop of blood and not the skin should be touched by the slide, cover-glass or paper. Neglect of this precau- tion will result in the admixture of epithelial scales, oil globules and micrococci with the film. (i) In this method a drop of blood is taken on the surface of a slide near one end. The edge of another slide is brought into contact with this drop, which then spreads out so as to fill the angle between the two slides along the whole extent of the line of contact. On pushing the upper shde towards the other end of the lower slide a film of blood will be left behind. The thickness of this film is easily regulated, as if the angle between the two slides is acute the film left behind will be very thin ; if the angle be nearly a right angle a thick film will be left. An angle of about 45° gives the desired thickness, but it is well to slightly vary the thickness of the film by alternately slightly increasing and diminishing the angle made between the two slides as the upper one is pushed along, so that different parts of the film will be suitable for examination for different purposes. A slight modification of the method is to take up the drop of blood on the edge of the upper slide and bring the drop of blood and the edge of this slide into contact with the upper surface of the. lower slide and proceed as above (fig. 20). (2) A drop of blood is taken on a slide rather nearer one end than the other, and the larger the drop the farther from the middle. Another slide, a glass rod, or, perhaps best, the shaft of a needle is then applied to this drop so that the blood spreads along the whole of the line of contact. The upper slide, glass rod, or needle is then drawn across the lower slide and an excellent film will be left (fig. 21). Fig. 20. 49 Fig. 21. 5° DRY FILMS (3) Cigarette paper, or gutta-percha cut in the form of a narrow slip, is used in this method. The lower surface of the slip is brought into contact with the drop of blood on the finger or ear. This drop adheres to the slip on removal. The edge of the slip is placed on the slide and the blood then spreads out between the tissue paper or gutta-percha tissue, and on pulling the free end of the slip a good but usually scratchy film will be left (fig. 22). Fig. 22. Fig. 23- These three methods can be used for cover-glass pre- parations, particularly if long cover-glasses be used, but are best for slides. (4) The last method advocated is not satisfactory with slides but is useful for cover-glass preparations. A small drop of blood is taken on one cover-glass and this cover-glass is then applied to a second so that the FIXATION 51 blood spreads out between them. The cover-glasses are arranged diagonally, so that the corners of each cover- glass can be taken hold of. The upper cover-glass is then drawn or slid over the lower, care being taken that it is not lifted off. A good film should be left on each -cover-glass (fig. 23). In case of emergency any piece of flat glass, broken window-pane, &c., can be used and good films obtained, but the best films are those in which the slides are of good quality, even in thickness and free from scratches, dirt, grease, or irregularities. The films, however made, should be dried rapidly by waving them to and fro in the air, but not heated ; other- wise crenation and distortion of the corpuscles will take place. Fixation. — If such films were placed in water or aqueous solutions of stains the haemoglobin would be dissolved out, and the corpuscles more or less destroyed ; it is therefore necessary to fix the films. Films can be fixed by heat, but a temperature above a certain point vacuolates and distorts the red corpuscles. As a general rule in blood work, fixation by heat should .be avoided, though for one method of staining — the .Ehrlich-Biondi — fixation by heat is necessary. Good results are more difficult to obtain in the Tropics by this method than in England, and, as the same information can be obtained by easier methods, it is not recommended for tropical work. Fixation by absolute alcohol, or by absolute alcohol ..and ether in equal proportions, gives good and reliable results. Fix for ten minutes or more and then dry in air. There are other methods of fixation, and of these ex- posure of the film to the vapour of 40 per cent, formalde- hyde (formalin) for two minutes is perhaps the best. Saturated solution of perchloride of mercury does not give good results with films of malarial blood, as the parasites do not stain well after the use of this reagent, .but for other blood work the results are fairly satisfactory. 52 STAINING OF FILMS Staining of Dried Films. — When fixed the film can be stained, and the number of stains that have been employed is very large. Of the methods most generally applicable, the following have the special advantages and drawbacks indicated. Often two or more methods can be employed with advantage on different slides, in order to bring out special features in the blood. HEMATOXYLIN. — Any good haematoxylin stain will stain most of the basic elements in the blood and most of the parasites. The number of preparations used is large. The formula recommended is composed of a mix- ture of — Hsematin 2.5 grammes. Absolute alcohol 50 cc. Alum 50 grammes or to saturation. Water ... ... ... ... 1,000 cc. The hasmatin is dissolved in the alcohol and added to the solution of alum in water. In warm weather this stain matures rapidly, two or three weeks being sufficient. When mature the sides of the vessel containing the mixture are deeply stained. Like all other hsemtoxylin stains, it must be tested before use to find the time required for staining. When properly mature this preparation requires about seven to ten minutes to stain blood well. It need not be filtered im- mediately before use. The stain may be placed over the film, or the slide with the film on it may be immersed in a pot of the stain, which should be well shaken before use. If the stain is placed on the slide do not pour off the stain, but flush it off. If well flushed, even when a dirty stain is used, little deposit will be left on the film. If the stain be poured off, however much the slide is then flushed or washed, dirt from the stain will adhere to the film. After flushing off the stain leave in ordinary tap water for five minutes. Drain and allow to dry. As a counter-stain eosin is useful. An aqueous i per RED CORPUSCLES 53 cent, solution of yellow eosin (soluble in alcohol) is used. It will stain in twenty to thirty seconds ; then wash and allow to dry. A film so prepared is in a fit condition for examination with an oil immersion lens. The oil can be placed directly on the films, but if it is intended to keep the film, it is simpler to mount in xylol balsam and then examine. Plate I. shows the appearances of the blood- cells stained in this manner. Red Corpuscles. — The red corpuscles are stained by the eosin, the depth of the colour varying according to the richness of the corpuscles in hasmoglobin. As in fresh blood, the size, depth of colour and shape of the red corpuscle should be observed. Among the rarer forms in normal blood are red corpuscles which hardly stain with eosin, the shadow or ghost corpuscles, poly- chromatic corpuscles, which are faintly stained with both stains, so as to have a purplish colour (Plate I., lo), and red corpuscles containing granules which stain deeply with the basic stain used, haematoxylin. The last-named cells are described as containing basophilic granules (Plate I., 9). Nucleated red corpuscles are very rarely present in the blood of healthy individuals, but are found not only in blood of patients markedly anaemic but also in some cases of malaria, &c. The nucleated red cor- puscles have a nucleus staining deeply, but not evenly, with haematoxylin (Plate I., 3 and 4). They have a sharply defined margin. Not unfrequently the nucleated red corpuscle itself is polychromatic, or contains baso- philic granules. Such nucleated red corpuscles may be larger (megaloblasts), smaller (microblasts), or the same size (normoblasts), as the normal. The Blood Platelets are stained feebly with both stains and have a uniform faint purple colour. In an overstained specimen the network of fibrin filaments, starting from either a single plate or a group, is plainly brought out, but in a normally stained specimen only the platelets and the bases of these filaments are revealed (Plate I., 5). 54 WHITE CORPUSCLES White Corpuscles. — The leucocytes have their nuclei stained deep blue. The protoplasm is stained differently in the different varieties of leucocytes, but granules, with the exception of those staining deeply with eosin, are not brought out by this method. They are, however, visible in the unstained leucocytes and can be demon- strated by other stains. Fig. 24. — a. Lymphocytes; b, large mononuclear leucocytes ; c, transitional leucocyte ; d, polymorphonuclear leucocytes ; e, eosinophile leucocytes. In normal blood four varieties of white corpuscles can be differentiated. Of these two have a single, more or less rounded, nucleus. These mononuclear leucocytes are of two classes, though it is not always easy to say WHITE CORPUSCLES 55 to which class a given mononuclear leucocyte belongs. Still, with practice the number of doubtful instances greatly diminishes. The points to be considered in the differentiation are the size of the corpuscle, the shape and staining reactions of the nucleus, the stain taken up by the protoplasm, and the relative amount of protoplasm as compared with the nucleus. (i) The small mononuclear leucocyte, or lymphocyte, is usually not much larger than a red corpuscle and varies from 7 /i to 12 /*. The nucleus stains deeply and forms the greater part of the corpuscle. The protoplasm is often reduced to a mere rim, and in any case is relatively scanty in proportion to the size of the nucleus (fig. 24, a). The protoplasm is stained faintly pink, much the same as the protoplasm in the polymorphonuclear leucocyte (Plate I., II). (2) The Large Mononuclear Leucocytes (fig. 24, b), sometimes called the hyaline cells, are variable in size, but some of them form the largest white elements in normal blood. The nucleus is not so deeply stained as in the lymphocyte. The protoplasm is relatively abundant and stains slightly with basic stains. It may be unstained or faint blue, or, if pink, is less so than the polymorphonuclear leucocyte. All these points have to be taken into account in the separation of these leucocytes (Plate I., 12). Some corpuscles are found with the nuclei deeply in- dented, or horse-shoe shaped. In staining reactions they resemble the large mononuclear and are probably ad- vanced forms of these, and not, as usually described, transitional forms between these and the polymorpho- nuclear leucocytes (fig. 24, c). The other two classes of leucocytes are much easier to distinguish. (3) The Polymorphonuclear Leucocytes (fig. 24, ' 2', of the male. These changes only take place when alterations in the blood occur, such as abstraction or addition of water. The'crescents lose their peculiar shape and become first 90 GAMETOCYTES oval and then spherical. Small portions, one or two, are extruded, the polar bodies, and remain usually adherent to the outer surface of the altered crescent. Of these altered crescents, a proportion which varies in different specimens throw out long, filamentous flagella, varying in number from two or three to six. These flagella are actively motile and lash about in the blood plasma, or over neighbouring red corpuscles for some time. Finally they break away, and can be seen moving rapidly through the plasma. The crescents which undergo this change and flagel- late are the males, and the flagella are equivalent to spermatozoa, and are known as MlCROGAMEXfes. The residue of the crescent is a small protoplasmic mass con- taining all the pigment. It soon dies, and is either broken up or devoured by a leucocyte. The other altered crescents do not flagellate. After the extrusion of the polar bodies they retain their spheri- cal shape, but the pigment in the interior is often in a state of violent agitation. They form the Macrogametes. Very rarely a flagellum that has broken off the male crescent is seen to enter this rounded body, and is ab- sorbed by it. These mature crescents which do not flagellate are the females, and the entrance of the flagellum is a process of fertilisation. After fertilisation further changes take place, the pigment becomes violently agitated, and the whole body changes shape, one end becomes conical, and the body becomes actively motile, moving steadily through the blood serum. This fertilised female is known as the "travelling vermicule" or ookinet, and passes into the outer wall of the mosquito's stomach, where it becomes encysted and forms the zygote. The male and female crescents can often be distin- guished in the freshly shed blood by the arrangement of the pigment. In the female there is usually a clear space in the middle surrounded by pigment, whilst in the male no such clear space is present and the pigment is less in a ring and more in a clump, than in the female. GAMETOCYTES ni The young forms of crescents are sometimes found in the brain, spleen and elsewhere; they can be distin- guished by their shape and the tendency of the pigment to be arranged in a central, irregular clump, and not in one mass. The same series of changes occur in the gametes of benign tertian and quartan malaria after the blood is shed. The gametocytes stain rather feebly with basic stains. The outlines of the red corpuscles which contain them can usually be made out, though, as all the haemoglobin is absorbed, the remnant does not stain, or only faintly, with eosin. The gametocytes contain chromatin in considerable quantity, but this chromatin, though it. stains with the red of the polychrome methylene blue, does not stain with haematoxylin or most basic stains. Stained by Leishman's method, the gametocytes of benign tertain and quartan are easily recognised, as the chromatin granules are collected in a clump surrounded by an unstained area free from pigment (Plate IV., 6). In the crescents the chromatin in the female is collected into a solid block in the centre, and round this the pig- ment is arranged. In the male there is no central block of chromatin, but numerous scattered particles mixed up with the pigment. In the males the chromatin may be very abundant (Plate IV., i8, 19). To make permanent preparations showing the changes that occur in shed blood, it is necessary to prevent the blood from drying and to examine at intervals of a few minutes. For this purpose the slides with the fluid film on them must be kept in a moist chamber. This is easily done by cutting windows in a folded piece of blotting paper and placing this blotting paper on a slab. The blotting paper should be moistened, and the windows each covered with a slide on which is the wet film face downwards. The evaporation from the damp blotting paper will render the air so moist that evaporation from the film will be very slow. 92 GAMETOCYTES One of these slides can be taken off and allowed to dry every five minutes, and in this way we have a series of blood films five, ten, fifteen, twenty minutes, or more, after the blood has been shed. To obtain stained specimens of flagellating bodies thick films can be used, and when allowed to dry the haemo- globin may be removed by placing the slide in water. After this the film is again allowed to dry, fixed in alcohol, and stained with a strong basic stain, such as carbol fuchsin. To observe the changes that occur in the arrange- ment of the chromatin these decolourised films cannot be used, but the specimen with a moderately thick film must, after drying, be stained by Leishman's method. In both male and female a portion of the chromatin will be seen to be extruded in the polar body. The remainder increases in amount, and most of it in the male will be seen in the form of nodules at the periphery of the altered crescent. Finger-like processes of the protoplasm will be seen to project from the vicinity of these masses, at first without any chromatin, and these processes elongate and form long, slender flagella without chromatin. Ultimately, however, the chromatin enters the flagella as a long, thin filament, leaving a mere residuum in the remnant of the crescent at the base of the flagellum. When the flagellum breaks loose it has this chromatin filament running nearly its whole length. Even when all the flagella have broken away there are still remnants of the chromatin in the protoplasmic residual mass left behind. In the female the chromatin forms a less compact mass after the extrusion of the polar bodies, and it is with this mass that the chromatin of the flagellum which fertilises it probably fuses. Minor differences in the crescents as regards shape, staining, reaction, and colour of the pigment, are described by those who subdivide the malignant or sub-tertian into three species. GAMETOCYTES 93 As regards the genesis of the gametocytes, suggestions have been made from time to time that they may be formed by the union of two young parasites in one cor- puscle. Two, or even three or four parasites are not uncommonly found in one red corpuscle. These para- sites may be in actual contact with each other, but there is no satisfactory evidence that conjugation or fusion of two such parasites ever take place. On general grounds there is little or nothing to support this hypo- thesis of the formation of the male and female sexual forms. The differences between the three main species of parasites are shown in tabular form on the next page (Table, p. 94). Mistakes can be made with every method of blood examination, but most of them after a little experience are easily avoided. In fresh fluid blood films the following are often mis- taken for non-pigmented parasites. (i) The normal lighter colour of the central portion of the corpuscle, due to the bi-concave shape of the red corpuscle. The gradual shading and the absence of any definite edge to the lighter part is usually sufficient to prevent this error, and familiarity with this appearance in normal blood is of importance. (2) Vacuoles or slits in a blood corpuscle are distin- guished by the very sharp, abrupt edge of such a vacuole, and by the oscillatory motion of the edge. It can be generally seen that whilst in a parasite there is a faint opalescence, in the vacuole the space is perfectly clear {vide fig 19, c). (3) Blood plates resting on a blood corpuscle are in some cases difficult to distinguish. Round such blood plates there is usually a ring where the hemoglobin has been pressed out of the corpuscle, and in some cases by focussing it can be determined that the body is one whic^i is on and not a part of the red corpuscle. (4) Small particles resting on a corpuscle will displace the hzemoglobin beneath them and cause a lighter- 7 Form of Gamete Rounded body Rounded body " Crescent "- or saus^e- shapedbody 6 Effect on Red Corpuscles The corpuscle be- comes swollen and pale. With special stains Schiiffner'sdots are found The corpuscle be- comes smaller and darker At first little change, but later corpuscle is de- colourised 5 Character of Figment Yellowish -brown fine granules Black, coarse granules At first fine and black, but aggre- gate into masses earlier than in the other para- sites 4 Selective Sites for Sporulation Common in the circulating blood, but most abundant in spleen In circulating blood In internal or- gans, brain, lungs, intes- tines, &c. Very rarely found in circulating blood 3 Activity of Movement Very active Usually slug- gish Very active 2 Number of Spores IS to 2S 8 to 12 Varies greatly in some cases; 7 or 8 are the common num- bers, whilst in other cases 20 or more spores are common z Length of Cycle 48 hours 72 hours Variable and difficult to de- termine ; pro- bably about 34 to 48 hours Tertian (Benign tertian) Quartan (Benign quartan) Malignant Tertian (Sub-tertian) Autumno-EEStival DECEPTIONS 95 coloured patch in the corpuscle. Such particles, if dark, are often mistaken for pigment, and the pale area is taken for the parasite. (5) Crenations, particularly when they occur as pro- jections on the upper or lower surface of a corpuscle, are frequent sources of error. The effect of focussing, or alteration of the illumination, will show the true natufe of these crenations {vide fig. 19, a, b). (6) Bent or twisted "buckled" corpuscles may cause confusion (fig. 19, d and e). Many effects are miistaken for pigmented parasites. Some of these are due to insufficient illumination, as refraction effects with a dim light closely simulate grains of pigment. Crenated corpuscles, leucocytes, &c., are thus sometimes taken for pigmented parasites. Full illumination will dispel this illusion. Particles of dirt, or epithelial fragments with specks of dirt adhering, usually overlap at one edge or other a red corpuscle on which they lie. If they do not, by focussing it can often be determined that they lie on or beneath the red cor- puscle. In most cases such fragments can be distin- guished by their sharp angular outline, the irregularity in the size of the grains of dirt they contain, and by their high refractive index. Flaws, specks of dirt, or grease on slides or cover- glasses may cause confusion, but may be distinguished in the same manner. In any case of doubt it is well to touch the edge of the cover-glass with a needle whilst observing the object, and in that way it will be seen that the movement of the object is independent of the cor- puscle that was supposed to contain it. In stained specimens there are similar fallacies, and in addition, dirt from the stain, precipitated grains of stain, yeast cells, or other micro-organisms, may be present. It is well in any case of doubt to examine some part of the slide where the stain has extended beyond the blood film, and see if the same appearances are presented there. 96 DECEPTIONS In the great majority of cases, if the appearances met with in normal blood have been carefully studied, par- ticularly the blood plates and the various forms of de- generation of blood cells and of stained precipitates, mistakes are rare. Very rarely we do get an appearance from stain precipitates deposited on a red corpuscle that is difficult to distinguish, and therefore we should avoid diagnosing malaria from a single body believed to be a parasite. It is better in case of doubt to look carefully for a second parasite. Crescents should never be diagnosed on the ground of the shape only. A crescent always contains pigment, and is longer than the diameter of a red blood cor- puscle, and stains with basic stains. Three blood plates arranged in a row may be about the same size and shape as a crescent, but do not contain pigment or stain like a crescent. A transformed or altered crescent can be mistaken for a quartan parasite. Groups of blood plates are sometimes taken for sporu- lating bodies, and if they surround, as they may, a mass of dirt, the mistake is easily made. Even in fresh fluid blood the peculiar appearance of the edges of blood plates should prevent this mistake, and in stained speci- mens the manner in which the blood plates stain will enable them to be recognised. Imperfect fixation is a cause of some errors. In specimens fixed by heat, or fixed in alcohol that has absorbed water, small round bodies, artificial vacuoles, water or air, are often found in the red cells. They may be numerous in each corpuscle, or only one or two may be present. The sharp edge and high refractivity of these bodies, as well as the variation in size, distinguishes them from parasites. Familiarity with the appearances of blood prepared in different ways is necessary, so that these appear- ances will cause no difficulty, and the recognition with certainty and rapidity of parasites will then be easy. DECEPTIONS 97 Leishman-Donovan bodies, which are described later, are found in some cases of kala-azar in the leucocytes of the peripheral blood. The large mononuclear leucocyte is the variety in which they are ordinarily found, but they also occur in the polymorphonuclear leucocytes. They can be distinguished by the double chromatin mass — a smaller deeply staining mass, and a larger more lightly stained one {vide Parasites in tissues). 98 CHAPTER V. Parasites found in the Blood of Animals. Birds harbour two well-known species of intracorpus- cular parasites — proteosoma and halteridium, and others occur. Proteosoma (Proteosoma grassi, fig. 32fl) is found in comparatively few species of birds. Amongst these the Indian sparrow is the most common. Working with this parasite, Major Ross first demonstrated the sexual cycle of the haemosporidia in a mosquito. Proteosoma in birds has many resemblances to the malaria parasite of man. In its earliest stage it is unpigmented ; with continued growth pigment appears, and as the parasite grows it displaces the nucleus of the affected red blood corpuscle, and the corpuscle becomes paler. All stages of develop- ment, from ring forms to sporulating forms, are found in the blood at the same time. The infection is readily transferred from one bird to another by inoculation, and also to other birds, such as canaries. It is naturally transmitted by mosquitoes (CMte/a/f^am). Halteridium (Plates I. and III.) is a common parasite of many species of birds ; it is found in pigeons, crows, jays, finches, parrots, &c. — often a high percentage of the birds of a susceptible species are found to be infected. The infection cannot be transferred from one bird to another by inoculation, and the method of its transmis- sion in nature is doubtful. It was with this parasite that MacCallum first demonstrated the fertilisation of the macrogamete by the microgamete. This parasite is HALTERIDIUM 99 characterised by its peculiar curved shape surrounding the oval nucleus, but not displacing it. It does not cause any change in the red corpuscle which contains it. Halteridium resembles proteosoma in producing pigment, but is easily distinguished from it by its position in the cell (fig. 326). Halteridium has attracted a good deal of attention recently in view of Schaudinn's remarkable observations on the life-cycle of Trypanosoma noduce, a trypanosome occurring in the blood of the little owl {Athene noduce). According to Schaudinn this flagellate attaches itself to the red blood corpuscle by means of its flagellum, and. a b Fig. 32. — a, Proteosoma; b, halteridium. gradually penetrating it, comes to lie by the side of the nucleus. In this process the trypanosome loses its flagellum and undulating membrane, and the blepharo- blast becomes closely applied to the nucleus. The flagel- late Trypanosoma nodua thus becomes transformed into the halteridium. The trypanosome stages are found at night chiefly in the internal organs, while the halteri- dium phase is found by day in the peripheral blood. When fully grown the trypanosome may divide asexually, or if the gametocyte forms are taken into the stomach of the mosquito Culex pipiens, develop further. Schaudinn's 100 PIROPLASMA observations still await confirmation. Some observers are of the opinion that probably both trypanosomes and halteridia were present in the birds, and that Schaudinn confused the changes occurring in these two classes of parasites. The frequency with which flagellates are found in insects adds to the doubt as to Schaudinn's hypotheses. Various other parasites allied to those of human malaria have been found in animals. African monkeys harbour a parasite (Plasmodium kochi) closely resembling the sub-tertian parasite of man, and some species of Asiatic monkeys, bats, and flying foxes harbour parasites which are not unlike those of quartan malaria. PiROPLASMATA. — Another class of parasite, of which several representatives have been found in the blood of animals, is piroplasma. These parasites differ from the Hcemamcebce, such as the parasites of malaria, in that they form no pigment, that the nucleolus does not fragment, that division is into two or four only, and in the frequency with which extra-corpuscular forms are found. All the parasites of this class which have so far been investi- gated, have been shown to have as an intermediate host some species of tick of the sub-family Ixodina. The first of these to be discovered was Piroplasma bigeminum (Plate II., 13), the cause of Texas fever in cattle. It is transmitted in America by Rhipicephalus annulatus, and in Africa and Queensland by R. australis. p. parvum causes Rhodesian fever of cattle. It occurs in the blood in bacillary, spherical and intermediate forms. It is transmitted by Rhipicephalus appendiculatus. An animal may harbour both Piroplasma bigeminum and P. parvum at the same time. P. bigeminum may be conveyed from one animal to another by inoculation, but similar experiments with P. parvum have been negative, and the offspring of infected ticks are not infective. P. canis is the cause of epidemic jaundice of dogs. It is carried by Hcemaphysalis leachi in South Africa, and PIROPLASMA lOI Dermacentor reticulatus in Europe, and Rhipicephalus sanguineus in India. Other species of piroplasma described are : P. ovis, found in sheep and carried by Rhipicephalus bursa; P. equi, P. muris, and an unnamed species found in the monkey in Uganda has been described by P. H. Ross. Development of Piroplasma. Asexual Cycle. — This parasite has a free and an intra- corpuscular stage in its asexual Hfe-history. In the free stage they are pyriform in shape, and in the intracorpus- cular at first rounded, afterwards dividing into two or more pyriform bodies by a process of budding. The newly found parasites are at first connected together by a thin process, but finally become separated, and escaping Fig. 33. from the corpuscle invade other corpuscles. They may enter and leave two or three corpuscles before again becoming rounded and undergoing division — ultimately, however, the process of division is undergone, and so the cycle is repeated. Sexual Stage. — Koch has described developmental forms of P. bigeminum in Rhipicephalus australis, R. evertsi and Hyalomma cegyptium, and Christophers has demonstrated similar forms of P. canis in Rhipicephalus sanguineus. From these observations it appears that in the stomach of the tick the parasite escapes from the red blood corpuscle and becomes elongated, one of its chromatin masses pas- sing to the blunt (? anterior) end, forming a projection — 102 H^MOGREGARINA the other chromatin mass remaining in the middle of the parasite. Next radial processes arise near the projection, the other end of the parasite becomes pointed, and the general contour of the parasite becomes angular. After the second day couples of these forms are seen apparently connected together, a process of copulation. The male element, after giving up its chromatin, is thrown off or stretched out over the enlarging fertilised female. The fertilised element after becoming round, oval or eventu- ally club-shaped, leaves the gut and passes to the ovary. In the ova of infected ticks large pear-shaped bodies are described. Christophers also states that he has found further developmental forms in the cells of the gut of unfed nymphs reared from infected mothers, and has traced them to the salivary glands of nymphs of the second generation, which were about to become adults. H^MOGREGARINA. — The haemogregarines are unpig- mented parasites, occuring both as intracorpuscular forms, in which they are bent upon themselves so as to form a tail (Plate III., figs. i6 and 19), and as free vermicular forms in the blood plasma (Plate III., figs. 17 and 20). They are non-pigmented, and in stained specimens the nucleus stains and contains abundant chromatin as numerous granules sometimes arranged in radiating lines. Sporulation takes place not in the blood, but in certain cells of the viscera. The parasites are common in the blood of cold- blooded vertebrates. The effect on the red corpuscle varies. Some species displace the nucleus, some do not. At least one species causes the formation of granules which stain like the Schiifner's dots in the human cor- puscle containing the parasite of benign tertian malaria (Plate III., 21). Several species have recently been found in the blood of mammals. None have been found in human blood. The first of these mammalian haemogregarines was discovered by Bentley in the leucocytes of dogs in Assam, H^MOGREGARINA 103 and described by James as Leucocytozoon canis. Soon afterwards similar parasites were described by Balfour as occurring in the red blood corpuscles of the jerboa, Jaculus goudoni (fig. 346), and by Christophers in the Indian field-rat, Gerbillus indicus. Later, Patton described another haemogregarine in the leucocytes of the Kathiawar squirrel, Funambuls pennantii, and Balfour a similar parasite in the leucocytes of the rat {Mus decumanus). Fig. 34a. Fig. 34*. L. canis occurs in the polymorphonuclear leucocytes. The parasite is enclosed by a capsule which is not easily penetrated by the stain. It is an oblong body rounded at the ends. The contents are the haemogregarine, which is sharply bent on itself, so that the nucleus is frequently horse-shoe shaped. The development of this parasite has been studied by Christophers. It has been found to reproduce itself by 104 H^MOGREGARINA encystment in the bone-marrow of the host, there form- ing merozoites (fig. 35). He also found that the sexual development of the parasite took place in the dog tick {R. sanguineus). When taken into the gut of ticks the parasite escapes from the capsule and shows active vermicular movements. Within twenty-four hours these, associated in pairs, have lodged themselves in the large cells of the gut. On the second day conjugation between two similar individuals takes place, and a globular body is formed containing a single large homogeneous mass of chromatin. This chromatin mass spreads out to form a reticulum, and the protoplasm increases in amount. The chromatin collects at the periphery into irregular star-shaped masses, and on the third or fourth day the whole body splits up to form eleven to fourteen sporo- zoites. When set free the sporozoites are in the lumen of the gut. The method of re-entry in the dog has not been discovered. H. balfouri occurs in the red blood corpuscles of the jerboa or desert- rat. As seen in stained specimens it is a slightly curved body, with rounded ends lying either apparently free or in the remains of a red blood cor- puscle. It is believed that the free forms owe their con- dition to the destruction of the red blood cell which contained them. A long oval nucleus, situated about the centre of the parasite, is present, and occasionally a few dots of chromatin in the pale polar areas. The stage of schizogony has been observed by Balfour to take place in the liver cells of the host (fig. 35), and is similar to that described in L. canis. H. gerbilli is a parasite similar to the preceding, occur- ring in the red blood corpuscles of the Indian field-rat. The stage of sporogony occurs in the louse. The complete life-cycle has not been discovered. L. funambuli is a parasite occurring in the large mononuclear leucocytes of the palm squirrel. It differs from L. canis in the absence of a cytocyst. A similar parasite has been found by Balfour in the Norway rat. Fig. 35. H^MOGREGARINA 105 Zoological Position of the Malaria Parasites. Protozoa i Class Sporozoa Sub-class Telosporidia Neosporidia I I Order (l) Gregarinoidea Hcemosporidia Genera : (2) Hmmogregarina Coccidiidea (l) Plasmodium (Ht^m- amceba) I P. vivax (Benign tertian parasite) P. malaria (Quartan parasite) P. falciparum (Sub-tertian parasite) P. pracox (Proteosoma) P. kochi (found in monkeys) &c. H. ranarum H. geriilli H. balfouri Leucocytozoon canis L. funambuli &c. (3) Piroplasma (4) Halieri- diiim (?) P. bigeminum P. parvum P. ovis P. equi P. canis &c. io6 CHAPTER VI. Parasites found in Blood Plasma. The most important animal parasites found in the plasma of the peripheral blood are trypanosomes and spirochsetae belonging to the Class Mastigophora, and embryos of various filaria, nematode worms. In some of the lower animals hsemogregarines are found during their motile stage, and the piroplasma pass a short period of their existence free and may be found in the plasma. The haemamoebidas are not found in the plasma, though for a brief period the " spores " must be free. As stated in the preceding chapter, according to Schaudinn the halteridium has a free stage in the plasma. In freshly shed blood trypanosomes are readily seen as actively motile, worm-like bodies darting about be- tween the blood corpuscles. Their movements are so rapid and they are so transparent that it is difficult to make out their form clearly in the living condition. The largest trypanosomes are found in fish (Plate I., 26), both fresh and salt water. They can readily be demonstrated in small fish by cutting off their heads and making a smear on the slide with the cut surface. These smears can be examined fresh by placing a cover-glass on the top of the exuded fluid, or the films can be dried and stained. Trypanosomes are found in some birds. The method of examination of the blood for these is the same as that required for the haemosporidia. If a small bird is to be examined it is held in the palm of the left hand and one leg is allowed to protrude between the fingers. A needle TRYPANOSOMA 107 is then inserted deeply into the vascular pad surrounding the root of the claw and left there for half a minute. On squeezing the leg so as to force the blood towards the claw the blood will exude in drops, and films can be made as with human blood. With larger birds an assistant is necessary to hold the bird, and the bird should be wrapped in a thick cloth for the protection of the assistant. The trypanosomes that have attracted most attention are those of the mammalia. Several species are known ; they closely resemble each other in their appearance, but differ in size, shape, and motility to some extent ; also in the positions they assume and the way in which they stain (Plate I., 26, 27, and Plate IV., 21, 22). They may be differentiated by their pathogenic action. By inocu- lating with the blood a series of animals, it will then be found which animals are immune and which are suscep- tible. For such inoculation the blood must be mixed with some fluid that will prevent coagulation. Sodium citrate solution, 10 per cent., may be used, and the blood should be diluted with one-twelfth of this solution. Others use a weaker solution of citrate of soda, i per cent., and dilute the blood more freely. Injection of such diluted blood into the subcutaneous tissues will lead to infec- tion with trypanpsomata of the animal which has been injected if it is susceptible. The more important of the trypanosomata are : — (i) Those found in a large proportion of the rats in both tropical and temperate climates. These are non- pathogenic to full-grown rats, and all other animals experimented on are insusceptible to the infection (T. lewisi, Plate IV., 21). (2) Nagana or "Tsetse-fly Disease." The try- panosoma of this disease (T. brucei) can be inoculated into a large number of wild and domesticated animals, but man is insusceptible. To cattle, horses, donkeys, dogs, guniea-pigs, rats, &c., this parasite is pathogenic, but the time required to cause death varies greatly in these I08 TRYPANOSOMIASIS animals. Wild game, and particularly the buffalo, harbour the parasite, which appears to be harmless to them (Plate I., 27). It is carried from animal to animal by biting flies belonging to the genus Glossina, usually by G. morsitans. (3) Surra {T. evansi). A disease fatal to horses ; cattle often recover. It occurs in India, Philippines, &c. It is carried by various biting flies, such as some of the tabanidae. In the living condition it can be distinguished "from T. brucei by its greater activity, as it not only moves but actually progresses. (4) DOURINE (r. equiperdum). Europe. Rats, dogs, rabbits, &c., are susceptible. Cattle, sheep and goats are refractory. Transmitted by coitus with the semen. (5) Mal de Caderas (T. equinum). South America. Cattle are immune ; most of the other animals are sus- ceptible. T. Theileri. South Africa. Cattle only are sus- ceptible. No other animal has yet been inoculated successfully. It is the largest of the trypanosomes found in mammals. (7) T. DIMORPHUM occurs in horses in Africa. Various animals are susceptible. The parasite occurs in several forms. (8) T. Gambiense (Plate IV., 22). Man is insus- ceptible to all the above-mentioned trypanosomes, but in Africa, on the West Coast and throughout a large part of Central Africa, another species has been found in man. The parasites are found in small numbers in the blood in most cases, particularly in chronic cases, but can be found in larger numbers in fluid drawn with an aspirating needle from a lymphatic gland. The symptoms are constant irregular pyrexia, enlarged lym- phatic glands, usually in the neck or above the clavicle, erythematous rash. Enlargement of spleen and liver have been noted. Monkeys are susceptible, and, though not so readily, white rats and guinea-pigs. After a longer or shorter period in man grave cerebral symptoms super- STAINING TRYPANOSOMES 109 vene, usually of the lethargic condition known as "sleeping sickness," and the patient dies. In this stage of the disease trypanosomes are found in the cerebro-spinal fluid. They are not numerous, and it is necessary to centrifugalise the fluid to demonstrate them. For the examination of blood for trypanosomes, films prepared as for malaria are the best, as the parasite will then be seen undistorted. When the parasites are scanty and for purely diagnostic pui-poses, thicker films de- colourised by the action of water may be used. In some infections the parasites can only be found by injecting a highly susceptible animal with the blood of a suspected case, as a large infection may then result in the animal which has been injected. This proceeding is necessary in many cases of dourine. In centrifugalised blood the parasites accumulate in the upper part of the mass of red corpuscles and can be found there more readily than by the ordinary method. Trypanosomes stain rather feebly with most basic stains, haematoxylin, methylene blue, &c. A stronger basic stain, such as carbol fuchsin, should therefore be used. Clearer specimens are obtained by diluting the stain with two parts of water and leaving to stain for ten minutes. Good results can also be obtained by overstaining with this stain and then decolourising with J per cent, solution of glacial acetic acid in water, but the parasite is often swollen and distorted, though quite recognisable. Leishman's stain, used, as for other blood work, gives excellent results with fresh specimens and shows well the various points in the structure. The body is elon- gated and the posterior extremity is bluntly truncated, whilst the anterior is prolonged into a long flagellum, rarely in two. The flagellum is continued as a definite curved rod in the body of the parasite. Slightly posterior to the termination of the flagellum is a deeply staining nodule — the centrosome. About the middle of the body is a rounded mass, larger but less defined — the nucleus. no MULTIPLICATION OF TRYPANOSOMES In fission forms the centrosome first divides, then succes- sively flagellum, nucleus and protoplasm (fig. 36). The p«-otoplasm with Leishman's stain is blue. The centro- some, nucleus and flagellum are red. The multiplication is by fission. These fission forms are rarely found in the peripheral blood. Occasionally there are two flagella, with no signs of fission in centro- some or nucleus. The human trypanosome is said to be carried by G. palpalis, but other species of glossina are also sus- pected of acting as carriers. The mode of transmission may be direct, the trypano- SPIROCH^TA 1 1 1 somes being taken from any infected animal, and without any further development in the fly enter the next animal bitten. No sexual phase has been observed in the try- panosomes. Further work is much required on this subject. The spirochaeta of relapsing fever — Spirochceta ober- meieri (fig. 37) — is now generally believed to belong to the Mastigophora. They can be seen in fluid blood films made as for malarial blood. The organisms are very transparent and can only be seen in fresh fluid prepara- tions with the diaphragm nearly closed. They are then seen as fine, transparent, thread-like bodies, which are in active movement and coil and uncoil themselves. They are also seen in the corkscrew-like forms which are com- monly drawn as representing them. Dried films are best thin. In such films the spirochaetae are seen in the undulating form (fig. 376). In thicker films they appear more frequently coiled up (fig. 37a). The spirochaetae stain with all basic stains, but not intensely, and are best demonstrated by the use of the stronger basic stains, such as carbol fuchsin (Plate IV., 23). They stain well by Leishman's method or with Giemsa. Ordinarily no definite structure can be made out. The disease can be reproduced in monkeys, and less readily in rats. African tick fever has been shown by Ross and Milne to be caused by a similar spirochaeta — S. duttoni. In appearance it closely resembles S. obermeieri, but is more readily inoculated into lower animals, rats, guinea-pigs, &c. The pathogenicity of the two parasites differs, and infection with one does not affect the sus- ceptibility to infection by the other spirochaetae. There is leucocytosis and marked relative increase of the polymorphonuclear leucocytes. This increase per- sists to some extent in the periods of apyrexia, so that a differential count of the leucocytes may exclude malaria. The spirillum shows no signs of longitudinal division in the blood, and in human blood has no tendency to 112 SPIROCH^TA great variation in length. It is found in the plasma, never in the red blood corpuscles. The spleen enlarges, and in fatal cases spirilla are found in large numbers in that organ. The organisms are found in greatest number during the first pyrexial period. In the apyrexial period they are not to be found, and in the subsequent pyrexial attacks they are found in femaller numbers than in the primary attack. In some cases the disease passes on into a chronic condition of irregular pyrexia — secondary fever — it is exceptional to find the parasites during that period. Spirochaetae are found in the mouth and sometimes in faeces. In various syphilitic lesions, in yaws, in scleros- ing granuloma and in many ulcers spirochaetae are found. The best known of these js S. pallida, found in syphilis. Spirochaetae are found in the blood of many of the lower animals. Schaudinn believed that the spirochaetae are closely LEISHMAN-DONOVAN BODIES 113 related to the trypanosomes, and therefore belong to the flagellata. He considered them to result from the repeated longitudinal division of the trypanosomes, the nucleus and centrosome becoming both elongated and attenuated. Fig. 38. — a. Single trypanosome much enlarged ; b, stage of fission ; c, parasites still attached by posterior ends ; d, same, both parasites com- mencing to divide, nucleus and blepharoplast divided- ; e, resultant stage of division ; /, one of the four spirilla into which e has divided. Leishman-Donovan Bodies. — These parasites are found in the blood, in some cases in the leucocytes, either in the large mononular or in the polymorphonuclear. Possibly a free flagellate form may, in time, be found in the plasma, as in cultures the bodies develop flagella. These flagellate forms have not been observed in man. The bodies are found in the spleen, liver, lymphatic glands, lungs and submucosa, and occur in these situa- tions, particularly in the spleen and liver, in enormous numbers. They are contained in the endothelial cells, and in masses imbedded in a hyaline matrix between the cells. They can be observed in smears from the organs taken after death, or by puncture and aspiration, with a hypo- 8 114 LEISHMAN- DONOVAN BODIES dermic syringe, of the spleen or liver. Fatal accidents have resulted from puncture of the spleen, and punctures of the liver should therefore be made, though the para- sites are not found so readily in the fluid drawn from the liver. A large all-glass syringe is convenient for the pur- pose. The needle should not be too fine. The skin must be carefully sterilised over the selected place and the syringe and needle sterilised dry. The needle should be plunged right into the liver, so that it moves virith the movements of the liver. It should be rotated and with- drawn a little, but still kept in the liver before aspiration. The aspiration should not be too forcible. If much blood comes this will so dilute the fluid con- taining the Leishman-Donovan bodies that a prolonged search may be necessary to find them. They swell up and break down on the addition of water, so that thick decolourised blood films cannot be used. It is for this same reason that the syringe used for aspirating must be dry. The bodies are small, round, or oval masses of proto- plasm, which stain faintly with basic stains, and contain two chromatin masses, which stain deeply with ordinary basic stains, and with the polychrome methyl blue a deep red. These two chromatin masses are unequal in size. The larger is oval,, is situated to one side of the parasite, and stains with Leishman a decided red, but not very deeply. The smaller is rod-shaped, and stains intensely red with Leishman. It usually is directed point- ing obliquely towards the nucleus. (Fig. 39.) These bodies are much the same size as blood platelets but the peculiar chromatin masses render them easy to recognise. They are frequently found in clumps. In the peripheral blood they may be found in thin films within leucocytes, but as many leucocytes as pos- sible should be present, as only i in 100 to 500 will contain the bodies. The edges and ends of the films contain most leucocytes, and the number can be in- Fig. 39. LEISHMAN-DONOVAN BODIES 1 15 creased by suddenly lifting the upper slide off the lower one, in making a film by the ordinary method. There IS always some anaemia, and degenerate red corpuscles are common. The leucocytes are scanty, only 2,000 to 4,000 per cubic millimetre. The mononuclear elements are relatively increased. Leishman-Donovan bodies were considered by Leishman to resemble, in the arrangement of the chromatin masses, degenerate forms of trypanosomes. Laveran suggested a resemblance to piroplasmata. Fig. 40. — a, Trypanosomes and the altered forms found in culture ; b, Leish- man-Donovan bodies and the altered forms found in culture. Rogers and others have shown that in cultures in sterile citric acid solution or in sodium citrate, 2-5 per cent, solu- tion, the bodies become much elongated and form a flagellum, showing that they are the resting stage of a flagellate (fig. 406, i, 2, 3). It had previously been shown that trypanosomes could be kept alive for some time in a medium rich in haemo- globin, and that forms (? degeneration forms) were pro- duced which had no flagellum, whilst the form of the organism became round and the centrosome was brought nearer to the nucleus (fig. 40^, i, 2, 3). It is now considered to be established that the bodies belong to Mastigophora or Flagellata, but not to the genus Trypanosoma, as there is no undulating membrane and the flagellum emerges from the end at which the centrosome is placed. It is probably a Herpetomonas. Il6 DELHI BOIL Flagellate forms may be discharged from the ulcers in the intestines in kala-azar, the disease due to the Leish- man-Donovan bodies. Delhi Boil. — Scrapings from the raw surface in these ulcers show a considerable number of bodies closely resembling Leishman-Donovan bodies. The two diseases are probably distinct, and therefore the parasites in Delhi boil are probably of a different species from those found in kala-azar. NEMATODA 117 CHAPTER VII. Parasites other than Protozoal found in Human Blood. Animal parasites are found in human blood belonging to higher orders of animal life. Schistosomum hcematobium (Bilharzia) and S. japonicum frequent the veins of the portal system and pelvis (vide Trematoda). Nematodes. — One species of filaria in the adult form has been once found in the circulatory system of man by Magalhaes in Brazil, but no further observations have been made. The worms were found in a blood clot on the left side of the heart. In the lower animals nematode worms are not un- common in the blood. Filaria immitis is found in the right side of the heart and pulmonary vessels of the dog, and in the East and in some of the Pacific Islands it is exceptional to find a dog free from these parasites. When the worms are in large numbers cardiac dilatation and death result. Various nematode worms in horses and other animals cause " verminous aneurisms." Of the human filaria the adults are found in various parts of the body, whilst the embryos may be discharged through an aperture in the skin, as in Filaria tnedinensis (guinea-worm), and probably also in Filaria volvulus, a worm found in a subcutaneous cyst in a patient in Sierra Leone. In those filaria in which we are at present more specially interested, the embryos find their way into the blood and circulate with that fluid. Filaria sanguinis hominis. — The filarial embryos, or Il8 MICROFILARIA microfilaria, as seen in fresh blood, are clear, transparent, worm-like bodies, which are in active movement. They are most readily found in a fresh fluid blood film, as the active movements and the disturbance in the red corpuscles set up by their movement catch the eye. An inch or two-thirds inch objective is quite sufficient magnification for the detection of the commonest filarise, but it is better to use a half-inch, as the smaller species may be overlooked with the two-thirds objective. The film must not be so thin as that required for examina- tion for malaria parasites. No special precautions are required, and sufficient blood should be taken to com- pletely fill the space between the slide and cover-glass. As the slide must be kept for a sufficient period to enable the movements of the worm to cease, the cover-glass should be ringed with vaseline to prevent evaporation of the blood. To examine the embryos in detail, higher powers, in- cluding a one-twelfth oil immersion, are required. At first the movements of the filaria are so active that it is impossible to examine it with these objectives, but after some hours the movements become much more sluggish, and finally cease. The best time for examina-, tion is just before the cessation of movement and the death of the embryo. The points to observe in the examination of the fresh embryos are : — (i) The character of the movement and whether active locomotion takes place or whether the move- ment, however active, leads to no progression. (2) The size of the embryo. This is of the greatest importance, as measurements of dried specimens vary greatly with the rapidity with which the film has dried. (3) The shape of the embryo and that of the two ends. (4) The presence or absence of a loose sheath. (5) Any details of structure, and particularly the presence, position and character of any contractile DIAGNOSIS OF MICROFILARIA 1 19 vesicles, the so-called V spots, and the cephalic movements and any appearance of armature. Embryos can also be readily observed in dried films. The blood films for diagnostic purposes should be as thick as possible. A convenient way of making them is to allow three or four large drops of blood to fall on a slide close together and smear them together into a space about two-thirds of an inch in diameter (fig. 41). Allow to dry, protecting the films from insects during the process. Such a film will be so thick as to be almost Fig. 41. opaque. It must not be fixed. When quite dry place in distilled water and leave in the water till the haemo- globin is all dissolved out. It is best to have the film side downwards in the water, but not resting on the bottom of the vessel. As the haemoglobin dissolves out it will fall to the bottom of the vessel. It will be found better after a few minutes to transfer the slide to clean water, so that it is easy to observe when the haemoglobin is all removed. Remove the slide from the water and examine .at once whilst still wet. The white corpuscles will stand out I20 STAINING OF MICROFILARIA from the film as refractile spots and the white colourless worms will also stand out brilliantly. If it is preferred to stain the specimen it should be allowed to dry and fixed in alcohol and ether. Any basic stain gives good results, and weak carbol fuchsin is per- haps the best of the aniline stains. Haematoxylin gives good and permanent results, but the sheathed filariae do not stain rapidly. If the hsematin mixture is used it should be warmed, and fifteen or twenty minutes will be required for satisfactory staining purposes ; the slide should then be left in water for ten minutes. A good many slides can be stained at the same time. For this the staining vessel (fig. 42) is convenient. Fig. 42. Counter-staining brings out nothing more, but eosin may be used for this purpose. The shape of the worm, and also the sheath if present, are well shown in a specimen stained with haematoxylin. The body of the worm is found to contain a core of deeply staining points or nuclei. These do not extend to either extremity, nor do they completely fill the worm, as a clear, unstained portion is left on each side. This un- stained portion must not be mistaken for the sheath. The sheath will be faintly stained and only clearly seen at the two ends, where it will be found flattened on itself and often folded sharply like a piece of ribbon. MICROFILARIA 121 In the nuclear core complete or incomplete gaps in the mass of nuclei will be seen in most filariae. For each species the position of these gaps is constant, or nearly so, and consequently the exact position of these gaps is important for the differentiation and identification of species from the examination of these embryos (Plate II., 17, 18, 19). The arrangement of the nuclear core at the blunt cephalic end is of particular importance, as it is an additional point used in the diagnosis of species. In the Microfilaria nocturna the nuclei are loosely arranged at this end, whilst in Microfilatia diurna they form a compact mass terminating almost as a straight line. Embryos of some species of filaria are not found in the same number all through the twenty-four hours. During a part of this period they may be found in numbers, whilst a few hours later they are found with difficulty or not at all. Thus one species has a periodicity which is called nocturnal, because the embryos are found in largest numbers in the peripheral blood at night ; in other species embryos are only found in the daytime and are said to have a diurnal periodicity. Embryos of other species are found in fairly equal numbers at all times of the day and night. In any investigation of the periodicity of filarial em- bryos it is essential that the blood examined should be measured. The periodicity can be altered in the case of Filaria nocturna by changing the habits of the host, and cases are fairly common in which the periodicity is reversed without known cause. It is still more common to find small numbers of F. nocturna during the day and larger numbers at night. The chief points of difference in the various embryo filaria are indicated in the subjoined table. These points require no detailed explanation. It is well to draw the embryos accurately with a draw- ing camera or camera lucida. By substituting a scale for 122 MICROFILARIA SPECIES the object a scale can also be drawn on the same paper and measurements made from this, which are easier and usually more accurate than measurements made with a micrometer eyepiece. Distance Adult (known or suspected) iiame of embryo Length Greatest thickness Sheath Shape of tail Periodicity of head gap from head mm. mm. mm. !aria nocturna •317 •0075 Present Sharply pointed Nocturnal in peri- pheral blood •052 F. bancrofti laria diurna . . . •317 •007 Present Sharply pointed Diurnal in periphe- ral blood F. loa. laria perstans •I9S ■004s Absent Blunt, truncated None •03 F. perstans. 'aria demar ■ ■21 •005 Absent Sharply None •03 F. demar ^uayt pointed quayi. laria ozzardi... •21 •005 Absent Sharply pointed None •03 F. ozzardi. Periodicity refers to the time of appearance of embryos m the peripheral blood. With regard to this periodicity, it was for a long time not definitely known what became of the embryos during the time they were absent from the blood. Post-mortem examinations, however, have shown that in the case of persons harbouring this filaria where death has occurred during the day, the embryos are found in greatest numbers in the lungs and large vessels, though some may be found in the vessels of other viscera. In sections of the organs of such a person the micro- filaria are found in numbers. The material may be imbedded in either celloidin or parafHn, and should not be too thin, or such short lengths of the filaria will be cut that they cannot be easily identified. Haematoxylin solution, two minutes, is quite sufficient to stain the embryos in section, and there is no need to counter-stain. Transverse and oblique sections of numerous embryos will be found. In places longer lengths, or even complete embryos, which were lying in the plane of the section, mav be seen. FILARIA 123 As far as is known, no developmental changes take place in the human filarial embryos in the blood or human tissues, but there is evidence that some degree of growth does take place in some of the avian microfilaria whilst they are circulating in the blood. In the case of the human filaria, the next stage of growth occurs in several species of mosquitoes of different genera — Culex, Anopheles, Mansonia, &c. — and when a certain stage of maturity is reached the embryos are in- jected by the mosquito into man. At this stage, the em- bryos in the case of F. nocturna are 1-5 mm. in length, the alimentary canal is complete, but the sexual organs are not developed. The further development in man has not been traced, but the adult forms of the species F. nocturna {F. ban- crofti) have been found by many observers always in, or in connection with, the lymphatic system. The other human adult filarias, F. persians, F. demarquayi, F. ozzardi and F. loa (the adult form of F. diurna), are found in connective tissue, either subcutaneous or in the subperitoneal tissues. The adult human filariae are not very readily found. F. bancrofti are found in lymphatics in almost any part of the body, but as a rule, in the cases of elephantiasis, the adults are long dead and the positions they once occupied only indicated by lymphatic obstruction. F. Persians, though smaller, are more readily found, as they occur, at any rate in greatest numbers, in subperitoneal connective tissue, particularly at the base of the mesen- tery. F. demarquayi has been found by Dr. Galgey in the same position, and F. ozzardi has been once found in the subserous connective tissue of the anterior abdo- minal wall. F. loa can be seen when it passes under the skin or conjunctiva. It is difficult to extract, for as soon as an incision is made in the skin it rapidly moves away. F. immitis, the " worm in the heart " of dogs, is found in the cavity of the right side of the heart and the pul- 124 FILARIA monary vessels. When only one or two worms are pre- sent they are usually in the snaaller pulmonary arteries. Avian filariae occur in many positions. Some species are found in loose connective tissues, as in the neck, others in the limbs, and particularly in thickenings about the claws ; others in the submucous tissues, as in the crop ; and others in the blood-vessels, and even in the pouches formed by the semi-lunar valves. Adult filariae are easily mistaken for empty blood-vessels, small nerves and shreds of fibrous tissue. They are more readily recognised with slight magnification, and for this purpose a watchmaker's glass of about five-inch focal length is very useful. Those mounted in horn are best, and should be perforated at the sides, otherwise moisture condenses on the lens. The advantage of these glasses is that both hands are free, and it is easy to learn the use of this simple lens. In searching tissues for filaria a dark surface, such as a slab of slate, makes a good background, and the rough surface of the slate prevents the specimen slipping about. The tissue should be kept wet with normal saline solu- tion, as this keeps it transparent. The dissection should not be made with the tissues floating in water or salt solution, as strands of tissue are much more readily twisted or ravelled out if floating. Description of Filaria. — Some authors construct a formula for the description of filariae based on the relative positions of various structures and the measurement of the worm at these places. The unit of measurement is the one-hundreth part of the length of the worm, so that the measurements are percentages of the length. Five measurements are taken by the author of the method — Cobb — commencing from the head : the base of the oesophagus, the nerve ring, the cardiac constriction, the fourth at the vulva in the female and the middle of the male, and the fifth at the anus (fig. 43). Many of these points are very difficult to make out in the human filariae. In living filariae the first of them is very variable in the same individual. As the head and FILARIA 125 neck are capable of considerable contraction, the head cannot be taken as a fixed point to serve as the basis of a series of measurements. Also the whole formula is based on the assumption that the proportions of various parts of the body are constant in different individuals, which, according to Shipley, is not certain; Though we do not consider that this graphic method is applicable in many cases, still, where possible, it may be given. The human filariae resemble each other rather closely in their adult forms, and some of them require very careful examination for differentiation. Measurements Fig. 43. of the head and tail, making due allowance for the con- tractility of the worms, are of great importance. Par- ticular attention must be paid to the transparent cuticle, as there are important differences in its arrangement in different species, and these differences are constant for the individuals of each species. The measurements should be made, where possible, in the fresh worms, as serious shrinking and distortion occurs with most reagents. Alcohol and spirit cause great distortion. This can be diminished by placing the specimen first in dilute spirit, i to 3 of water, for a few hours, and then gradually increasing the strength, but however carefully this is done the distortion is great. Much less distortion is caused by spirit if the specimen is first hardened in formalin 2 per cent. 126 FILARIA Fig. 44. Head of Filaria bancrofti, S , Fig. 45. Head of Filaria ozzai-di, ? . FILARIA 127 A general method for the treatment of nematodes is as follows : — (i) Place the worms in a i per cent, saline solution and shake up. 1 (2) Have ready alcohol (60 to 70 per cent.) which has been heated to the boiling point. This is best done in a porcelain dish over a flame protected by wire gauze. Transfer the worms from the saline solution to the hot alcohol, dropping them in one at a time, the worms will die in an extended position. Fig. 46. Tail of Filaria hancrofii, s • Fig. 47. Tail of Filaria ozzarai, ? . They may be preserved in 70 per cent, of alcohol till till required for examination. (3) For examination they are placed in a mixture of 95 parts alcohol and 5 parts pure glycerine. The alcohol is then allowed to evaporate. In pure glycerine the worms become transparent. They may be studied in glycerine or mounted in glycerine jelly. If placed at once in glycerine without any pi-eliminary treatment, there is, at first, some swelling, though when 128 FILARIA F. bancrofti F. Persians F. ozzardi Female Male Fen^ale Male Female Male Length Greatest breadth Diameter of head Character of cephalic end Distance of genital pore from head (female) Diameter at point of genital pore Distance from tail of anus Cuticular thickening on tip of tail Spicules (male) Papillae (caudal) .. Habitat Geographical distri- bution 80-90' mm. •23 mm. ■055 .. ■66-75 ■"""• '14 mm. ■225 .. None None 44 mm. •OS „ None Two unequalr anterior and posterior, both retrac- tile None Lymphatic system... In most tropical regions 70-80 mm. ■12 mm. •07 ,. '6 mm. •07 „ •145 .. Double ter- minal cuti- cularthick- ening None 45 mm. '06 mm. ■04 „ Two unequal spicules Four preanal and one postanal. Very close to opening of cloaca Connective tissue, usually Africa (West Coast and Central), British Guiana 81 mm. '21 mm, •05 » •71 mm •12 „ •23 » None .., None ... 38 mm. '19 mm, ? ? None. None ... subperitoneal British Guiana. FILARIA 129 F, demarquayi F. loa F. magalhaesi nale Male Female Male Female Male Not known . . . 5 mm. 19 ,. lar thick' ; over Knobby iregular tline 50-55 mm. ■55 mm. 30-35 mm. 2-35 mm. •3 mm. No thickening over tip. Two lateral alse. Cuticu- lar bosses not found at tip, but over the greater part of the body ritoneal connective tissue [ndies 175 mm. Thickening over tip. The " bosses " so abundant over the cuticle in the body of the worm are not found at the tip Two unequal, anterior and posterior Three preanal pairs and two postanal. The last are very small Connective tissues, subcuta- neous, subconjunctival, or in the deeper parts of the limbs West Africa 155 mm. •6-7 mm. •o5 „ 2'56 mm. •58 .. •13 .. None 83 mm. •3- -4 mm. •04 >, None. Two spicules Four preana and fou postanal. Left side of heart. Brazil. 13° FILARIA left long in the glycerine there is a return to a more natural condition. The specimens so prepared are much softened and can very readily be flattened out, and whilst thus gently compressed between two slides, be hardened in methylated spirit and finally in alcohol, and mounted after clearing in oil of cloves. Such specimens are very Fig. 48. Head of Filaria demarquayi, ? . Fig. 49. Head of Filaria Persians, ?. transparent and do not show much detail ; if, however, they are slowly stained with very dilute solutions of stains, such as dilute borax carmine, before placing ih glycerine, many details of structure are brought out well. They can also be stained with well diluted haematoxylin, FILARIA 131 and subsequently slightly decolourised with dilute acid spirit ^ per cent, to show in situ eggs and embryos. Many filariae show fairly well when mounted direct in glycerine jelly, but these, after a time, became distorted. If previously hardened by placing first in i per cent, formalin for two days and then in 2 per cent, formalin for two days, and then kept in 5 per cent, formalin for Fig. so. I^'g. 51. Tail of Mlaria demarjitayi, ? . Tail of Filaria Persians, 5 . some days, they can be mounted in glycerine jelly, or even in Farrant's solution, and retain their natural size and appearance. The different points enumerated in the tabular form are usually made out, but to see either the genital pore or the anal opening that portion of the worm must be viewed in profile. It is therefore necessary to turn the worm gently before mounting so that they can be seen. 132 BACTERIA IN BLOOD This can generally be effected by slightly moving the cover-glass by pressure of its edge with a needle so as to roll the worm over slightly. The points of difference and resemblance are shown in the table (pp. 1 26-131, and in figs. 44 to 51) for the known adult human filariae. The description of F. ozzardi is from a single specimen. The embryos or microfilarise known as F. ozzardi and F. demarquayi correspond in every respect. As the single adult found of F. ozzardi differs from the specimens of F. demarquayi in some respects, the worms cannot be considered as the same, but further specimens are required before the question of the identity of the species can be considered to be settled. Examination of the Blood for Pathogenic Bacteria. — Most of the organisms found in blood films are due to contamination with skin organisms during the preparation of the film.. To avoid this the finger, which for this purpose is the most convenient part to examine, should be well washed writh 2 per cent, lysol and then wrapped in a i in 500 sublimate compress covered with gutta-percha tissue for twelve hours. The first drop of blood should be rejected as the most likely to be con- taminated. Thick and thin films should be taken and rapidly dried. The thin films can be stained by Louis Jenner's and Leishman's stains, or the film can be fixed and stained by any of the methods used for bacteria. In thick films, after drying, the haemoglobin can be removed by placing in sterilised water, and afterwards fixed and stained. In most cases organisms are present in such minute numbers that they will not be found by this method, and the drops of blood obtained should be used for making cultures. The organisms of plague, septicaemia, tubercle, &c., may be found in the blood, but great caution must be exercised as mistakes are frequent. Cultivation of the organisms from blood drawn from a vein by a hypo- dermic syringe is more satisfactory. For this purpose BACTERIA IN BLOOD 1 33 a few drops of blood should be placed in each of a series of flasks containing broth, so that great and rapid dilution of the blood takes place. The leucocytes will fall to the bottom of such flasks, and will not destroy so many of the organisms which may be present in the blood. 134 CHAPTER VIII. Certain Properties of Blood Plasma and Blood Serum. The living blood in the body is composed of a fluid element — the plasma — in which are suspended the solid elements, red and white corpuscles, blood platelets, and at times parasites. After death, and when the blood is shed, coagulation occurs, the blood plasma being con- verted into a jelly-like mass which soon contracts, and a fluid — the blood serum — separates. Parasites are usually included in the coagulum as well as the more solid ele- ments of the blood, such as the corpuscles. Blood Plasma. — For certain purposes it is desirable that the blood should be kept fluid and coagulation prevented. This is requisite when we wish to inject blood containing living parasites, such as filariae, or try- panosomes, &c., or to obtain certain constituents of the blood, such as the white blood corpuscles. If the blood is allowed to coagulate, parasites contained in it are usually entangled in the blood clot. To prevent this, the blood should be quickly mixed with a lo per cent, solution of citrate of sodium. One part of this solu- tion, if rapidly and thoroughly mixed, will prevent the coagulation of twenty-five parts of blood. Twice the quantity of a 5 per cent, solution is somewhat easier to work with and equally effective — others use a normal citrate of soda solution, a much larger quantity being required. In these mixtures the blood corpuscles are not destroyed. To obtain blood in quantity such as is required for injecting into animals, it is best to plunge a hypodermic needle into a distended vein. The median basilic or BLOOD PLASMA I 35 median cephalic will be found most convenient. An all-glass syringe should be used. No powerful suction is required ; if the point of the needle is in the vein, by holding the syringe horizontally very little suction is necessary. When possible injections should be made at once with unmixed blood, if this is not possible the blood should be citrated. To study the characters and special properties of either the red or white Corpuscles, coagulation must be pre- vented in the same way and the corpuscles rapidly separated by centrifugalising the blood. Such corpuscles may then be transferred by means of a pipette to normal saline solution, and will retain their properties for a considerable time. Coagulation Time. — Blood varies greatly in the rapidity and firmness with which it coagulates, and the time re- quired is influenced by various diseases. Methods of estimating the coagulation time for clinical purposes are not very satisfactory, and all determinations must be made at a constant temperature. Wright's method is to draw up blood into a series of capillary tubes of uniform calibre, and attempt, by blow- ing at intervals of half a minute, to dislodge the blood. When it cannot be dislodged it has coagulated, and the time it has taken is the coagulation time. This simple > method appears to give as good results as any. Specific Gravity. — The specific gravity of the blood is another variable element, and is not easily determined accurately with the small amounts of blood that can be used for clinical purposes. Blood is dropped into a series of fluids of known specific gravity varying from 1035 to 1068, and the specific gravity of the fluid in which the blood neither sinks nor rises is of the same specific gravity as the blood. The fluids chiefly used are glycerine and water, or chloroform and benzol in varying proportions. Chemical Reaction. — The reaction of the blood can be determined either by using glazed htmus paper previously 136 BLOOD soaked in chloride of sodium solution, or a plaster of Paris disc soaked in neutral litmus solution. Spectroscopic Examination. — In addition to haemoglobin we may have in the blood in some cases derivatives or modifications of haemoglobin. A small direct vision spectroscope is the most satisfactory method of deter- mining the presence of these substances. The blood should be laked by the addition of distilled water to render it sufficiently translucent. If it be desired to determine the presence or absence of haemoglobin from the serum another specimen of the blood should be allowed to coagulate, and when the serum has separated that should be examined separately. The diluted blood should be placed in a small vessel with two plane sides inclined towards each other at an acute angle, so that varying thicknesses of the fluid can be examined. Either ordinary daylight or a lamp can be used, and the spectrum should first be focussed as sharply as possible and the slit closed as much as is convenient to bring out Fraunhofer's lines distinctly. The spectra of oxyhaemoglobin and reduced haemo- globin can be readily obtained from the same specimen, either by shaking up with air to oxidise or reducing by the addition of ammonium sulphide (fig. 52, i and 2). Methaemoglobin gives two additional lines, as seen in the diagram, and the two lines between D and E are further apart and faint ; on the addition of alkali the spectrum changes and becomes more like that of oxy- haemoglobin (fig. 52, 4 and 5). Methaemoglobin is of considerable importance, as it colours the urine brown and not red. If no spectroscopic examination is made it will usually be overlooked. In some of the mildest cases of " Blackwater Fever " methaemoglobin and not haemo- globin may be found. Urobilin is shown by the single faint band between E and F (fig. 52). Bile in human urine causes no definite banding. The opposite table gives the spectra of haemoglobin and its derivatives ; some of these are only formed under TONICITY aBC D ETj F Fig. 52. — I, Oxyhseraoglobin ; 2, reduced haemoglobin; 3, CO. hijemo- globia ; 4, methaemoglobin ; 5, methaemoglobin (after addition of alkali) ; 6, alkaline hsematin ; 7, acid bsematin (etherial solution) ; 8, hasmatopor- nVivrin ltirit{\ • o. haematoDorohvrin (alkaline! : 10. bilirubin. \ 138 TONICITY artificial circumstances and consequently are of little practical clinical value. The colouring matter of blood is haemoglobin, it forms some 90 per cent, of the red corpuscles, and is not found in blood plasma, nor, when the blood coagulates, in the serum. It can, however, be readily removed from the red corpuscles by the addition of water either to the fluid blood or to the freshly dried blood. Advantage is taken of this property when thick films are made, as in examining for filaria, in order to render a thick film transparent. To prevent the occurrence of this solution in making preparations for the examina- tion of thin films, " fixing agents," such as alcohol, per- chloride of mercury, formalin solution or vapour, or heat, are employed. Estimation of Tonicity. — Different specimens of blood vary in power of the corpuscles in retaining their haemo- globin. Though distilled water will remove the haemo- globin completely from the corpuscles in fluid blood, saline solutions over a certain strength will not remove it. The resistance or "tonicity" of the blood corpuscles is measured by the strength of saline solution, which is just sufficient to prevent the solution of the haemoglobin. Such a solution is said to be " isotonic." Normal saline solution '75 per cent, prevents the solution of haemo- globin in blood. A series of weaker solutions differing in the amount by -02 per cent, is made, and a drop of blood is dropped into each, and after shaking allowed to stand. The weakest salt solution that does not cause solution of the hemoglobin is the index of the " tonicity " of the blood used. The strength of that solution gives the isotonic strength, which is the measure of the resistance of the blood, normally 0-46 to 0-48 per cent. A less accurate but more convenient method is to mix a measured amount of the blood with a measured amount of a solution, such as a 3 per cent, solution, which is well above the isotonic TONICITY 139 strength, and add "gradually measured amounts of water till the solution of the haemoglobin takes place ; from this the strength of the solution which just causes solution can be calculated. Wright's tubes with the air and mixing chamber are convenient for the purpose. The tonicity of the blood is of considerable importance, as a decrease in the tonicity often precedes a haemolytic attack. This occurs in black- water fever, and persons whose blood is of a low degree of tonicity should not be allowed to live in countries where this disease is endemic. These methods assume that the red corpuscles in the blood are all equal as regards their tonicity, or nearly so. This is not the case, as even in healthy blood an occa- sional corpuscle will be found, that will be decolourised in a stronger solution of salt than the others, and also a few will retain their haemoglobin when all the others are decolourised. With healthy blood the great majority of the corpuscles may be equal, but with blood in some diseases, a much larger degree of red corpuscles are of a lower degree of tonicity than the average. To determine the range of variation, of tonicity in the corpuscles the blood should be first well diluted with a strong salt solution, 4 per cent., which is hypertonic to all corpuscles. The mixing can be done in one of Wright's tubes. The tube is allowed to stand and the corpuscles will fall to the bottom, and by heating the air chamber can then be expelled into a clean watch glass. Hanging drop preparations of this blood, diluted with one, three, or seven parts of distilled water, will be equiva- lent to 2 per cent, and i per cent, and -5 of salt solution. An examination of these hanging drops will show if any considerable proportion of the corpuscles have lost their haemoglobin. If none or very few are decolourised with the I per cent, salt solution the remainder of the red corpuscles in the watch glass in 4 per cent, salt solution should be diluted with three parts of distilled water in 140 TONICITY one of Wright's tubes and well mixed in the mixing chamber. The fluid can be expelled into a clean watch glass and a series of dilutions, as hanging drops, made. One part diluted with one of water will give "5 per cent., with two of water •33 per cent. Two parts of the diluted blood with one of water will give •66 per cent., and so on. In this manner, by examining a series of these hanging drops, and determining the proportion of the "shadow" corpuscles which can be easily seen with an oil immersion if the light is cut off, the proportion of corpuscles of lower index of tonicity than represented by these solutions can be determined. When the haemoglobin is dissolved in the serum the blood is said to be "laked." Dissolved haemoglobin is found in the serum in acute haemolytic processes, but appears to be rapidly removed either by the hepatic or renal cells, converted by the liver into bilirubin, or deposited in the subcutaneous tissues. The yellow tint of skin and conjunctiva in some diseases which simulate jaundice is of this nature, and is called haematogenous jaundice. The yellow tinge round old bruises is due to the solution of the haemoglobin of the extravasated blood. Normal blood serum is " hypertonic," that is, not only is it sufficient to prevent the solution of haemoglobin from the red corpuscle, or is isotonic, but is considerably above that strength. This excess of tonic value is not simply due to the amount of salts ; it varies consider- ably and is estimated by determining the dilution with water required to render it isotonic as regards normal red corpuscles. This can be determined either by using a series of dilutions and dropping (with distilled water, or as a series of hanging drops in Wright's tubes) blood into each, or by diluting to a known extent a mixture of blood and serum. Blood Serum. — Blood serum is required for several purposes, notably for the demonstration of the presence or absence of specific agglutinins. SERUM 141 The glass tubes devised by Wright afford a ready method of obtaining and diluting serum. The simplest form is to draw out a piece of glass tubing of about one- quarter inch diameter. In drawing out the tube it is well to rotate it in the flame until it is quite soft at the required place, then removing it from the flame, pull steadily. In this way tubes of more uniform size are obtained than if the traction be exercised while the tube is still in the flame. The thin tube thus formed should be broken, and at a convenient distance another portion of the tube should be heated and pulled out in the same manner, or bent as shown in fig. 53.* One of the capillary extremities should be sealed. A puncture with a broad needle or small knife should be made in the skin and the upper half of the unexpanded tube, that towards the sealed end should be heated in the flame of a spirit lamp, which is lighted and placed close at hand before the skin is punctured. Holding the lower part of the tube which has not been drawn between the fingers to make sure that it is not too hot, the open drawn-out end is placed in the exuding blood. As the air in the tube cools and con- tracts the blood will be drawn up into the tube. If there is not enough or the blood is not drawn entirely up into the thick part of the tube by the time it is cool, the sealed * We are indebted to the kindness of the proprietors of the Lancet for the use of fig. 53. 142 SERUM end can be broken off and the upper end of the thick tubing again heated and the same end again sealed. The contraction of the air will be sufficient to draw more blood up, and the blood already in the tube higher up. When sufficient blood is in the tube and the tube is cool, the lower end through which the blood entered can be sealed. The tube is now placed on its side, horizontally, till the blood coagulates, and is then placed vertically, so that as the serum is expressed by the contraction of the clot it will run down into the narrow part of the tube. In this way clear serum, free from blood corpuscles, can be obtained without using a centrifuge. Capillary vac- cination tubes can be used to collect the blood, but will require to be centrifugalised to obtain clear serum. _/^ Fig. 54. Serum, however prepared, requires dilution for most purposes, and the degree of dilution is important. The graduated pipette of a haemocytometer may be used for this purpose. A very convenient method of obtaining any degree of dilution is by Wright's tubes. A piece of glass tubing is drawn out sharply in the middle so as to make a short, sharp qonstriction. About an Inch and a half from this constriction on each side the tubing is drawn out into a long, thin capillary tube. One of these is broken off and sealed, and the other, preferably the more uniform and thicker, is touched with a file, broken off square and left open. The part above the middle constriction with the sealed capillary tube attached is called the air chamber, and that below in connection with the open capillary tube is called the mixing chamber. A narrow mai'k is made on the open capillary tube with a grease pencil about an inch or less from the open end. This distance depends on the calibre of the tube, the DILUTION OF SERUM I43 greater this is the shorter the distance from the open end to the mark, as the volume of the column of fluid between the open end and the mark is the unit of measurement. The finger or ear is pricked, and from the blood obtained the serum is allowed to separate as above and blown out into a sterile watch, glass. The air chamber is then well heated, and the open end of the tube is placed in the blood serum till the serum runs up to the mark. The tube is then removed from the serum and a little air enters the tube as the air in the hot air chamber contracts, the open end is then placed in the diluting fluid and withdrawn as soon as the fluid reaches the grease pencil mark ; it is then withdrawn, but as soon as air has entered the tube it can be replaced in the fluid and again withdrawn when the mark is reached. This can be repeated as long as the air in the air chamber contracts. If repeated nine times there will be nine parts of the diluent to one of the blood serum. As the fluid by con- traction of the air in the air chamber is all drawn up into the mixing chamber, it can there be well mixed by rapidly rotating between the palms of the hands. This procedure would give a dilution of i in 10, and by continuing the process greater dilutions would be obtained ; but it is better, if high dilutions are required, such as I in 100 or i in 1,600, after well mixing the serum and diluent to expel a part of it into a sterilised watch glass by heating the air chamber. The expelled diluted serum is further diluted in a second tube in exactly the same manner as the first dilution, and from this a third, and in turn a fourth, dilution can be made. The tubes with the diluted serum may be sealed up and kept for some time if necessary. If it be desired, a known amount of a broth culture of an organism can be in the same manner drawn up into the mixing chamber and there mixed with diluted or undiluted serum. Wright uses india-rubber teats to draw up the fluid {vide fig. 55), but as in the Tropics india-rubber does not 144 SERUM keep well, the air chamber drawn out into a long capillary tube is more satisfactory. Wright, when using an air chamber, blows it out into a bulb so as to have a larger volume of air, but a smaller air chamber is sufficient in most cases, and if it cools too rapidly, so that the air ceases to contract, the sealed end may be broken off, and whilst the tube is still open the air chamber can be heated and the tube again quickly sealed. This can be repeated as often as one wishes if a large volume of serum or blood is required in the mixing chamber. Fig. 55. Instead of serum the blood itself can be mixed with a diluent in a similar manner, and the diluted blood used for counting leucocytes or red corpuscles. It is necessary that the diluent should be one that will prevent coagula- tion and will not cause destruction of the red corpuscles. Gower's solution is fairly satisfactory, or if it be desired to stain the leucocytes, Toisson's fluid may be used. The mixing for uniform and successful results must be done quickly, otherwise part of the blood may coagulate or the corpuscles adhere together in masses. In addition to agglutinins other substances may be formed in serum, as a result of infection by micro- organisms. These include the toxins and antitoxins, i.e., the poisonous products of the growth of organisms and substances that neutralise these poisonous products ; also haemolysins or substances that have a destructive action on blood corpuscles. Precipitins. — A class of substances which promise to be of much practical importance are the precipitins. It is found that if blood of one animal, as, for instance, man, be repeatedly injected into a rabbit, the constitutional disturbance set up by the injections becomes less and PRECIPITINS 145 less, and after a few injections they cease to cause any disturbance. It is further found that the blood serum of this rabbit, immunised as to human blood, will give a precipitate when added to a solution of human serum or of closely related animals, such as the ape, when much diluted, but not with solutions of serum of other animals, such as the Rodentia. Similarly, if a rabbit be immunised by repeated injec- tions of the blood of any animal of a different genus, horse, rat, pig, &c., the serum of the rabbit will give a precipitate with solutions of the serum of the horse, rat, and pig respectively, or animals closely related to them. This gives a new means of grouping animals arid pro- mises to be of practical and medico-legal value. The immunised or test sera can only be made where proper appliances are available, but appear to keep well. The application of the test is easy. Clean white filter paper is soaked in the fresh uncoagulated blood of the animal and allowed to dry in the air. A portion of this paper is treated with normal saline solution. The clear solution thus obtained is placed in a small test tube (Durham's tubes are suitable) and a few drops of the test serum are added. A precipitate indicates that the blood was either that of the animal against whose blood the rabbit was immunised or a closely related one. Dr. Nuttall has made observations on a large series of animals, and the results obtained have been con- sistent, and in many ways have thrown light on the relationship of different animals. The filter papers, soaked in blood and well dried in the air, keep well and give the reaction after many months. It is im- portant that blood should be obtained from any rare animal and examined in this way to aid in its classifica- tion. The filter paper or white blotting paper should be soaked in the blood and clots removed. The blood must be fresh. The paper should then be allowed to thoroughly dry and, if possible, wrapped in wax paper and sent to England to be tested with the prepared sera. 146 OPSONINS Culture Medium. — Blood serum makes an excellent cul- ture medium for many organisms, and some will not grow, or only grow with difficulty, in more artificial media. For the cultivation of trypanosomes the blood serum obtained after the blood has been " laked " is the most satisfactory medium. Such a fluid is a solution of haemo- globin in diluted blood serum. Opsonins. — Another property of blood serum is tWe power it possesses of acting on pathogenic micro- organisms, and so altering them that they can be taken up and destroyed by the leucocytes. The substances to which the blood serum owes this property have been named by their discoverer, Wright, opsonins (Latin opsone, I prepare food for). Wright believes that the leucocyte is a constant factor in the phenomena of phagocytosis, and that the variable and most important factor is the amount of opsonin in the blood serum. It is further con- sidered probable that these opsonins have a high degree of specificity, that is to say, that there is a corresponding opsonin for each organism ; that for the tubercle bacillus, for example, has no action on staphylococci and vice versa. In order to estimate the amount of opsonin present in a blood serum for a given organism there is required: — (i) The serum to be tested. (2) The serum of a healthy person to be used as a control. (3) An emulsion of the given organism in normal saline solution. This emulsion may contain either living or dead organisms, and they should be present in such numbers as to admit of their being readily counted in the leucocytes. (4) Leucocytes washed free from plasma. To obtain these allow about i cc. of blood from a healthy person to drop into 10 cc. of normal' saline solution containing i^ per cent, of sodium citrate. This mixture is now centrifugalised until the corpuscles have OPSONINS 147 fallen to the bottom. The clear supernatant fluid is then pipetted off and replaced by normal saline solution. After further centrifugalisation the supernatant fluid is again removed, and the leucocytes, which form a creamy layer on top of the red blood cells, are ready for use. (5) Two pipettes prepared as described on p. 144 (fig. 55). As described under serum dilutions, draw up equal parts of (i) emulsion of organisms, (2) emulsion of leuco- cytes, (3) serum to be tested. Expel these on to a glass slide and mix thoroughly. Afterwards draw the moisture into the pipette and seal off the tip. Remove the rubber teat and place the pipette in an incubator, or in a vessel of water at 37° C, carefully noting the exact time. Now prepare a second pipette in the same way, using this time the normal serum instead of the serum to be tested. Place this under precisely the same temperature conditions. At the end of fifteen minutes in the case of each pipette expel the contents on a slide and prepare smears. The smears are then fixed and stained by a method suitable for the demonstration of the organisms. The stained smear should then be examined with a lens of lower power, and a part selected where leucocytes are present in greatest numbers. On examination with the oil immersion lens, it will be found that organisms have been taken up by the poly- morphonuclear leucocytes. The number of organisms in eighty such leucocytes should be counted, in the one case where the serum to be tested was used, and also in the other case where the control serum was used. The ratio between these two figures gives the opsonic index. For example, if in the smear prepared from the mixture in which the serum to be tested was used, it was found that 80 polymorphonuclear leucocytes contained 95 organisms, and in the other smear the same number of polymorphonuclear leucocytes contained 190 organ- isms, then the opsonic index of the serum under examina- tion for that organism is -^^ — "5. 148 CHAPTER IX. ARTHROPODA — INSECTA. With the progress of the enquiry into the causation of disease it has come to be of importance that the investigator of tropical diseases should possess some knowledge of the blood-sucking flies, mosquitoes, ticks, fleas, &c., which are known or suspected to be con- cerned in the transmission of various infections. A brief account is here given of the more important members of the zoological division Arthropoda, to which these carriers of disease belong. The Arthropoda are bilaterally symmetrical segmented animals with a thick chitinous cuticle. To some of the segments are attached articulated ambulatory limbs — it is from this feature that the name of the phylum was derived. The segments vary in number, and in some arthropods the segmentation is not obvious externally, but is inferred from the arrangement of internal structures, such as nerve ganglia. The cephalic segments, three in number, are always fused into one mass, the head — the cephalic appendages are modified for purposes of mastication or suction and sensation. The three or four segments behind the head are often fused more or less completely to form the thorax, which may be united to the head to form the cephalo-thorax. To the thorax are attached the legs. The abdominal segments are usually distinct, but may be fused and united to the cephalo-thorax, as in some of the Arachnoidea. The abdominal appendages are essentially those connected with excretion and repro- ARTHROPODA 149 duction, but may be so modified as to form "stings," or weapons of offence and defence. The sub-kingdom or phylum Arthropoda is divided into five groups : — (i) Prototracheata. (2) Myriapoda (centipedes, millipedes, &c.). (3) Insecta (horse-flies, butterflies, mosquitoes, &c.). (4) Arachnoidea (spiders, ticks, &c.). (5) Crustacea (cyclops, &c.). The Insecta and Arachnoidea include the most im- portant carriers of disease, and will be considered first. Class Insecta or Hexapoda. — The term Insecta was originally employed to embrace all those animals whose body is externally divided into segments, including butter- flies, beetles, centipedes, scorpions, &c. It is now used in a much more restricted sense to apply to such arthro- pods as have six walking-legs. The Insecta have the body distinctly divided into three regions — head, thorax and abdomen. They take in air by means of tracheae, a system of tubes ramifying throughout the body and opening externally by means of orifices placed in pairs at the sides of the body. The appendages forming the mouth-parts are paired, and consist of mandibles, maxillae and labium, the pair in this latter part being combined to form a single body. To the head are also attached a pair of antennae. There are two pairs of wings, but one or both of these may be rudimentary or modified for purposes other than flight : sometimes even rudimentary wings appear to be absent. The wings are always placed on the thorax, and to this region also are attached the three pairs of legs. Insects in many of the orders undergo a variety of changes of form in the course of their development. In the system here adopted the insects are divided into nine great groups or Orders, the characteristics of which we give in brief. By "mandibulate mouth" is meant one in which the mandibles or maxillae, or both, are fitted for biting, crushing or grasping food. The 150 INSECTA term "suctorial" implies that some of the mouth-parts are modified to form a tubular suctorial apparatus ; this is frequently protected by a modification of other parts, which act as a sheath. (i) Aptera. — Wingless insects. All wingless insects do not, however, belong to this order. Mouth mandibulate, or very imperfectly suctorial. Two sub-orders — (i.) Thysanura ; (ii.) Collembola. (2) Orthoptera. — Four wings, the front pair being leather- like, and usually smaller than the hind pair, which are of a more delicate structure and contract after the manner of a fan. Mouth mandibulate. This order includes earwigs, cockroaches, grasshoppers, crickets, &c. (3) Neuroptera. — Two pairs of membranous wings, frequently with much network ; the front pair similar to the hind ; the latter with little or no fanlike action in closing. Mouth mandibulate. This order includes dragon-flies, may-flies, termites, &c. There are some parasitic wingless forms, such as "bird- lice." Adult dragon-flies are destructive to other insects, and their larvae are very destructive to aquatic larvae, such as those of mosquitoes. Attention to the breeding of some species of dragon-flies, particularly of the Agrionidce, is important, as these breed in places similar to those in which larvae of Culicidae are usually found. The larvae of AgrionidcK have short rounded bodies. Their mouth-parts are modified to form a protrusible mask with which they seize their prey. These larvae lurk at the bottoms of pools, puddles and streams, and are the most important of the natural enemies of mos- quito larvae. The adult Agrionidce can be distinguished by the peculiar position of the wings, which are always inclined backwards, never at right angles to the body as in other dragon-flies. (4) Hymenoptera. — Two pairs of membranous wings, the front pair larger than the hind, which are always small, and do not fold up in repose. Mouth mandibulate, sometimes also provided with a tubular proboscis. INSECTA 151 This order includes bees, wasps, ants, &c. (5) Coleoptera.—Tv!0 pairs of wings, the front pair shell-hke and forming cases which meet, together over the back, so as to lose entirely the appearance of wings and to conceal the delicate membranous hind pair. Mouth mandibulate. This order includes the beetles. (6) Lepidoptera. — Two pairs of large wings covered with scales. Mouth suctorial. This order includes butterflies and moths. (7) Diptera. — One pair of membranous wings. Mouth suctorial, but varying greatly, sometimes penetrating as well as suctorial. (8) Thysanoptera. — Two pairs of very narrow fringed wings. Mouth imperfectly suctorial. This order includes thrips. (9) Hemiptera. — Two pairs of wings, the front pair either leather-like, with membranous apex, or entirely parchment-like or membranous. Mouth perfectly suc- torial, and modified into a definite beak, which is bent so as to be flattened out under the head and thorax. This order includes bed-bugs, scale-insects, lice, &c. Metamorphosis. — Conspicuous changes after birth, or metamorphosis, is one of the most striking phenomena of insect life. The more highly specialised insects, such as Lepi- doptera, Diptera, Hymenoptera, Coleoptera, and most Neuroptera, undergo profound changes of form known as indirect or complete metamorphosis. For example, the egg of a mosquito or house-fly produces a larva. The larva feeds and grows, casting its skin from time to time, but not changing markedly in form till it becomes a pupa. From this pupa, in which internal development only takes place and no food is taken, the imago or adult sexual form emerges. Thus these insects, at dififerent stages in their life-history, assume very dissimilar forms. In other less highly specialised insects, such as Orthoptera and Hemiptera, the imago or adult resembles 152 INSECTA in external form the young at birth, except that the latter is devoid of wings and mature sexual organs. This is known as direct or incomplete metamorphosis. As there is no distinction between larva and pupa, it is usual to employ the term " nymph " to describe the stage between egg and imago in such insects. In cases of complete metamorphosis, it is usually possible to determine the order to which a larva belongs, though the actual form of the larvas and adults is so dissimilar. Most dipterous larvas have no legs ; they may be aquatic, as in the case of mosquitoes, or head- less maggots, as in those of the Muscidze. Coleopterous larv^ have three pairs of legs, whether they are like maggots, worms, or wingless insects. Lepidopterous larvae have not only three pairs of legs, but also supple- mentary legs or pseudopoda on the abdominal segments ; these differ in appearance of the true legs and are not jointed. 153 CHAPTER X. DiPTERA. Many members of the order Diptera are known to be concerned in the causation of diseases of man and of animals. Diptera may be harmful to man and animals in a variety of ways, by biting, by living as parasites especially in the larval stage, either internally or externally, and by carrying disease germs either as direct agents or as intermediate hosts for parasites. (i) Certain species are noted for their virulent bite ; such insects apparently secrete a poisonous or irritating saliva. Whether the virulence of flies' saliva varies at different times is not known ; the dissimilar effects pro- duced by particular species upon man at different periods and upon different individuals may be due to varying susceptibility. There is no doubt, however, that at certain times biting insects are more venomous than at others. Diptera feed both by night and by day ; as a rule each species, often each family, has its particular feeding time. The gadflies (Tabanidce) , for instance, only feed during the day ; Culicina usually feed at night, but some species are day-feeders, and some may feed either by day or by night; Anophelina chiefly but not exclusively by night. Fleas, or Pulicidce, are almost exclusively nocturnal. (2) Internal Parasitism is fairly common in this order of insects, and man may be the host. Human dipterous parasites are nearly always found as such in their larval state. There are some notable exceptions in which the 154 DIPTERA adult is the parasite, as the Jigger Flea (Sarcopsylla penetrans). The larvae of diptera parasitic in man and in animals produce what is technically called Myiasis. These parasitic larvae may be situated internally (internal myiasis) or externally, or under the skin (cutaneous myiasis). Grubs such as the horse-bots, or larvae of Gas- trophilus equi, may live and develop in the stomach and intestines of the horse, the horse forming a definite host. Grubs may exist in the intestines of man, as the Antho- tnyia larvae, by chance occurrence and not normally. There are no known dipterous larvae which live and develop only in man's intestines. Cases of internal myiasis in animals are common, in man rare. Cutaneous myiasis is much more common in man. The eggs of various diptera are deposited on sores and wounds, and the grubs feed in such places {Lucilia and Calliphora), or the larvae may live under the skin {Der- matobia), or even penetrate the organs of sight. External myiasis refers not only to the skin but includes cases of insect invasion of the external openings of the body, such as in the nose, orbits, ears, vagina, rectum, &c. (Screw-worm, Compsomyia macellaria). Cases of internal myiasis require the most careful in- vestigation, as diptera may deposit not only eggs but living young on fasces directly they are voided, and these maggots may be thought to have been passed per anum. There are, however, well-authenticated cases of internal myiasis, even in England. (3) Diptera often feed indiscriminately upon man and animals. In this way a biting fly may carry germs of some disease from animal to man, such, for instance, as anthrax, or from man himself to a fellow-creature. Another source of infection of disease in man in which diptera play a prominent part is not due to biting diptera alone, but to germs being carried from faecal matter in latrines, &c., by all kinds of carrion and foul-feeding flies, to man's food and drink (typhoid fever, &c.). An important fole is played by diptera as definitive DIPTERA 155 hosts of human parasites, such as the malaria parasites, and as intermediate hosts for the Filarice. Order Diptera. — Flies with the anterior pair of wings membranous, except in the cases of fleas and certain ^^%^^^ Fig. 56. — Antennae, i, Culicid ; 2, Simulid ; 3, Asilidse; 4, Hsematopota ; 5, Tabanid ; 6, Muscid. other parasitic forms, as the sheep " tick," which are wingless. The posterior pair of wings is transformed into a pair of club-shaped processes, the halteres or balancers. The head, thorax and abdomen are distinct. The head is very variable in shape. There are usually 156 ANTENNiE two large compound eyes, and ocelli may be present. The antennae are very variable and present important characters which are of importance in classification. Certain families have antennae which consist of a number of segments approximately similar to one another and arranged in a linear manner (fig. 56, i and 2). The number of the segments in this division varies in the different families, from eight to sixteen. Diptera having this form of antennas are called Nemocera or Nematocera. The majority of insects popularly known as "flies" have antennae of another form, namely, three segments, the third of which is of different form according to genus or species, and bears on its front a fine projecting bristle frequently feathered, the arista (fig. 56, 6). Between the two forms of antennae described there exists a variety of intermediate forms. In these latter there are one to three segments, and a terminal appendage, which is fre- quently annulated (fig. 56, 5, Tabanidce), or may be hairlike (fig. 56, 3, Asilidce) . Flies with these forms of antennae are called Brachycerous. Exceptional forms of antennae are found in the parasitic flies of the series Pupipara. The mouth is suctorial, and in some the parts are adapted for piercing. The normal mouth-parts are : — (i) The labrum or upper lip. (2) The mandibles. (3) The maxillae. (4) The labium or lower lip. (5) The hypopharynx. Jointed appendages, the maxillary palpi, are also present ; the labial palps are represented by the labellae. The form, and sometimes the function of each part, varies in each group. The labium in many species is more or less fleshy and acts as a sheathing organ ; the labrum and hypopharynx are often much elongated, and together may form a more or less perfect tube. The space between the eyes is called the vertex, that part in front of the eyes the frons, the part behind the occiput ; the sides the gence, or cheeks ; and the mouth parts arise from a projection in front, the clypeus. MOUTH PARTS 157 Many diptera have a peculiar structure in the form of a vesicle on the head called a " ptilinum." This is a bladder-like expansion in front of the head, which appears as the fly emerges from the pupa. It serves to rupture the hard shell in which the fly is enclosed. This ptilinum becomes completely inverted in the mature fly, being represented externally by a space, the " lunula," under an arched suture, extending over the point of insertion of the antennae. Fig. 57. — Mouth of an Empis. a. Lower lip or labium ; *, labella ; c, stylets or maxillas ; d, hypopharynx ; f, upper lip or labrum ; g, maxillary palp. (After Meinert.) The head is joined to the thorax by a narrow neck ; the back of the head is called the na'pe. The thorax may have all three segments distinct, or the pro- and meso- thorax may fuse ; the former is usually small, the latter large ; the metathorax, more commonly known as the metanotum, is small. The prothorax is most pronounced in the Nematocera and forms either two prothoracic lobes or a narrow collar ; a portion of the mesothorax is cut off behind by a depressed line, forming the scutellum; a transverse suture may sometimes be seen on the meso- thorax running across from the base of the wings, and there is also a prominent groove above the root of the wings, along which there are often characteristic bristles. The pleura or sides are built up of several pieces, and lie below the meso- and metanotum. The wings have a variable number of veins, which are IS8 WINGS both longitudinal and transverse. The figure given here is of a Daddy-long-legs (Tipula) . In the centre will be seen a space surrounded by veins — the discal cell (fig. 58, 9). On the fourth longitudinal vein that bounds this cell in front will be seen a shor* connecting vein — the anterior cross-vein ; this always connects the fourth longitudinal vein behind with the third in front, and the cell behind is always the discal cell (9) ; between the second and third longitudinal veins are the marginal cells. The other cells are shown in the figure. Fig. 58. — Wing of Tipula. a, Costal vein ; b, mediastinal vein ; c, first longitudinal vein ; d, second longitudinal vein ; e, third longitudinal vein ; f, fourth longitudinal vein ; g, fifth longitudinal vein ; h, sixth longitudinal vein ; i, seventh longitudinal vein. I and 2, Mediastinal cells ; 3 and 4, sub-marginal cells ; 5, anterior basal ; 6, posterior basal ; 7, anal ; 8, posterior marginal; 9, discal cell. (After Loew.) The longitudinal veins ar© known as follows : — The costal (a) ; Auxiliary, mediastinal, or subcostal (6) ; First longitudinal vein (c) ; Second longitudinal vein or radial (rf) ; Third longitudinal vein or cubital (e) ; Fourth longitudinal vein or discoidal (/) ; Fifth longitudinal vein or postical {g) ; Sixth longitudinal vein or anal Qi) ; Seventh longitudinal vein or axillar rib {i). On the hind margin of the wing near the base there is often a more or less free lobe, the "alula," and still nearer the base or placed on the sides of the body two HALTERES 159 other lobes, the one nearer the alula called the " anti- squama," the other the " squama." The halteres or balancers may be hidden by the squamae, as in certain Muscidce. In such cases the fly is said to be calyptrate. Fig. 59. — Base of wing, calyptrate diptera. c, Haltere hidden by (a) squama. Fig. 60. — Puparium of a "Screw-worm" (enlarged). The legs are attached to pro- meso- and meta-thorax ; they usually terminate in ungues or hooks, and pulvilli at the base of the ungues, in the form of two pad-like fleshy l6o CLASSIFICATION cushions, but the latter may be absent. In certain flies we find between them the empodium, a median appendage in the form of a pad, bristle, or spine. The abdomen is composed of nine segments, but they are not as a rule all shown. The male genitalia or hypo- pygium is of importance for differentiation of genera and species. All parts of the body may bear bristles (chcetce) which are important in classification (chcetotaxy). The larvae of all diptera have neither true nor false legs, and may or may not have a distinct head. The pupae may be either naked or enclosed in the hardened larval skin or puparium. The production of living young occurs in some groups. In the forest-flies and sheep-ticks, or " keds," the young may be born as fully matured larvae in a puparium case. Classification of Diptera. A satisfactory classification of Diptera is not yet agreed upon by entomologists. At one time it was suggested that the order should be divided into two great groups, Nemocera and Brachycera, according to the structure of the antennae, as described above. Later Brauer pro- posed a classification based mainly on development as shown by larval and pupal characters, into two sub- orders, Orthorrhapha (straight^seamed) and Cyclorrh'apha (circular-seamed). The characters of these groups were defined as follows : — Sub-order i. Orthorrhapha. — Larva with a distinct head; pupa either free or encased in the larval skin (puparium) ; the pupal skin always bursts, for the exit of the pupa or imago, in a T-shaped opening on the back of the anterior end, rarely in a transverse slit between the eighth and ninth segments. Imago without the frontal lunula and ptilinum. Sub-order 2. Cyclorrhapha. — Larva without any distinct head ; pupa always in a puparium (fig. 60) ; CLASSIFICATION OF DIPTERA l6l and (fig. 79, a) ; imago always escapes via a more or less cirqular opening at the anterior end (fig. 79, c). Frontal lunula always present ; ptilinum usually so. It is proposed to adopt here a classification in which the Diptera are divided into six groups, as follows : — (i) Orthorrhapha nemocera. — Antennze with more than six segments, not terminated by an arista ; the segments of the flagellum, i.e., the part beyond the two basal segments, more or less alike. Palpi slender, four- or five-jointed. In this group are included the families Culicidce (mosquitoes), Chironomidce (midges), Psychodidce (owl- midges), Simnlidce (sand-flies), &c. (2) Orthorrhapha brachycera. — Antennae variable, but never resembling those of the Nemocera, as the joints differ markedly from each other, nor like those of Cyclorrhapha. Arista, if present, usually terminal ; if not present flagellum terminates as an appendage con- sisting of a number of indistinctly separated segments. Palpi only one- or two-jointed. In this group are included the families Tabanidce (gad- flies), AsilidcE (robber-flies), &c. (3) Cyclorrhapha aschiza. — Antennae composed of not more than three joints and an arista ; the latter not usually terminal, the front of the head without any definite arched suture over the antennae, but frequently with a minute area of different colour or texture there. Syrphidce, or hover-flies, Conopidce, Pipunculidce, Phoridce and Platypezidce. (4) Cyclorrhapha schizophora. — The antennae consists of three joints and an arista, which is usually dorsal. In certain members of this group the halteres are not covered by the squamae, hence they are said to be acalyptrate. A group of flies, the Muscidce acalyptratce belong to tliis class. The. calyptrates in this group include the families Anthomyiidce, Tachinidce, Sarcopha- gidcE, Muscidce, Oestridce, &c. (5) Pupipara. — Many of the flies belonging to this group are wingless or have wings greatly reduced in size. l62 ORTHORRHAPHA NEMOCERA The young are produced alive, full-grown, but having still to undergo a metamorphosis. In this group is included the family Hippoboscidce. (6) Aphaniptera. — Fleas are included here, as by many they are considered a sub-order of Diptera. ORTHORRHAPHA NEMOCERA. Family Cecidomyid^ (Gall Midges). — Small, slender flies with long antennae, with bead-like segments ; pro- boscis short, elongated in one genus only. Abdomen composed of eight segments. Wings usually hairy ; no alula J never more than five longitudinal veins, usually only three, the first, third and fifth ; fourth and sixth may be present. Costal vein encloses entire wing ; fifth Fig. 6i. — Wing of a Cecidomyia. vein forked ; only one basal cell. Larvae vegetable , feeders ; most produce galls. A few live as parasites in society of plant lice. Larvae with fourteen segments and possess ah "anchor process" under the head end of body. The proboscis is elongated in the genus Clinorrhyncha (Loew), and directed downwards. They are often injuri- ous to crops, but only exceptionally cause annoyance to man by biting. Family CuLiciDiE (Mosquitoes). — Proboscis elongated for piercing. Eyes renifoum ; ocelli wanting. Antennas ^ usually plumose in the male (except Sabethes, Wyeomyia, &c.). Thorax with large mesothorax, narrow scutellum, rounded metanotum. Abdomen composed of eight seg- ments. Wings (figs. 62 and 63) with six longitudinal NEMOCERA 163 veins, exclusive of the sub-costal, and two fork-cells ; veins clothed with scales ; costal vein continued round the border of the wing, fringed with scales. Head, thorax and abdomen usually but not always scaly. Palpi short or long in the female and male. The females are bloodsuckers in many species. The larvae and pupae are aquatic. This family is dealt with in more . detail in a subsequent chapter. Fig. 62. — Wing of Anopheles fiiaculifennis. Fig. 63. — Wing of a Culex. Family Blepharocerid^. — These little flies have broad wings and long legs. The proboscis is elongated, and the females in some species (Curitpira) are blood-suckers. The thorax has a distinct transverse suture. The hind legs are longer than the front ones and there are no pulvilli. The broad wings are quite bare, there is no discal cell, they are iridescent, and have a secondary set of fine network of veins. They perform aerial dances like midges, especially near the spray of waterfalls. The larvse live in rapidly running water fixed to stones by suckers. Some forms of larvae {Curupira) are composed of only six or seven segments, with widely projecting side lobes and small tracheal gills near the suckers. The pupje are flattened, inactive, and enclosed in a semi- oval shell, the anterior end having horny erect breathing tubes and suckers on the ventral surface. 164 CHIRONOMID^ Family Chironomid^ (Midges). — This family includes the majority of midges which are frequently taken for Culicida or mosquitoes. They are all small, delicate, gnat-like flies, with small head, partly concealed by the cowl-like thorax. The antennae in the female are thread- like and composed of from six to fifteen segments ; in the male they are densely plumose. Ocelli wanting or rudi- 5"tg. 64.^ — Wing of Ckironomus. Fig. 65. — A Ceratopogon. mentary. Proboscis short. The oval thorax has no transverse suture, is bare, and projects more or less over the head. The long, narrow abdomen is composed of segments and is often semi-transparent and pilose. The legs are slender and rather long and not spinose, but very hairy in some tropical species. The wings (fig. 64) are narrow, long, and bare or hairy, never scaly ; CHIRONOMID^ 165 the anterior veins darker than the rest; the sub-costal vein complete but small ; second longitudinal vein small or wanting ; third longitudinal vein sometimes forked close to its origin, the upper branch often rectangular ; fifth long vein forked, sometimes the fourth ; the costal vein always ends near the tip of the wing. Great numbers of this family occur in all parts of the world. The members of one European genus (Cerato- pogon) (fig. 65) bite severely. They often occur in swarms, dancing in the air. When at rest they wave their forelegs in the air. Ceratopogon occur in most countries. They are known as "punkies" or " no-see-um," and cause great annoyance by their bites. Many tropical genera bite severely, and on account of this and their small size are frequently misnamed " sand-flies." Fig. 66. — Wing of Ceratopogon. (After Leonard!.) The larvae of Chironomidce are mainly aquatic and worm-like, often red in cblour, and the pupae are active, and the respiratory tubes frequently plumose ; they also live in damp earth and in decaying vegetation, and in the Tropics in stumps of bamboos, pitcher plants and other open accumulations of water in plants. Those of Ceratopogon and allied genera live in the sap of trees, under fallen leaves, and in decaying vegetation, or are aquatic, and are long, slender, delicate, whitish creatures. In the genus Ceratopogon the dorsum of the thorax is not pro- duced over the head ; the palpi are four- jointed ; the wings are usually spotted (figs. 65 and 66). Family PSYCHODiDiE (Owl-midges). — Small, densely hairy, thick-set insects. Proboscis usually short, but in one European genus {Phlebotomus) it is long and horny ; l66 PSYCHODID^ palpi hairy and composed of four segments. The short abdomen is composed of six to eight segments, hairy. The legs are often short and densely hairy and the claws small. The wings are broad and when at rest lie roof- shaped over the body ; they are densely covered with long hairs and are fringed with hairs ; neuration mostly com- posed of longitudinal veins ; the first longitudinal vein near the costa, the second arises near the origin of the first and is usually twice forked, third vein simple, fourth forked, fifth, sixth and seventh usually distinct, the latter sometimes wanting. These small flies can at once be told by their moth-like appearance. They run well, but their flight Fig. 67. — Phlebotomus, sp. (From Giles' " Gnats or Mosquitoes.") is weak. Owl-midges are found frequently on windows and in out-buildings. The genus Phlebotomus and some tropical genera bite severely. The larvae live in stagnant water and decaying vegetation. They are cylindrical and have a short terminal breathing tube. The inactive pupas have two long tubular stigmata. Family SiMULiD^ (Sand-flies). Usually called sand- flies, black flies, brulots, buffalo and turkey gnats, and sometimes mosquitoes. All small, with oval thorax devoid of any suture. Cylindrical abdomen composed of seven or eight segments. The eyes are holoptic, ix., the two eyes meet in the middle line in the male, and there SIMULID^ 167 are no ocelli. The male is darker and more velvety than the female. The short antennae are composed of ten or eleven segments, the two basal ones distinct, the rest closely united. Palpi composed of four segments, the basal joint short, the next two equal, the last longer and narrowed. The legs short, thick ; femora broad and flat. Wings (fig. 68) large and broad, the anterior veins thickened, remainder delicate ; the sub-costal terminates in the costa about half the length of the wing ; first and third longitudinal veins lie close together ; fourth vein forked nearly opposite the anterior cross-vein ; forks terminate near the tip of the wing. Proboscis short with strong horny lamellae, consists of two resisting bristles for puncturing, and on its sides two maxillary four-jointed Fig. 68. — Wing of Simulium. palps. These small flies bite very severely and cause much annoyance. They especially attack the eyes, nostrils and ears of both animals and man. Sand-flies occur in all climates. The larvae are all aquatic and some live in rapidly flowing water ; they attach themselves to stones, plants, &c., and form elongated cocoons, open above. They are soft skinned, with thickened ends, a cylindrical hSad, and on the first segment a prominence with bristly hooks. The end of the abdomen is provided with appendages, by which the larvae attach themselves. The pupae have the anterior end of the body free, and from it pass out a number of thread-like breathing tubes. The flies are accused of propagating anthrax and septic diseases. Their punctures give rise to severe inflammation, which sometimes results in depilation in animals. l68 BRACHYCERA Orthorrhapha Brachycera (Antennae short). Family Tabanid^ (Gad-flies). — This family includes a number of genera, the popular names being gad-, breeze- or horse-flies, brimps and sneggs. They are mostly large Fig. 69. — Head of Tabanus. and stout ; the head (fig. 69) large ; eyes very large, contiguous in the male, the upper facets larger than the lower, usually with green and violet markings when alive. The antennae composed of three segments, third segment composed of six to eight rings ; no stylet. The Fig. 70.- -Wing of a Tabanus. proboscis prominent, often greatly elongated, fleshy, with pointed horny ^processes ; the female with six, the male with four stylets ; the former only suck blood. Palpi two jointed, the second joint large. The abdomen is broad, often flattened, never slender, composed of seven seg- ments {vide fig. 71). The legs are rather thick, mid-tibiae always with spurs; tarsi with three membranous pads at the tip. There are never any bristles. The third longitudinal vein forked. Two submarginal and five TABANID^ 169 posterior cells present ; anal cell closed at or near margin of wing. Tegulse large. Mostly large flies which occur during hot weather and have remarkable powers of flight. The bite of the female is often severe. The eggs are spindle-shaped and dark, and are laid on leaves and stems of plants, and on water plants. The larvae are carnivorous and feed upon snails, insect larvae and also roots ; elongated, composed of eleven segments, jointed, often with retractile fleshy protuberances ; the last segment has a breathing pore, or the last two segments may form a breathing tube. The pupas are free, and live in earth and water. The worst biting species are found in the following genera : Pangonia, Chrysops, Hadrus, Hcematopota, Therio- plectes, Afylotus and Tabanus. 1 There are two sections, distinguished as follows : — Hind tibiae with spurs at the tip Pangonince. Hind tibife without spurs Tabaninm. The following characters separate the above-mentioned genera.: — Pangonina;. Third joint of antennse eight ringed, the first ring slightly the longer ; the fourth posterior cell open ; proboscis often very long Pangonia. Third joint composed of five rings, the first of which is much longer than the following ; the second joint of antennse as long as the first ; wings with dark areas ; three ocelli ; brilliant eyes with purple lines and spots Chrysops. Tabanince. Third joint of antennae without or with only a rudi- mentary basal process ; thorax and abdomen with iridescent tomentum ; tibiae dilated Hadrus. Thorax and abdomen without iridescent tomentum ; front of 2 as broad as long Hcematopota. Third joint of the antennos with well-developed basal process. First antennal joint short ; body broad. Eyes pubescent, small ocelligerous tubercle present Theriophaes. Eyes pubescent, but no ocelligerous tubercle Aty lotus. Eyes bare Tabanus. p,(;_ ^l,^Tabanus dovinus. YiQ, >jz.—Hamatopota piuvialis. CHRYSOPS 171 ThePangoitia are found in woods, forests and pastures ; their flight is rapid. The proboscis may be greatly elon- gated- so that they can pierce through even thick clothes. An epizootic of anthrax in Pine Islands, New Caledonia, was traced to this genus. The genus Tabanus (fig. 49) is world-wide ; the short, thick, salient proboscis and the last joint of the antennae being annulated and notched in crescentic form and their large size render them easily Fig. 73. — Head of Hcematopota. Fig. 74. — Wing ai Hamatopota pluvialis. identifiable. The genus Hcematopota (fig. 72) has no crescentic antennal notch (fig. 73), and the wings (fig. 74) overlap ; the abdomen is also narrower than in Tabanus, and the wings have hyaline spots. The genus Chrysops can usually be told by their wings (fig. 75) being marked with dark areas and their eyes with purple lines and spots. They bite severely and usually attack round the eyes. An example of Hadrus 172 ASILID^ is the Motuca fly {H. lepidohis) of Brazil, which causes deep wounds. Family AsiLiD^ (Robber-flies). — Mostly large flies, usually more or less elongated in form, and often thickly hairy and with strong bristles. Head broad and short with a freely movable neck ; eyes separate in both sexes, Fig. 75. — Chrysops Distinciipennis. with a deep notch between. Antennae composed of three segments, the third elongated, generally simple, with or without a terminal style or bristle; style sometimes thickened and forming one or two apparent antennal joints. Proboscis firm; upper lip horny, used for piercing; labella not fleshy. Legs strong and bristly. Wings when closed lying parallel over the abdomen ; three long basal cells, two or three sub-marginal cells and five posterior cells ; third longi- LEPTID^ 173 tudinal vein forked. These flies usually feed upon insects. Some reach as much as two inches in length. The larvae live in rotten wood and in the soil, and feed upon other larvae. There do not seem to be any authentic records of these Robber-flies biting man, but some of the larger tropical species are said to do so ; Fig. 76. — Lepiis scolopacea. animals are also attacked by them. There are over 150 genera in this family. Family Leptid^. — This family includes" a number of elongated flies of moderate or large size (fig. 76). The veins of the wings distinct, not crowded anteriorly ; 174 EMPIDID^ third longitudinal vein forked, basal cells large ; five posterior cells usually present. Third joint of antennae complex or simple, with or without a terminal or dorsal arista or a terminal style. One genus only (Sympho- romyia) bites, the rest being predaceous upon insects. The section Leptince, in which the biting genus occurs, has short antenna with simple third joint, with a ter- minal or dorsal arista or a terminal style ; the proboscis is short, and some or all the tibiae have spines. The larvae live in the earth and in decaying wood, sand, water, and the nests of wood-boring beetles ; they are predaceous ; usually cylindrical, and may have fleshy abdominal legs ; the anal segment has a transverse cleft, and often two posteriorly directed processes, and two stigmata between them. The genus Symphoromyia has a single spur on the third tibiae ; the third joint of antennae kidney-shaped, and the arista nearly dorsal. Fig. 77.— Wing oi Empis. Family Empidid^. — This family is a large one and includes many genera. The flies have a piercing mouth, being all predaceous, feeding upon other insects. Prob- ably some attack man in the Tropics. They are mostly small to moderate-sized species, with small head, provided with either a short or long proboscis. The proboscis (fig- 57) consists of two stylets (c), a hypopharynx {d), and an upper (/) and lower lip (a). The antennae three jointed, the first two joints often small, third joint very variable, with or without a terminal arista or style. Abdomen of from five to seven segments ; male genitalia very promi- nent. The legs have peculiar structures, the femora CYCLORRHAPHA 175 thickened and spiny; metatarsi flattened. Neuration (fig. "J^) of wing variable; there are three or four posterior cells ; the anal cell is closed remote from the border, sometimes wanting, at other times it is closed near the border {Hilarimorpha) ; then the discal cell is wanting. Tegulae small. The larvae are cylindrical, with small ventral swellings on the mesothoracic segments; they live \n earth and amongst decaying vegetable matter. The pupae have two points at the anterior end. CYCLORRHAPHA — ASCHIZA. This section does not contain any members that bite or annoy man. The chief family, the Syrphidce, or Hover-flies, are noted for the good some of their larvae do in destroying Aphides. CYCLORRHAPHA— SCHIZOPHORA. I. MUSCID^ ACALYPTRAT^. — Mostly small flies with the antennas composed of three segments bearing a non- terminal bristle ; halteres never covered by a squama or basal scale ; nervuration of wings simple, few cells. This group contains a large number of sub-families. None annoy man to any noticeable extent. The follow- ing families are of economic importance, agriculturally and otherwise : Chloropidce, Trypetidce, Psilidce (as vegetable feeders), Scatophagidce (dung-flies). II. MusciD^ Calyptr^. — Halteres covered with a squama. Family Oestrid^ (Warble-flies) (fig. 78). — Flies of large size, thick-set, and often very hairy. Mouth small, parts rudimentary ; eyes rather small, bare. Head large ; the antennae small, composed of three segments, more or less hidden ; arista simple or plumose. Thorax broad, with distinct transverse suture. Abdomen short and thick. Legs of moderate length, the hind pair often longer than the rest. Wings with or without markings ; anal Fig. 78. — Dermatobia noxialis. Fig. 79. — Dermatobia noxialis. A and B, Larv^ ("bots"); c, puparium. (After Brauer.) SARCOPHAGID^ 1 77 cell, small discal cell, may be absent. The larvae or bots (fig. 79, A and b) are provided with circles of spines, two hooked mandibles and anal breathing pores ; parasitic. They live in three ways — (i) under the skin, (2) in nasal and pharyngeal cavities, and (3) in the alimentary canal. Man as well as animals may be attacked {Dermatobia). The larvae of Dermatobia (fig. 79) live under the skin of man, apes, cattle, dogs, &c. In the adult Dermatobia the arista is plumose on the upper side and the tarsi slender ; the proboscis is bent at the base and is con- cealed in the buccal cavity ; squamae large ; first posterior cell closed ; body hairy. Larvae club-shaped, slender posteriorly, and surrounded with rows of prickles on the borders of the segments of the apical half. The chrysalis stage is formed in a hard puparium case (C). The common species, D. noxialis (Goudot), occurs from Mexico to Brazil, and is known as the "macaw worm," " ura," " torcel," and " moyoquil worm." Family Sarcophagid^ (Flesh-flies). — Usually thick- set and of variable size. Abdomen composed of four visible segments, with bristles which are confined to the anal end, but sometimes elsewhere. Arista plumose to the middle, apex always bare. Some are metallic {Cyno- myia). Larvae feed on decaying animal and vegetable matter, and may live as parasites in the flesh of animals and in the orifices of man, also in wounds and ulcers. Those of Sarcophaga often occur in wounds in man, and are sometimes produced alive. The larvae are rounded, and thin anteriorly ; abdominal segments distinct, each with a circle of spines; mouth with two-curved mandi- bles; posterior stigmata placed in a deep cavity, and there are two pointed anal swellings. The pupa lies in a brown oval puparium. The genus Sarcophaga (Meigen) has the first posterior cell open ; the tibiae with a few bristles ; the mid and posterior cross-veins nearly in the same line. Sarcophaga carnaria, the common British flesh-fly, may be taken as an example. 1 78 MUSCID^ Cynomyia (Desvoidy) has a metallic coloured abdomen and the tibiae with short hairs. Cynomyia mortuormn is a bright blue fly about the size of a blow-fly, and, like it, lays its eggs in decaying animal matter, and may possibly do so on wounds. Sarcophila (Rondani), like others in the Sarcophagidce, are viviparous. The females deposit their larvae in wounds in animals and man. The larvae of the genus Ochromyia are also parasitic under the skin of animals and man — Cayer or Senegal fly (0. anthropophaga) . Family MusciD/E (House-flies, Tsetse-flies, &c.). — A large family, easily told from the former by the arista being plumose at the tip (now and then it is bare) ; there are no bristles on the abdomen except at the tip, and the first posterior cell is very narrow. The eyes of the $ contiguous, bare or hairy in both sexes. Abdomen composed of four visible segments. This family con- tains the house-fly {Musca), blue- and green bottle-flies {Lucilia and Calliphora), stable or " stinging flies " {Sto- moxys), horse-flies [Hoimatobia), and tsetse-flies (G/oss/n«). The larvae are variable ; some live in decaying vegetation, in decaying animal matter and faeces ; others, as the screw-worm (Chrysomyia), as parasites in animals and man : so also may Calliphora and Lucilia. The Stomoxy- ina3, which include the stable-fly, tsetse-fly and the horn- fly, have elongated, piercing probosces, and are blood- suckers. The following characters will separate the more im- portant genera : — Proboscis long, used for piercing ; palpi shorter than proboscis Stomoxys. Palpi nearly as long as proboscis Hamatobia. Proboscis very long, straight Glossina. Proboscis short, not adapted for piercing ; arista plumose on both sides ; curvature of fourth vein angular ; mid tibiae without bristles on inner side ; abdomen non-metallic ; blackish species with more or less yellowish markings Musca. STOMOXYS 179 Mid tibiae with bristles on the inner side ; abdomen, &c., with metallic colours. Thorax blackish Calliphora. Thorax black with whitish stripes, more or less metallic Chrysomyia. Thorax unicolorous, metallic Lucilia. Fig. 80. — Stomoxys. Genus Stomoxys. In the genus Stomoxys the soHd, elongate proboscis, joi-nted at an angle near its base, is the obvious char- acteristic. The type species of the genus is Stomoxys calcitrans (fig. 80) which is very like the common house- fly in general appearance. Mouth-parts in Stomoxys. — The proboscis beyond the angle projects horizontally. The palpi are very slender and scarcely one-third the length of the proboscis ; they spring from above the angle and are not in contact with the proboscis. i8o STOMOXYS The proboscis consists of three parts — labium, labrum and hypopharynx. The labium is the only part seen in the ordinary resting position. It is a solid-looking chitinous structure, ovoid in cross-section, with a narrow, shallow groove on its Fig. 8i. — Wing of Stoinoxys calcitrant. Fig. 82. — Cross-section of proboscis of Stomoxys (from Giles), h. Hypo- pharynx ; /, labium ; Irm, labrum ; vi, muscle ; t, tracheae. anterior or upper surface, within which lie the labrum and hypopharynx. The lateral walls of the labrum are incurved below to form a tube, with a slit along the lower side. This slit is closed by the apposition of the hypo- pharynx, a delicate rod within which lies the excretory tube of the salivary ducts. DISSECTION OF STOMOXYS i8l Dissection. — To expose the structures in the thorax in such flies snip off with sharp scissors the legs with a little of the ventral wall of the thorax. There will then be seen a number of vertical bundles of muscle fibre (the coxal muscles). Separate these muscle bundles in the middle line and expose the great thoracic ganglion, a sausage- shaped mass of considerable size situated opposite the first pair of legs. Clean this away, and just under its anterior end will be seen a nodule about the size of a small pin's head, this nodule is the proventriculus. Ex- tending backwards from it is a glistening tube, the mid- gut, and lying upon it three delicate tubes, the crop duct centrally placed, and at the sides the commencement of the salivary glands. The abdominal viscera can be exposed by snipping through the chitinous covering of the dorsum in a longi- tudinal direction and floating the contained structures in normal saline solution. Internal Anatomy. — The buccal cavity, which is a narrow tube, is contained in the base of the proboscis. From it the fharyn.v runs almost vertically upwards in the head, then bends sharply backwards to become the oesophagus, the latter being continued backwards as the mid-gut. At the junction of the oesophagus and mid- ..gut is situated the proventriculus, into which opens also the duct of the crop, a large hollow sac situated in the anterior part of the abdomen between the mid-gut and the salivary glands. The mid-gut runs backwards into the abdomen as a narrow tube until it reaches nearly to the posterior border of the crop, where it becomes dilated. This dilated portion has three simple coils, which lie superposed in the middle of the abdomen. The tube then gradually narrows and into this region the four Malpighian tubes open. The alimentary canal is con- tinued as a uniformly narrow tube to the rectum. The narrow lower intestine has variable bends but is not coiled. The rectum is a dilated cone-shaped cavity with its apex towards the anus ; within it are four rectal papillae. 1 82 DISSECTION OF STOMOXYS Below the dilatation the rectum is continued as a short narrow tube to the anus. The appendages of the alimen- tary canal are the Malpighian tubes, the crop and the salivary glands. The salivary glands lie in the abdomen ventral to the crop, they are continued forward into the thorax and become the salivary ducts at the anterior end of the thorax. These ducts join in the head, forming the common salivary duct which passes into the hypopharynx. Fig. 83. — Dissections of the abdomen of Stomoxys, after Lieut. Tulloch, seen from above, with the alimentary canal unravelled. CD, Common seminal duct ; D, seminal duct ; mt, Malphigian tubes ; o, junction of distal intestine and metenteron ; R, rectum ; RP, rectal papillae ; so, salivary glands ; TT, dilated ends of left Malphigian tubes ; vs, vesicular seminalis. In the genus Ha;malobia the proboscis is similar to tha|: in Stomoxys, but the palpi at once separate it. Hcema- tobia serrata, the "horn-fly" of North America, causes much annoyance to cattle and bites man, but in Great Britain it seems harmless. Genus Glossina. In the genus Glossina the proboscis is long and straight. Palpi are the same length as the proboscis and form a GLOSSINA 183 sheath for it. Arista plumose to the tip, the hairs being compound. The wings when at rest are crossed over one another "scissors hke," and project well beyond the abdomen. The wing venation is characteristic, especially in the course of the fourth longitudinal vein, which makes two bends, one before it meets the anterior transverse vein and another before it reaches the costal margin. The females of this genus produce their larvae full grown, the larvae changing to pupae at once. The larvae are deposited in the neighbourhood of decaying vegeta- tion, particularly about the roots of certain trees, such as the banana tree and others of its class. Glossina are found in belts usually in the vicinity of streams or rivers on the edge of forest land. In such situations they may be present in large numbers for a few hours, at other times none or very few can be found. Month-parts in Glossina. — The maxillary palpi project horizontally and act as ensheathing organs for the proboscis. The proboscis is expanded at the base, the remainder being proportionately very slender ; it reaches almost to the end of the palpi and is slightly curved, with the con- vexity beneath. It consists of three parts — labinm or lower lip, labrum or upper lip, and the hypopharynx. The labium consists of a large basal bulb and a terminal long, slender, chitinous rod. It is deeply grooved on its upper surface and bent upwards and inwards. The labrum is a narrow chitinous rod, the prolonga- tion of the exterior wall of the pharynx. Its edges are turned downwards and inwards, so that it forms about two-thirds of a tube. It terminates in a sharp point. The hypopharynx is a solid rod, semilunar in cross- section, with a rib running down its ventral surface, within which is the salivary canal. As will be seen from the figure, the labium and labrum together constitute a tube up which the blood passes to the pharynx. The convex dorsal surface of the hypo- pharynx fits closely to the sides of the labrum. 1 84 GLOSSINA The internal anatomy in Glossina is in general very similar to that in Stomoxys. The mid-gut is, however, longer and larger. Fig. 84. — Transverse section of the proboscis of Glossina palpalis at almost mid-length. Fig. 85. — Glossina morsitans. Synoptic Table of Species. Hind tarsi dark, or at least all the segments more or less dark (in the 2 of G. tachinoides the basal half of the first joint and the extreme bases of the other segments are usually pale). GLOSSINA 185 (i) Ground colour of abdomen ochraceous bufif, with interrupted dark brown deep transverse bands and sharply defined pale hind borders to the segments, a very conspicuous square or oblong pale area in the centre of the second segment ; small species, not exceed- ing 8 mm. in length, exclusive of proboscis, the males much smaller tachinoides. (2) Abdomen very dark, or for the most part uniformly brown, hind borders of segments if lighter extremely narrow and cinereous ; pale area in centre of second segment usually triangular, with the apex directed backwards and continued into a cinereous median stripe ; larger species. \ci) Third joint of antenna dusky brown to cinereous black. (a) Thorax, pleurae and coxae more or less uniform in colour or with stripes only palpalis. (i8) Thorax with elongated trans- verse black spots ; pleurse, coxas and femora with conspicuous black spots maculata. {b) Third joint of antennje pale (orange- buff) pallicera. II. Hind tarsi not entirely dark ; last two joints alone dark, remainder pale. A. Smaller species; length rarely reaching 11 mm., often considerably less ; wing expanse not exceeding 25 mm. (a) Last two joints of front and middle tarsi with sharply defined dark brown or black tips. Generally distinctly larger ; head wider ; front darker and nar- rower in both sexes, sides parallel in $ ; abdominal bands deeper, leaving hind margins only narrowly pale ; hypopy- gium in $ smaller, darker and more hairy ; tip of abdomen more thickly clothed laterally with short black hair, bristles on sixth segment finer and less prominent longipalpis. l86 LUCILIA Usually smaller ; head narrower ; front paler and wider ; eyes in ^ as well as in 2 distinctly converging towards vertex ; abdominal bands i less deep ; pale hind margins of segments therefore deeper ; hypopygium in the $ larger, paler, some- what more oval in outline, and clothed with fewer fine hairs ; tip of S' abdomen less hairy laterally ; bristles on the sixth segments in S stouter and more conspicuous morsitans. {b) Last two joints of front and middle tarsi without sharply defined black or brown tips ; front and middle tarsi entirely yellow, or last two joints of former faintly tipped with pale brown pallidipes. B. Large species ; length at least 1 1 mm., wing expanse at least 25 mm. Dorsum of thorax with four sharply de- fined small dark brown or oval spots arranged in a parallelogram, two in front of and two behind transverse suture ; bulb at base of proboscis brown at the tip longipennis. Dorsum of thorax without such spots, though with more or less distinct longitudinal stripes ; bulb at base of proboscis not .brown at the tip fusca. Genus Lucilia (fig. 86), the so-called green bottle-flies, have a soft proboscis (fig. 87). They are all of metallic colour and the abdomen is short and round ; the third seg- ments of the antennae are quadruple the size of the second. The ova and larvae are often deposited on wounds and ulcers in animals and man (L. sericata). This species causes the well-known " inaggot " in S'heep. Genus Chrysomyia. — This germs also contains metallic- coloured flies which differ from Lucilia in that the thorax is striped. The screw-worm fly (C. macellaria, fig. 88) is found in North and South America and the West AUCHMEROMYIA 187 Indies, but does not attack man farther north than Kansas. Genus Aiichmeromyia.— The blood-sucking larva of one species, A. luteola, has been described under the name of "The Congo Floor Maggot." The perfect in- Fig. 86. — Lucilia cmsar. Fig. 87. — Head of Lucilia casar. sect is about 11 mm. in length, of a pale yellow colour. The head is broad, equal in width to the thorax, and the proboscis lies in a deep groove. The dorsum of the thorax is flattened and marked by longitudinal dark stripes. The abdomen consists of five segments, the second being the longest and broadest. This segment is marked by a Fig. 88. — Chrysomyia macellaria. Fig. 89. — Auchmeromyia Luteola. ANTHOMYID^ 1 89 dark brown or black line down the centre and a similar line along its posterior edge. The third and fourth seg- ments are dark brown to black in colour. The fifth segment is small and contains the genitalia. The larva is dirty white in colour, about 15 mm. in length, the body comprising eleven segments. The anterior segment is conical in shape and carries the mouth, which is armed with two black booklets and paired teeth. They live under mats or in the cracks of the eai-thern floors of native huts. They feed at night. This fly has a wide distribution from Northern Algeria to Natal, and is especially common in the Congo and about Lake Chad. Family Anthomyid^. — These are mostly moderate- sized, dull-coloured flies, resembling the common house- fly. The arista is plumose, pubescent or bare. Abdo- men composed of four or five segments ; sometimes there are no bristles on the body, but they are usually present. The first posterior cell of the wings broadly open ; tegulas of considerable size. Male eyes usually contiguous. It is closely connected on one hand with the Muscidcv and on the other with the Sarcophagidce. None are metallic. The open first posterior cell is the chief character. The following genera have been connected with man either as parasites or by causing other annoy- ance, viz., Hydrotcea (Desvoidy), Homalomyia (Bouche), and Hylemyia (Desvoidy). They may be told as follows : — Eyes of 3" close together ; tegula large* ; fore femora of $ with processes, tubercles, &c., below ; arista always somewhat pubescent ; eyes bare ; black or blue- black, and pilose Hydrotaa (Desvoidy), Eyes of $ close together, bare ; tegula large ; abdomen nearly bare, unspotted: head almost composed of eyes ; antennae short, * Larger than ante-tegula. 1 90 HOMALOMYIA third joint elongated ; arista bare ; mid- legs of $■ often with peculiar structures ; black and grey Homalomyiai^oMf^'^. Arista plumose ; eyes bare ; elongated species, grey or black Hylemyia^Q^vtcAAy). The genus Homalomyia (fig. 90) has often occurred in human beings in the larval state in the intestines, being passed alive in the faeces. Most larvae in this family are vegetable feeders. They are normally slender and cylindrical, or flat and oval, with four rows of Fig. 90. — Homalomyia canicularis. thread-like processes on the segments, and have two mouth hooks. The puparium may be oval or flattened. In Homalomyia they have curious branched processes (fig. 91). The genus Hydrotcea also occurs in the larval form in human beings. The characteristic neuration is shown in fig. 92. Hylemyia larvas have also occurred in TACHINID^ PUPIPARA 191 human excreta, having been passed per anum. Some are dung frequenters and produce living young. Family Tachinid^.— Like Anthomyidce, but always bristly. Arista bare. Palpi formed of one segment. All veins of the vi^ings simple ; basal cells large ; three pos- FiG. 91. — Larva oi Homalomyia. Fig. 92. — Wing of Hydrotcea ciliata. terior cells ; first posterior cell closed or only just opened. Squamae large. Larvae parasitic in insects, especially in the larvae of certain Lepidoptera. PUPIPARA. Blood-sucking, parasitic on vertebrates (except Braula). Family HiPPOBOSCiD^. — Parasites upon birds and mammals when mature. Proboscis may be long and sharp. Palpi absent ; antennae placed in pits, composed Fig. 93. — Hippobosca equina (enlarged four times). Fig. 94. — Melophagus ovinus (enlarged twelve times). NYCTERIBID^ 193 of one segment, with or without terminal bristle or hairs. Eyes round or oval, often very small. Thorax flat, leathery ; scutellum broad and short. Abdomen leathery, inflated, no sutures visible. Legs short, strong ; claws large and dentate ; empodia distinct. Wings present or absent {vide figs. 93, 94). The larvae are born nearly matured in the puparium case, passing most of their development in the body of the parent. Of general louse-like form. This family contains the forest-fly (Hippobosca equina) (fig. 93), and the sheep ked {Melo- phagus ovinus) (fig. 94). The proboscis is composed of elongated, hard, closely applied flaps and an inner tube' between. They live on horses, cattle, and birds, and now and then attack man. Family NYCTERiBiDiE. — Found exclusively on bats. Spider-like ; no wings. Eyes and ocelli indistinct or wanting. Legs long, femora and tibiae flattened. Two other families, Braulidce and Streblidce, occur ; the former live on bees, the latter on bats. Aphaniptera (Fleas). These are parasites of fowls, dogs, rats, and human beings. The wings are reduced to mere rudiments ; the body is flattened laterally to permit their gUding about among the hairs of the host; the antennae are short and thick, and lie in pits in the head; the eyes are represented by two ocelli or may be absent. Contrary to what obtains in other Diptera, the three segments of the thorax are distinct, pro-,, meso-, and meta-thorax. Abdomen of nine segments, and in some species capable of eriormous distension. Legs powerful and adapted for jumping. The mouth-parts are piercing and suctorial. This sub-order is treated in some detail in a later chapter. 13 194 CHAPTER XI. Mosquitoes. Mosquitoes are the hosts of many parasites, and some of these are injurious to man or the lower animals. As these diseases include malaria, yellow fever, at least one of the human filariae, as well as the Proteosoma of birds and the Filaria immitis so fatal to dogs, a good working knowledge of the structure, life-history and modes of classification of these insects is required for tropical work. Mosquitoes, or Culicidce, belong to the order of dipterous insects, as they have the anterior pair of wings mem- branous, whilst the posterior pair are represented by a pair of club-shaped processes, the halteres or balancers. The insect is divided naturally into three regions : (i) head, (2) thorax, (3) abdomen. To the Jiead are attached the sensory and biting organs, consisting of two com- pound eyes, two antennae, two palpi, and a complex suctorial and piercing organ, the proboscis. To the thorax are articulated ' a pair of wings, a pair of balancers, and three pairs of legs ; whilst the abdomen is segmented and terminates in the anus and external organs of generation. In the Culicidce the head, thorax, abdomen and legs are thickly covered with scales in most of the genera, whilst on the wings, scales are found only at the edge and on the veins. The character and arrangement of the scales are important points in the differentiation of genera. The absence of scales on the wings or the presence of scales on other parts of the wings than those mentioned, or the substitution of hairs for scales, are valuable aids MOSQUITOES 195 in the differentiation of other insects from the Culicidce. The type of venation of the wings and the characters of the cephalic appendages are of value both for identifica- tion of the family Culicidce, differentiation of genera, of species, and of sexes. Scales lose their colour and become too transparent for proper examination unless the specimens are mounted dry, so that for identification of species it is advisable that the mosquitoes should be mounted dry and so arranged that all the surfaces can be examined. It is best to examine or send for examination young mos- quitoes, as in older specimens many scales are rubbed off and the insects otherwise injured. Mosquitoes hatched out from pupa should be kept alive for at least twenty-four hours, as if killed when too young they become much distorted when they dry. They should be kept in a dark place, as they then are less liable to have the scales rubbed off. The mosquitoes must be killed rapidly, and a cyanide pot is invaluable in this connection, though chloroform vapour, formalin, or even tobacco smoke, may be used. The dead mosquito must be mounted without delay, as the limbs soon lose their pliability. To mount they should be placed on their backs on a piece of cork felt. A small square or a circle of thin card should be taken, and on one side of it the date and place of capture of the mosquito should be written, with a distinguishing number if the name is not known. A fine entomological pin (No. 20) should be taken up with forceps near the paint, and the piece of card with the blank side downwards should, be placed on a piece of cork felt. The pin, still held in the forceps, should be pushed through the card. The hold on the pin should then be shifted higher, and the pin pushed still further through the card till about half of it is through. The pin, still held in the forceps, with the card transfixed on it should then be pushed through the thorax of the mosquito. On lifting- out the pin the mosquito, which 196 MOUNTING MOSQUITOES has been transfixed, will remain on the pin, and on turn- ing the card upside down the legs and wings can, with a. few touches of a clean needle, be arranged so as to be readily, visible, and will not hide any part of the back of the insect. A stout pin should then be run through /^ ^s; Fig. gS-—a, Forceps; i, pin ; c, disc; d, cork; m, mosquito ; e, large pin to carry disc. the, corner of the piece of card into the cork felt floor of the collecting box. To prevent insects attacking the specimens some powdered naphthalin enclosed in cloth should be placed in the box, or melted naphthalin may be poured, in the corners of the box. EXAMINATION OF MOSQUITOES 197 To examine such a specimen a low power, one inch or two-thirds of an inch, is required. With sucji a power the character of the scales on each part of the insect can be examined. The examination should be made by re- flected light and the insect so rotated that the part to be examined is horizontal. This can be done best by alter- ing the inclination of the large pin and using a strip of cork felt as a slide (fig. 96). In this way each part of the upper surface can be examined in succession. Fig. 96. To examine the under surface a second mosquito mounted with its back towards the card is required. The main types of scales found in the Culicidce are represented in the drawing (fig. 97).* These scales can for descriptive purpose be reduced to the small number of types represented : — (fl) Broad, flat, spade-shaped or tile-shaped scales. (6) Broad, expanded, asymmetrical scales. (c) Narrow, asymmetrical scales. {d) Narrow, hair-like scales. ((?) Narrow curved scales. (J &• g) Spindle-shaped scales. {h & i) Upright fork scales. (j) Long twisted scales. {k) Pyriform scales. * Figs. 97 and 99 — loi are reproduced by kind permission of the Editor of tla^ Journal of Tropical Medicine. 198 TYPES OF SCALES Fig. 97. — Types of scales, a to k. Head and scutellar ornamentation, I to 5 ; forms of clypeus, 6. i, Head and scutellum of Stegomyia, &c. ; 2, of Culex ; 3, of ^des, &c. ; 4, of Megarhinus, &c. ; S, of Cellia and other Anophtlina ; 6, clypeus, a, of Culex ; b' , of Stegomyia ; <:', of /'oblolia. (Theobald.) APPENDAGES • 199 On the wings other types of scales, either lanceolate, long, narrow scales pointed at the free end, or long and narrow and with square free ends, are met with (fig. 97). Head Appendages. — The head appendages can be easily seen in most specimens mounted as described, but for more minute examination it is better to cut off the head and mount it in a shallow cell either as a dry specimen or in glycerine jelly. In this way the parts are not much distorted, and if a thin slide be used both sides of the specimen can be examined. Canada balsam can be used, for the examination of the scales, hairs, &c., but not satisfactorily, as they become too transparent. Proboscis. — To examine the component parts of the proboscis it is better not to use the shallow cell, but to forcibly compress the head so as to cause the various component parts of the proboscis to separate ; in one or more specimens all the elements can be seen. Palpi. — The points to be noted in the palpi are their length relative to the proboscis, the number of joints, and the colour, shape and arrangement of scales and hairs. To determine the number of joints it is necessary to remove the scales from the palpi. The Antennce. — Their length, and the relative lengths of the different joints. The number, length and arrange- ment of hairs, and the presence or absence of scales. The different regions of the mosquito are shown in the diagram (fig. 98). To the head are attached the append- ages already mentioned, and, in addition to these, the back part of the head, or occiput, requires close examination. The thorax is composed of three segments fused together. The greater part is formed by the second segment, or mesothorax. Anteriorly on each side are two rounded projections, the prothoracic lobes, the rem- nants of the anterior segment. The posterior edge of the mesothorax is a narrow, overhanging, trilobed plate — the scutellum. The scales on this part of the thorax are of generic value. 200 ANATOMY OF MOSQUITO Proboscis — Proboscis Palpi Antennae Basal lobes ofanlennae Frons Verte) Eyes Occiput. Nope .-4?t'tarsal S^l; tarsal Fig. 98. (Theobald). METANOTUM 201 Partly overlapped by the scutellum is a rounded mass connecting the thorax and abdomen, known as the metathorax or metanotum. This is the third seg- FiG. 99.— Types of metathorax (Theobald), a, Culicina ; b, Dendro- viyina ; c, Joblotina. ment of the thorax. On each side of the metathorax are the halteres, which arise from the sides of the mesothorax. 202 THORAX AND ABDOMEN The abdomen is segmented and has no lateral append- ages. The last segment terminates in the external genitalia. These are of specific but rarely of generic value. Thorax and Abdomen. — In the examination of the dry mounted specimen by this method, each part of the piosquito should be examined in turn. By altering the angle in the manner described, the different parts repre- sented in the diagram can all be clearly made out and the character of the scales covering these parts investigated. Metathorax. — It is well to first examine the metathorax or metanotum. This part is nude in all sub-families except Dendromyince, in which there are hairs, in Joblotince including Limatus, which have both hairs and scales (fig- 99)- Scutellum. — Overhanging the metathorax is the scutel- lum, bordered by a row of stiff hairs and covered with scales. These scales are not necessarily of the same type as those covering the thorax, but are often the same as the scales covering the middle of the occiput, with the exception that there are no upright fork-scales. The scales on the scutellum are of great generic itaportance (fig- 97)- The character of the scales on the abdomen and thorax are used by Mr. Theobald to subdivide the old genus of Anopheles, and in the further subdivision of the Culicince. Occiput. — The scales on the occiput should next be examined. They vary according to genera. The up- right fork scales (h and i) are found only in this situa- tion. In some genera no other scales are found on the head. In most genera the scales at the side of the occiput are tile-shaped scales (a), but whilst in some genera {Megarhininai) these scales extend all over the middle line of the occiput, and are the only scales found, in others, as Stegomyia, they are found -also with fork-scales ; in others, again, they are not found in the middle of the occiput, but are replaced by spindle scales (/), either alone as in ^des, or with narrow-curved and upright fork-scales as in Culex, Mansonia, &c. (fig. 97). WINGS 203 Wings. — The type of wing venation can be seen in a specimen mounted as described above, but is better seen in the wings when detached, flattened out and examined dry. The character of the scales covering the longitudinal veins on the wings must be observed, as these are of generic importance (fig. 10 1). The wing venation of the Culicidce is comparatively simple. Here we follow closely in this, as in other respects, the description by Mr. Theobald. It is an easy one to work with (fig. 100). The thickened edge is called the costa, it forms the free edge of the wing. The scales on it are of no generic pt jny Fig. 100.— Neutation of Wing (Theobald). value; these may differ greatly from those on the longi- tudinal veins. The scales on the costa in all genera are mainly lanceolate. They are of unequal length, arranged in two tiers with, at their bases, a third row arranged obliquely; these last are more like the scales on the longitudinal veins. The straight edge of the wing, with the wing expanded, is the anterior edge, and is therefore so described. Next to the costa is a vein running^from the base or attachment of the wing to rather more than half-way to the tip, terminating in the costa, this is called the sub-costal vein (sc). The other veins running from the base towards the tip are known by numbers, the most anterior being the 204 WING SCALES Anopheles. Cyclolepteron. y anthinosotna. 4 Mansonia. Stegomyia. Eretmapodiles. , 7 Culex. 8 Mucidus. Psorophora. Fig. ioi. — Various Forms ofWing-scales (Theobald), i, Scales on veins and on costa in Anopheles; 2, scales on veins in Cydolepteron ; 3, scales on veins and on costa in Janthinosoma; 4, scales on veins in Mansonia; 5, scales on veins in Stegomyia ; 6, scales on veins in Erelmapodites ; 7, scales on veins and on costa in Culex : 8, scales on veins in Mucidus ; 9, scales on veins and on costa of Psorophorxu WING VENATION 20$ first longitudinal. This is a single vein running the whole length of the wing and terminating at the tip. It is covered with scales in its whole extent. The second longitudinal arises from the first nearly half-way from the base, and bifurcates before reaching the tip. The space enclosed in the bifurcation (Dj is known as the first fork-cell. The third longitudinal arises in the base of the wing, but is not covered with scales for nearly the first two- thirds, and therefore appears merely as a yellowish line. It does not bifurcate. The fourth longitudinal arises from the base, is covered with scales in its whole extent, and bifurcates near the tip, forming the second fork-cell (G). The fifth longitudinal arises at the base, is covered with scales in its whole extent, and bifurcates half-way up the wing, enclosing the third fork-cell (K). The sixth does not bifurcate, and terminates in the costa about the middle of the posterior border of the wing. There are markings or thickenings on the wing between the fifth and sixth longitudinal and posterior to the sixth, which have no scales and are not regarded by Mr. Theobald as veins. The most constant of these is scaled in some species of mosquito, which are placed by Theobald in a separate subfamily Heptaphlebomyina. Con- necting the second and third longitudinal veins is the transverse vein. The third and fourth are connected by the middle transverse vein, and from the fourth longi- tudinal to the anterior division of the fifth is the posterior transverse vein. There are definite bands of considerable thickness and often contain air. They are not scaled. The relative positions of these three transverse veins is of some importance in the separation of species. Varia- tions cannot be relied on implicitly for this purpose, as in some species the arrangement of the transverse veins varies considably in different individuals. 206 classification Classification of Mosquitoes. In a work of this kind it is not necessary to consider more than the identification of the commoner and more important genera into which the Culicidce are divided. For fuller details the reader is referred to the systematic works on the subject, particularly Mr. Theobald's " Mono- graph on the Culicidse." The Culicidce, as has been indicated in the previous chapter, constitute a family in the sub-order Orthorrhapha nemocera of the order Dipiera. Their systematic classi- fication has been attempted by several writers, but that of Mr. Theobald, which is based largely on the scale char- acters of the mature insect, now finds most general acceptance. The main characteristics of the family have been already given (p. 162). In brief, it may be stated that the Culicidce may be told by the venation of the wing and by the arrangement of scales on the head, body and veins of the wing. Other insects which are often mis- taken for these have a different form of wing venation, and the wings are either bare or ornamented with hairs. The male external genitalia are by some considered to be of great importance in differentiation of genera. The external genitalia vary greatly in different species. The general type (see fig. 97) may be described as con- sisting of two large fleshy basal lobes each with a terminal chitinous clasp segment, always curved and often orna- mented with spines. Between the claspers, arising in- ternally and ventrally to the claspers, are other chitinous processes, the harpes, which may be well developed, formed of two segments, or rudimentary. Between the harpes and the claspers are a pair of clasping organs, the harpagones. The chitinous lobes above the cloaca, the setaceous lobes, are part of the rudimentary eighth segment. The variations of each part are great. They should be examined both in the dried specimen, in order to see the relative portions of the various parts, and after treatment SUB-FAMILIES 207 with liquor potassse, or in flattened specimens, rendered transparent in order to make out the details. The family Culicidce is divided into various sub- families. (i) Sub-family Corethrina. — Proboscis short and not adapted for piercing, palps dependant. These insects are incapable of biting man and animals, and play no part in the transmission of disease (fig. 102 E). (2) ^vib-i2iva\\y Megarhinince. — Large, brilliantly coloured insects. Proboscis long and bent downwards. Palps thin towards the extremity and bent upwards, those of the male being composed of five segments, those of the female varying according to genera. Head clothed with flat scales only (fig. 102 F, and fig. 97 4). These mosquitoes are found as a rule only in the vicinity of the jungle. They rarely attack man. Larvce are larviverous. They have a very short respira- tory siphon. Head, thorax and abdomen thickly covered with flat, square-ended scales. Scales of wings small and square- ended. First sub-marginal cell much shorter than second posterior. Caudal tuft of hairs on each side of abdomen in some species. (3) Sub-family Anophelince. — Proboscis straight and adapted for piercing. Palps very nearly as long as the proboscis in both sexes, composed of five segments in the male and four in the female ; the last two joints in the male are expanded. Antennae plumose in the male and pilose in the female. , Head has numerous upright forked scales, a few narrow curved scales and flat, square-ended scales at the sides in most of the genera. Thorax and abdomen ordinarily have few scales as compared with other Culicidce. Scutellum simple, never trilobed. The varied covering of thorax and abdomen is the basis of the subdivision into genera. Wings in most genera marked with black or brown patches ; wing scales long and lanceolate or fusiform. Fig. 102. A — Lateral view of Anoplieline. B — Lateral view of Culicine. C — Anopheline viewed from above. D — Culicine viewed from above. E— Head of Corethra. F— Head of Megartunina. Fig. 103.— I, Culicine male ; 2, Culicine female ; 3, Anophelina male ; ., Anophelina female. 2IO SUB-FAMILIES' The female has only one spermatheca. The eggs are laid singly, are more or less boat-shaped and float in the water, owing to the presence of lateral air chambers. LarvcC have no respiratory siphon, and when at rest lie horizontally on the surface of the water. Pupae have trumpet-shaped respiratory siphons. The adult mosquito appears very narrow when viewed from above, and as seen in profile the long arcs of the proboscis, thorax and abdomen form an almost straight line. When at rest on a flat surface the insect commonly presents an appearance as if standing on its head. (4) Sub-family Culicince. — Palps in the ,? as long or longer than the proboscis ; in the S always much shorter than the proboscis ; metanotum nude. Viewed from above these mosquitoes appear much broader than Anophelince, and from a lateral aspect have a hunch-backed appearance, which is very different from that of the Anophelince (fig. 102). The eggs differ markedly in the different genera, both in shape and the manner in which they are laid ; in the genera Stegomyia and others they are laid singly, but never resemble those of Anophelince, while in Culex and others they are laid in rafts. The larvae ai'e provided with a respiratory siphon, and when at rest lie obliquely in the water with the head downwards. (5) Sub-family ^dince. — Palps short in both sexes. Antennas in the males not always plumose. The pro- boscis may be very long, and is sometimes clubbed at the extremity. Metanotum nude, without hairs or scales in most, but in the sub-division Dendromyince there is always a tuft of hairs on the metanotum. Blanchard makes a sub-family, Sahetince, of this division.* This sub-family has been less studied than Anophelina * Sub-family Sabetince, Blanchard, corresponds to Dendromyince, Theobald. Palps short in both sexes. Metanotum adorned with hairs but no scales. Sabethes, Dendrotnyia, Sabethoides, Wyeomyia, Phoniomyia, Binotia, are genera in this sub-family. SUB-FAMILIES 211 or Culicina. Most of its members are jungle, mangrove or forest mosquitoes. Many of them bite by day. They breed in natural collections of water, often in the hollow axils of leaves, in pitcher plants or in perforated bamboos. They may also breed in swamps, slowly moving water, roadside trenches, or in streams. Little work has been done to .show whether or no they can act as carriers of disease, and it is quite possible that it may be found that some of the Mdince can carry malaria, as the nuni^ber of Anophelines in some jungle country does not seem to be sufficient to explain the great prevalence of malaria in those districts. Many of these mosquitoes die very quickly in a dry atmosphere, and few of them will feed or live in captivity. The remaining sub-families are represented by a few rare species and are of little importance to medical workers. (6) Sub- family Joblotince {Trichoprosoponina;, TheohaXd). Head, thorax and abdomen covered with sqtiare-ended scales. Palps short in both sexes. Metanotum adorned with hairs and square-ended scales, (7) Sub-family HeptaphlebomyiucE. Head, thorax, scu- tellum, metanotum and abdomen as in Culex. Wings have a seventh scaled longitudinal vein. Four species known. H. simplex (Theobald, 1903) from Central Africa (Mashonaland) was the first described. Theobald's latest grouping of the Culicidce is modified from that of Lutz, and differs in some respects. He divides the Culicidce as on page 212. There are many points in favour of this classification : — (i) Corethrina are separated off from the Cidicidce and considered as a distinct faniily, the Corethridce. (2) Anophellnce and MegarhiitiJtce, two groups which differ at all stages of their development from the rest of the Culicidce, are separated off from the other groups, which have many points in common. (3) The old group ^deomyince, containing several markedly dissimilar sub-groups, is broken up so that 212 CLASSIFICATION the JEdince, Uranotcenice and Dendromyince, which are naturally distinct groups, are now widely separated. Though we consider that the scheme is one which will be of practical value and a useful aid in the subdivision of the Culicidcc, we have not thought it advisable to adopt it in the present unsettled condition of the classification of these insects, as it has not received the general approval of those working with mosquitoes. Culicida 1 I I ,.!.■■ Anophelina. Orthorrhynchce. , Megarhimna. oboscis straight, palpi Proboscis straight, palpi short Proboscis curved, palpi sh long in ? and j in ? , long or short in $ or long in ¥ , long in d I Metanotopsila Metanototricha \ I I I Heleropalpie. Micropalpa.' Heteropalpa. Micropalpa Ipi short in ? , long Palpi short in ? Palpi short in ? , Palpi short ir in i and S long in S and S I I I \__ cince. Heptaphlebomyince. ^dina. Uranotcenina. Trichoporo- Dendromyina. Lim gs, 6 Wings with First fork- First fork- sosopina. Proboscis Pro udinal 7 longitudinal cell large cell large straight. elbc 1 veins scaled veins (i) Genera of sub-family Corethrince. Metatarsus longer than first tarsal joint Corethra. Metatarsus shorter than first tarsal joint Mochlonyx. (2) Genera of sub-family Megarhinince. This sub- family is divided by Theobald into two genera. Palpi long in both sexes Megarhinus. Palpi short in the female , Toxorrhynchites. The differences between these two genera are com- paratively slight, and a division founded on the absence or presence of lateral caudal tufts would be simpler, but would not quite correspond with Theobald's division. (3) Generaof sub-family ^7'jo/)/je/wcE (Theobald). Theo- bald now subdivides this sub-family into sixteen genera. The practical value of the subdivision of the Anophelince ANOPHELIN^ 213 into so many genera is disputed by many medical men. There are few branches of natural history in which such enormous advances have been made as in our know- ledge of the Culicidce, and each year there are additions to this knowledge. Of the Anophelina; about one hundred species are now known. This is too large a number to be conveniently grouped into one genus. A subdivision is necessary, and some grouping will be made automatically by any one working with these insects. It is better that a uniform system should be adopted than that each worker should make his own groups. The subject is so large that it is now a special branch, and we propose merely to deal with selected parts of it and select such groups as are represented by common species, and by species known to carry malaria. It is unfortunate that the grouping into genera based on the external characters of the adults does not corre- spond with grouping according to the power of carrying malaria, or with the class of breeding place or the habits of the mosquitoes. In one genus, Myzomyia, are included the harmless M. rossi and the harmful M. funesta, the first essentially a foul or stagnant water breeder, the second breeding in fresh water, preferably in streams. In another genus. Anopheles, are included A. maculi- pennis, which hibernates through an English winter, whilst the larvae are killed by cold, and A. bifurcatus, which is killed by cold though the larvae can survive an English winter. Anophelina. Of the eighteen genera, eight are represented by one or two species only; one of these, Stethomyia, is repre- sented by three species, including Stethomyia fragilis {Anopheles treacherii). This genus is very similar to Anopheles, in that the scales on the wings are all the same colour. The flat scales on the head on which the separa- tion is now based are very scanty. The mammilation 214 GENERA OF ANOPHELIN^ of the prothoracic lobes, which was formerly described as the characteristic, is not present in all species. The eight genera, represented each by a single species and in some cases by a single specimen, are separated off on various grounds. They are : — Feltinella, in which the basal lobe of the male genitalia is divided into two segments. One species, F. pallidapalpi. Myzorhynchella. — Head is clothed with long, flattened, outstanding scales not closely applied to the head, but not so erect as the upright fork-scales. Chrystya, similar to Myzorhynchus, but separated by the possession of long lateral tufts of hairlike scales on the abdomen. Lophoscelomyia, resembles Nyssorhynchus, but differs in that there are long tufts of scales on the femora of the hind legs. Bamboo breeder. Kerteszia. — Intermediate between Myzorhynchus. and Cellia. Bironella. — Male only known. Chagasia. — Antennae of female have whorls of scales as well as hairs and dense outstanding scales at the side of the thorax. Aldrichia. — Thorax and abdomen scaled as in Culex. One specimen. The more important of the groups or genera of Anophe- lince are Anopheles, Myzomyia, Cydoleppteron, Stethomyia, Pyretophorus, Myzorhynchus, Nyssorhynchus, Cellia. 6 , (i) Only upright fork-scales on head ; wing j3 -g ^ scales lanceolate and uniform in colour Anopheles. ,^^•1 (2) Some flat head scales Stethomyia. rt-s"l XT a.u J 1 f(3) Wing scales mostly " & ., No flat head scales. i ■, ,, 2=-^ Scales on wines-, long and narrow.. Myzomyta. oS-i ,^^ °" "^'"^^ (4 Wing scales partly J3 c of two colours. , j ■ a J ^ , , , , , f-( V large and mflated Cydoleppteron. (5) Thorax with some narrow curved scales ; abdomen hairy Pyretophorus. (6) Thorax with hair-like curved scales, some narrow curved ones in front ; abdomen with apical lateral scale tufts, scaly venter; no ventral tuft Arribalzagia. ^ r^ "i ° O M cij GENERA OF ANOPHELIN^ 215 (7) Thorax with hair-like curved scales ; ab- dominal scales on venter only, w(ith a distinct ventral apical tuft Myzorhynchus. (8) Abdominal scales as lateral dorsal patches of small flat scales ; thoracic scales narrow and curved, or spindle- shaped Nyssorhynchus. (9) Abdomen nearly completely covered with irregular scales and with lateral tufts... Cellia. (10) No lateral tufts and smaller wing scales Neocellia. This subdivision is in the main as given by Theobald, and now that the known species belonging to the Ano- phelince are so numerous, a subdivision is necessary, James argues that as in any one country the number is comparatively small a complete description of the species found in each country would suffice for practical pur- poses, but with this argument we do not agree. Some of Theobald's points are very difficult to make out, and this is particularly so as regards the wing scales. Genus Anopheles. — Thorax and abdomen clothed with hairs only ; the palpi in the female are thin, not densely scaled, and generally unhanded. Wing veins covered with long lanceolate scales, which may or may not form spots. These spots, if present, are never so numerous as in other genera, and are not formed by different colouring of the scales. In the female a single spermatheca only is present. These mosquitoes may be said to be characterised by their extreme baldness. They are of comparatively large size. The genus includes two well-known carriers of the parasites of malaria in sub-tropical and temperate climates, A. niaculipennis and A. bifurcatus. Anopheles maculipennis, the type of the genus, is easily recognised. It is a yellowish-brown mosquito ; neither legs, proboscis, nor palps are banded. Four black spots on the wings formed by accumulation of scales. It does not assume the Anophehne position as markedly as most \ Anophelines. 2l6 SPECIES OF ANOPHELINyE This mosquito is widely distributed in Europe, from the borders of the Mediterranean to Scandinavia. It is found also in Algeria, Palestine, the United States and Canada. In England it is common ; according to Nuttall its distribution in England agrees to some extent with the old malarious districts. A. maculipennis is the most active propagator of malaria in Europe, Algeria, Tunis, and the United States. It has been shown to be capable of serving as a definitive host for all forms of the malaria parasite. It is also an inter- mediate host of Filaria immitis. In the winter in temperate climates the larvae die, but the imago can hibernate all' through an English winter. Anopheles bifurcatus is easily distinguished from the preceding species by having no spots on the wings. Its distribution corresponds in a general way with that of A. maculipennis, but is a less common species and does not frequent houses. This mosquito is also an active carrier of malaria, and has been shown to be more easily infected experimentally than even A. maculipennis. In an English winter the adult forms are killed, but the larvae remain alive even if the water be frozen throughout the winter. Genus Myzontyia. — Thorax and abdomen with hair- like curved scales. The wings are spotted, and have mostly long, thin or narrow lanceolate lateral vein scales. They are usually small or moderate-sized mosquitoes. Head scales. Myzomyia rossii is distinguished by the ornamentation of the wings. Along the costa are four large patches of dark brown or black scales. The large, middle spot has a small dark spot below it in the centre, giving it a T- shaped appearance. This mosquito is the " large dappled-winged mosquito " which Major Ross, in his work in 1899 in Calcutta, failed to infect with malaria. It is a common mosquito in I various parts of India and the East, chiefly in the neigh- bourhood of towns. It breeds in muddy pools or shallow tanks, even in cess-pools. SPECIES OF ANOPHELIN^ 2 17 This mosquito has never been found infected with the parasites of human malaria in nature. It may serve as an intermediate host for Filaria bancrofti. Myzomyia funesta. — A smalJ, rather dark mosquito. The black costa is marked by six pale spots ; there is always a pale costal spot near the base. The black scales upon the wing veins are also interrupted by white spots. There are pale spots on the fringe of the wings at the points of insertion of the longitudinal veins. This mosquito is widely distributed in Central and West Africa, and is an important carrier of the parasites of human malaria in those regions. It frequents houses, but does not leave them in the daytime, hiding in dark corners high up out of the reach of the breeze from doors and windows. It feeds in the early hours of the evening by preference, but at other times as well. Myzomyia cullcifacles. — Has unhanded legs, and the largest light costal area near the base of the wing. There are only three light areas on the fringe of the wings. It appears to be a carrier of malai-ia in India. Pyretophorus. — Thorax with narrow curved scales, not hairlike as in Myzomyia. Abdomen with hairs. Wings with small, short, lanceolate or narrowish scales, much spotted as a rule. Legs banded, sometimes spotted. Pyretophorus costalis. — Told by the curious mottled character of the femora and tibiae. The wings may be said to be white spotted with black, in contrast to M. funesta, which are black spotted with white. This species is also widely distributed over the vs^hole of Central Africa and the West Coast. The laryas are found '\y\ abundance during the rainy season in puddles in West Coast towns. It is the most common Anophe- line on the sea-coast. Like A. maculipennis it serves as a definitive host for the parasites of all forms of human malaria. It also is an occasional intermediate host of Filaria bancrofti. Genus Myzorhynchus. — Thorax with hair-like scales. Abdomen with ventral scales and a ventral apical tuft. 2l8 SPECIES OF ANOPHELIN^ Wing scales moderately broad and lanceolate. Palpi and proboscis densely scaled. In some species this scaling is so thick that the proboscis and palps appear to be very thick as seen with the naked eye. Myzorhynchus sinensis. — A brownish mosquito. Thorax, slaty-grey background with purplish-brown longitudinal stripes, adorned more or less with pale golden scales. Found in China, Formosa, Malay Archipelago, &c. Myzorhynchus barbirostris. — In this species the palpi are densely covered with deep black scales. The pro- thoracic lobes have dense tufts of large black scales projecting forwards. Found in Malaya and Old Calabar. Both these species can carry filaria. They are difficult to infect with parasites of malaria, and are probably not important carriers, as they may be numerous in towns where malaria is not prevalent. Genus Nyssorhynchus. — Thorax with narrow curved and spindle-shaped scales. Abdomen with ventral scales, and sometimes dorsal patches. Wing scales bluntly lanceolate. (The legs are always banded or spotted with white). N. lutzi is said to be a malaria-carrier in Brazil. N. fuliginosus. — Probably the " small dapple-winged mosquito " of Ross. Genus Cellia. — Thorax with flat spindle-shaped scales. Abdomen scaled, the scales irregularly disposed on the dorsum and on the venter, forming dense bifid tufts on each segment. Palpi densely scaled. Wings covered with large bluntly lanceolate scales. Cellia pharoensis is found in Africa, C. kochii in Asia, C. argyrotarsis and C. albipes in the West Indies and South America. These mosquitoes may breed in fairly dirty water. C. kochii is commonly found in outlying villages or suburbs of towns. They are night biters as a rule. C. argyrotarsis is one of the most important carriers of malaria in Tropical America. C. kochii probably acts as a carrier in the Malay Peninsula and Archipelago. CULICINA 219 These mosquitoes are common in the most malarial settlements in these countries. CULICINA. Genera of the sub-family Culicina. — This sub-family in- cludes a large number of mosquitoes. Numerous genera have been created by Mr. Theobald with a view to simplifying the identification. It is not proposed to deal here with all of these genera, but only .such as are known to be of importance from the medical point of view. Mr. Theobald's table is appended, but those who wish to go into the matter more fully should consult his monograph. The type, Culex pipiens, has narrow curved scales on the head and scutellum. On the head there are also upright fork-scales at the back, and flat tile-shaped scales at the sides (see diagram, p. 198). The lateral scales on the veins of the wings are long and narrow, those running along the vein are shorter and broader. 1. Certain genera can be told at once by the char- acters of the scales on the wing, though in other respects they may resemble the genus CuU.v. (a) Wing scales broad and asymmetrical (see fig. loi, p. 204) Mansonia. {b) Wing scales broad-ended or pyriforni, sym- metrical, and often parti-coloured (see fig. loi, p. 204) Mucidus. {c) Wing scales thick and elongated, ending either diagonally or convexly, more or less bluntly pointed Taniorhynchus. 2. Other important genera are characterised by having the head and scutellum entirely covered with flat, square-ended scales arranged like tiles on a roof (see fig. 97, i). {a) Palpi of ? short, of $ thickened apically and tufted Stegomyia. {b) Palpi of ? longer than in Stegomyia, of $ long, thin, acuminate and without tufts Desvoidea. 3. Other genera have head and scutellar scales of the Culex type, but are differentiated on various grounds. 220 . CULICINA («) Wing scales long, narrowly lanceolate, and collected in spots ; palpi clubbed in the $ Theobaldia. {b) Wing scales at apex of veins dense and rather broad ; femora swollen. Small dark mosquitoes Melanconion. (c) Wings with short, thick, median scales, and short, broad lateral ones on some of the veins ; scales mottled Grabhamia. 4. Peculiarly twisted scales arranged in whorls on the sixth, seventh, eighth, and sometimes ninth joints of the antennae in the males. The females show no similar scaling. Fairly com- mon mosquitoes throughout Malaya and in Brazil Lophoceratomyia. For purely diagnostic purposes Theobald arranges the genera somewhat differently. The table subjoined is based on this arrangement. A. Legs more or less densely scaled. (a) Head not entirely clothed with flat scales. 1. All the legs densely scaled. Wings with large pyriform scales ... Mucidus. Wings with narrow scales Psorophora. 2. Hind legs only densely scaled Janthinosoma [b) Head entirely clothed with flat scales. Wing scales long and rather thick. Hind legs of $ with apical scaly paddle Eretmapodites. B. Legs uniformly scaled with flat scales. {a) Head and scutellar scales all flat and broad. 1. Palpi of ? short, of $ thickened apically and tufted Stegomyia. 2. Palpi of 2 longer than in Stegomyia, and in $ thin, acuminate, simple ... Desvoidea. (b) Head scales mostly flat, but a median line of narrow curved ones ; scutellar scales flat on mid-lobe, narrow curved on lateral lobes; i? palpi longer than probosis Macleaya. (c) Head scales mostly flat,, irregular, narrow s curved ones behind ; mid-lobe scutellum with flat scales, lateral lobes with narrow curved ; Fio. 1 12. — a. Eggs of Culex ; 6\ V, eggs of Anopheles ; c, egg of Stegomyia ; d, egg of Mansonia ; e, egg of Psorophora. The eggs of Psorophora are not unlike those of St&go- myia in shape, but are rather more pointed. According to Dr. W. N. Berkeley they are " prickly." Eggs are best obtained by collecting adult female mosquitoes and keeping them in a small cylindrical vessel, a wide-necked 4-oz. bottle is suitable, containing water. It is well to have some twigs or fragments of grass floating on the water on which the mosquitoes may rest. The top of the vessel should be covered with mos- BREEDING FROM LARVAE 255 quito netting to allow air to have free access. They should be fed on blood as often as they will feed. Larvae can be obtained by keeping the eggs in water at at suitable temperature. When first hatched they are small and quite white, but they soon increase in size, and either in part or as a whole change colour. They are voracious and require abundance of food, but with many species of mosquitoes, particularly with some of the Anophelines, the water must not be putrid or peaty. A white, flat dish, such as a half-plate or full-plate photographic tray, is as good a breeding place as any. Some earth should be placed at the bottom, and it is well to place some grass with the roots and earth attached in two or three places, both along the edge and also towards the middle, so as to form at least one islet. The dish should be filled so that there is about three-quarters of an inch depth of water, and these dishes are best prepared a few days before the larvae are placed in them. A little of the " green slime " or other algae found growing in fresh water should be added and a few grains of dry rice may be scattered about the bottom. Abundant food will thus be supplied, but the water must not be over- stocked with vegetation, as if this decomposes the water will be unsuited to many larvae. Great care must be taken that none of the natural enemies of mosquito larvas are introduced into the water. Those most frequently in- troduced are the larvae of Agrionidce, one of the groups of the dragon-flies. These are short, squat, six-legged larvae with the characteristic protrusible prehensile mask. They are often introduced with mud or in muddy water and are most destructive to other larvae. Cannibal culicid larvae should be looked for, as they also are very destruc- tive. They can be recognised by the stiff row of curved bristles instead of fine hairs on each side of the mouth. These dishes must not be kept in the dark, must be well lighted, and are best exposed for short periods to direct sunlight if there is sufficient grass growing to 256 LARViE provide shelter for the larvae. They must not be left long enough in the sunlight to warm the water. When pupae haive formed they must not be exposed at all to direct sunlight. The water must not be overstocked with larvae, as they are all at times carnivorous. The. larvae should Fig. 113. Fig. 114. all be about the same age, but may be of different species. Some large larvae will destroy the young of both their own and other species. The tops of the dishes should be covered to prevent the entrance of dust, a plate of glass, or better, a larger glass dish inverted over the dish containing the larvae, will suffice. COLLECTION OF SPECIMENS 257 Larvae can either be raised from the eggs or caught from natural waters by scooping up the water in any receptacle. When large numbers of larvas are required any receptacle from a bucket downwards will do, but where larvae are scanty they are best caught by using a dipper. An ordinary white enamelled coffee-cup serves the purpose well, but in some situations a longer handle is better and this can be fitted on to the cup, or a soup ladle may be used. Some larvae are most numerous at the edges of pools or streams in the shady places. In using the dipper the open mouth should be turned towards the bank and plunged in inclined so that the water from the edge rushes in. The dipper should, as soon as the rush of water has ceased, be turned upright and removed from the pool. (Figs. 113 and 114.) It should be allowed to stand for a few minutes till the mud has settled and then examined. A hand lens is useful as the very young larvce can easily be overlooked. In obtaining the specimens care must be taken not to disturb the water in any way before using the dipper, as larvae readily take alarm and dart to the bottom. In shallow pools and small puddles, larvae can be readily seen by looking rather obliquely at the undis- turbed surface of the water. When they occur in such situations they are usually numerous. In running water and in larger masses of water they can rarely be seen in this manner, and unless a dipper is used will be over- looked. In such situations it is not common to find them in large numbers in any small surface of water, and consequently the dipper may have to be used fre- quently to demonstrate their presence. Though in any small area of water examined they may be scanty the total areas of this class of breeding place is so great that these places are of the highest practical importance. Some species of mosquitoes are more easily found as larv» because the adults do not frequent human habi- tations. During certain seasons, particularly cold and 17 258 BREEDING PLACES dry seasons, larvae of all species will be found more i-eadily than adults. In making collections of mosquitoes it is well both to breed from adults collected in as many different classes of place, houses, cattle-sheds, grass and forest, as possible, and also to rear adults from larvae or eggs found in still and running waters, natural pools, small and large, and also in artificial collections of water. No water, even that in cess-pits, is too foul for some species, whilst others will not breed in water that Euro- peans consider fit to drink. The pupae are found in the same situations as the larvae ; they appear as small black objects which are usually motionless unless disturbed. The larvae and pupae can be transferred from the dipper to a wide-mouthed bottle for carriage. Both larvae and pupae are easily destroyed if the water is kept in motion, as they do not rest on the surface sufficiently long for proper respiration. If it is necessary to carry them for long distances it is well to make frequent halts every half hour to an hour and place the bottle contain- ing the larvae upright in a shady place for a quarter of an hour or so. The character of the breeding place must be carefully noted. The special points are : (i) Whether it is fresh, . or foul, or brackish. (2) If the water is still or in'motion. (3) Vegetation in the water. (4) Other larvae or animals present. Special attention should be paid to animals that may prey on the mosquito larvae, especially fish, coleopterous and neuropterous larvae, &c. (5) Any special features either in the natural or artificial receptacle for the water. (6) Exposure to light, wind, &c., of the surface of the water. Many of the Stegomyia larvae will thrive in darkness, ^deomyina and Megarhinine larvae often have peculiar breeding places, such as the cups of pitcher plants, the hollows in trees, or the interior of bamboo joints or crab-holes. Some species will only be found in one kind of breeding place. ANATOMY OF LARVAE 25c Duration of larval stage varies with the amount oi food, temperature, and the species of the mosquito, Under the best conditions of food and temperature, with many mosquitoes the larval stage is seven or eight days, but may be indefinitely retarded by cold or insufficient food. Other mosquitoes, under the most favourable conditions, require several weeks for their development, Of these, Megarhinine larvae are instances. The pupal stage is not affected by the food supply, as the pupa does not require food. It is prolonged by a low temperature. The pupal stage is about two days with most species, but with Megarhinine pupa and a few others is six days. Anatomy of Larvce and Pupce. — The larvae vary in colour in different species, but even in the same species varia- tions occur according to the degree of exposure to light and the nature of the food. In the more transparent larvae the colour of the intestinal contents, green or brown, is more obvious than that of the larva itself. The larvae of the CuUcidce conform to a general type. The head is joined to the thorax by a narrow neck. In the head are a pair of compound eyes and two simple eyes or ocelli. There are a pair of short antennae and a mouth composed of an upper lip, a pair of mandibles, a pair of maxillae, and a lower lip or labial plate. ' The thorax is composed of three fused segments. There are no ambulatory legs, but sensitive and balancing hairs are abundantly supplied. The abdomen is long, and composed of nine segments. The last is smaller, and inclined at an oblique angle down- wards. At the termination of this segment is the opening of the anus, surrounded by four retractile papillae, prob- ably respiratory in function — anal gills or branchiae. On the upper surface of the eighth segment are the spiracles or openings of the two respiratory tubes, which run the whole length of the body, and supply the larvae with air. The openings of these tubes are direct in the Anophelina, which can therefore be described as asyphonate. In the 26o ANATOMY OF LARV^ other subfamilies the tubes are continued into a conical tube jointed on to the upper surface of the eighth segment, and at the apex of this tube are the external openings of the respiratory tubes. This projection is known as the respiratory syphon, and the larvae of all the Culicidce, except the Anophelina, are therefore said to be syphonate. The head is composed of many chitinous plates, which are thicker and darker on the dorsal surface. The opening of the mouth is directed slightly down- wards in all, and almost directly downwards in many. The upper lip consists of a middle portion or palate supported on either side by lateral plates covered with bristles. The pair of mandibles are placed beneath the upper lip, and are usually toothed. The movements are lateral. The pair of maxillze are below and behind the man- dibles ; their movements are obliquely upwards and inwards. The inferior lip, or labium, is a triangular plate, usually very dark in colour, and with a more or less serrated edge. On each side of the mouth are chitinous plates attached to the mandibles and maxillae, and from these arise the " brushes " or masses of long, stiff hairs, fme in most of the Culicidce, but thick and curved in the larvivorous larvae, which are so arranged that they can be moved laterally and folded completely over the mouth or thrown back so as to form a very oblique angle with each other. These brushes in life are in constant movement, and cause a sufficient current in the water to wash solid suspended particles to the open mouth of the larva. There are great variations ip the different parts which are of use in distinguishing larvae of one mosquito from those of another. Much attention has been paid to the shape of the inferior lip plate, which is a conspicuous object, and varies in closely related species. The antennae are articulated to the head. They move slightly. They are not truly jointed, but in some there is an abrupt variation in thickness, probably indicating a ANATOMY OF LARV^ 261 joint. They vary in length and in the number and arrangement of the hairs and spines ornamenting them. They are of vakie in differentiating the larvae of dif- ferent species. The head is very mobile in some species, and in the Anophelines so much so, that they can turn the ventral surface upwards and feed in that position. As the ventral surface is light, and the dorsal black, a larva may at one moment appear to have a dark head, and at the next a light one. The thorax is v^^ell supplied with simple or compound hairs. The longer and more conspicuous are arranged on each side. In a few species there are in addition strong curved spines slightly below the lateral hairs. One or two pairs of these spines may be present. On the abdomen are also hairs, varying greatly in genera and species. In the Anophelines are peculiar palmate hairs, the shape of which vary in different species. In many of the JEdeomyina compound hairs are very numerous. In the Stegomyia hairs are scanty and in- conspicuous. The shape of the respiratory syphon attached to the eighth segment in all but the Anophelines is of great importance. It varies in length, so much so, that attempts have been made to classify the Culicidce on the " syphonic index," or the relative length of the syphon. Generally speaking, all the Megarhina, all larvivorous larvas, Stego- myia and Desvoidea, as well as some of the ^deomyina, such as Uranotcenia and many other genera, have short respiratory syphons. Most of the Culex in the restricted sense, and many others of the Culicina and Jideomyina have long respiratory syphons. A re-classification on the basis of the syphonic index would break up the present classifications founded on adult characters, whether those adult characters were on the character of the palps, pro- boscis, or scales. It would be less convenient than the classification founded on adult characteristics. The openings of the respiratory tubes at the end of the 262 ■ PUP^ syphon are often guarded by mobile flaps, and hairs and spines are present on the syphons in most species. In some of the ^deomyina these are very numerous. The alimentary system of the larva consists of a tube, apparently structureless and of uniform calibre, running from the mouth to the anus. In the more fully grown specimens this tube is seen to be contained inside the true intestine, which is arranged as in the adult. In the space between is clear fluid not containing any ^ood particles. The Malpighian tubes and other appendages of the alimentary canal of the adult are present at this stage. The intestinal system, including the inner tube con- taining the food, and the outer tube and appendages, can be pulled out of the larval case in a manner similar to that by which the intestine is removed from the adult, but it is more easily done by extraction through the anterior part of the larva than from the tail. The space between the temporary and permanent intestine may' contain gregarines and numerous micro- organisms, and it is to this space that attention should be paid in investigating the conveyance of parasites acquired by larvae. The respiratory system of the larva is comparatively simple. At the end of the respiratory syphon, if there be one, or from the dorsal surface of the eighth abdominal segment, are the openings leading into the two main tracheae, which pass up the abdomen, giving off branches to each segment and inosculating freely in the thorax. They send off branches to the various parts here and to the head. The Pupa. — When the larva has reached its full stage of development the thorax becomes swollen, casts its cuticle with all the appendages, and becomes a pupa. The organs are already formed. The pupa differs most materially from the larva in that there is no longer a mouth opening externally, and the respiration is conducted through two tubular openings arising on each side of the compound head EGGS, LARV^ AND PUPvE 263 and thorax. The change in appearance is great, the head and thorax are fused, and the only external append- ages are the two respiratory tubes. The abdomen is still segmented and is usually curved, so that the termina- tion is under the compound thorax. It terminates in two large fins. The pupal stage is a comparatively short one. There is no possibility of feeding and the pupa remains quiet, breathing through the respiratory tubes unless disturbed, whilst the more complete development of the imago takes place within its sheath. The duration of the pupal stage is affected by the temperature, but is usually from two to five days. The pupa of some species will not remain alive longer than a few days if the conditions are not favourable for development. In the examination of eggs, larvae and pupae, the points to be observed are as follows : — Eggs. — (i) The size, shape, colour. (2) The manner in which the eggs are arranged and where deposited. (3) The character of any thickenings or other external markings. (4) The length of time required under stated conditions, temperature and so on, between the deposi- tion of the eggs and the hatching of the larvae, and any variations noted with variations of conditions. (5) The effect of desiccation, immersion and temperature on the vitality of the eggs. Larva. — In the larva the relative sizes and shapes of the different divisions — head, thorax and abdomen. The character of the head appendages, the antennae, mouth apparatus, &c. Any marked colouring. Much work has been done on the differences in the appendages of the head of Anophelina larvae, and it has been shown that the differences are so marked in their arrangement that many of the species can be distinguished as larvae. In the thorax the character of the lateral hairs and any characteristic markings must be noted. In the abdomen the points of greatest importance are the appendages on the eighth and ninth segments. 264 LARViE The presence or absence of a respiratory syphon attached to the eighth segment is one of the most important generic dififerences. Where present it varies in length and shape in different genera. In different species it varies in colour and in the distribution of colour so markedly that it is often easier to distinguish between different species by the character of the syphon than it is to distinguish between the adults. Varying positions of larvae are associated with the differences in length or the absence of the syphon. The arrangement of bristles and hairs on the eighth and ninth segments present marked differences in the different species. In Anophelina on the other segments, in addition to the lateral hairs there is on each side a row of stellate or palmate hairs. These are nearer the middle line than the simple bristles, and the stellate portion forms a kind of cup. This adheres to the surface film of the water and aids the larva in maintaining its horizontal position. Colouring of larvae is of less importance, as in some species the colour may vary from yellow to green, brown, or even black. In others variations are comparatively small, these are usually dark under all circumstances. In noting the colour any conspicuous marking must be mentioned, the conditions under which the larvae was grown, and whether or not change of conditions, such as greater light, different food, &c., results in a change of colour. The nature of the food can be determined by the examination of the contents of the intestine, or by watch- ing the larvae feed in water containing a mixture of natural foods. It will be found to vary. The kind of food on which they thrive best should be noted. The duration of the larval stage under as many diverse conditions as possible, including exposure to light, heat, and cold, and any observations as to the conditions pre- disposing to death or leading to an undue proportion of males in the imagines, should be noted. BREEDING PLACES 265 The natural enemies of the mosquito lai'vse, fish, larvae of other insects, &c., are of great importance. Where possible the species should be determined. If the larvae are caught as larvae and not reared from eggs particular care should be taken to observe the nature of the places in which the larvae were found. Breeding places of the known carriers of disease, such as Anophelines, Stegomyia and Culex, require very detailed consideration. It is convenient to divide these into permanent waters such as will withstand a con- siderable period of rainless weather, and temporary waters, which require frequent renewal. They may be natural or artificial. Of permanent waters, rivers, large ponds and the edges of lakes under certain conditions are of the utmost iniportance. In such situations the larvae are usually widely scattered, and without the repeated routine use of a dipper such places, often the most im- portant, are usually overlooked. The conditions favourable are the growth of grasses, reeds or sedges in the water. These growths check the stream, provide food, and protect to some extent the larvae from their natural enemies. There are two main classes of growths important : — (i) Those growing from the bed of the river or lake, in the shallows and on shelving banks. The height of the water greatly affects the area suitable. The taller and thicker sedges are not so suitable as the lower and thinner ones, probably on account of the absence of light and too great stagnation of the water (fig. 1 1 5). (2) Those growing from floating masses of roots and attached to the earth only near the edge of the river. The raft formed by the closely interlaced roots is sub- merged by the weight of the grass growing in the air, and in the shallow water lying above this raft of roots Ano- pheline larvae breed freely. No alteration in the level of the water makes any material difference to this, a com- mon class of breeding place (fig. 116). In flood times islets of this floating grass are torn off and carried down 266 BREEDING PLACES the stream, carrying with them larvae, and in this manner they may be carried long distances down the river. It is not improbable that the cutting of the sudd in rivers may result in. larvae of mosquitoes being carried for long distances down the river and thus extending the area of distribution of these mosquitoes, but according to Balfour no such result seems to have taken place on the Nile. Rivers are dangerous when variations in level are not too great or too rapid. Such streams as have a constant Fig. 115. Fig. 116. supply independent directly of the rainfall are particu- larly dangerous. Such sources are the melting of the snow from snow-covered mountains and the effluents of large lakes. Springs which often arise on the slopes of hills are other important permanent breeding places. These usually commence as a small pool with a surrounding swampy area. The grasses round are often of different species or grow more luxuriandy than elsewhere, and these places can therefore usually be identified with ease. The streams arising from such springs are not of much BREEDING PLACES 267 importance during heavy rains, but when the water supply is diminished, wherever the streams spread into swampy areas, or form pools fringed with vegetation, or in backwaters, larvae are usually to be found with the aid of the dipper. In some of these situations they are carried by the stream from the springs or other breeding places. In others the eggs may be deposited and hatch in the place in which the larvae are found. Amongst the easiest places to find larvae are the pools left in the bed of such a stream when the spring commences to dry up, par- ticularly if a small current connects the pools and keeps the water fresh. Some springs will dry up after a month's dry weather, others in three or four months, but some are usually permanent though the water may be scanty from one wet season to another. Swamps unless kept supplied by fresh water are not suitafjle breeding places for some species. For other species they are suitable provided the vegetation is not too rank. A high-level subsoil water may lead to formation of natural permanent pools. On the sandy shores of great lakes the sand is usually thrown up into a ridge with a hollow behind it, and in this hollow as long as the lake level is high water will be present and forms a suitable breeding place. As the lake level reaches its greatest height at the end of the wet season and very slowly falls in the di-y season, these pools may persist in the vicinity of such lakes for some months after the rains have ceased. Temporary breeding places are of many different classes. Almost any hollow or hole that will contain water is a suitable breeding place during continuous rains. If the rain be intermittent only such places as can retain water during the periods of intermission are suitable. Such places require a frequent and heavy rainfall and an im- pervious soil, and are not often found except under these conditions. The " Anopheles' pool " most often described belongs to this class and is exceptional in many places whprp AnnhhAina are abundant. 268 ARTIFICIAL BREEDING PLACES A place that is frequently flushed is not a suitable breeding ground, but irrigation trenches or natural hollows are good breeding places for some species if the area of the trenches is such that the water supplying it is insufficient to flush it in its whole extent. Artificial Breeding Places. — Borrow pits at the sides of railway embankments, the trenches so often made in the course of road-making, and hollows or furrows made in native or other gardens, are common breeding places of Anophelines and some other mosquitoes. A high level of the subsoil water is necessary for these places to be of importance. Irrigation systems where the water supply is con- tinuous but insufficient to flush are important places. In any case, even with a well-designed system, if the source of the water be from a natural breeding place larvae will be conveyed all over the irrigation system. Instances occur in which larvae are conveyed for over a mile by such a trench from a natural permanent breed- ing place to a European settlement. Obstructions in the course of a stream, such as Irish crossings, dams, &c., may convert an inferior natural breeding ground into an excellent one. Badly graded gutters, broken bottles, water-butts, empty tins and any artificial receptacle that will hold water are preferential breeding places for some species of mos- quitoes, particularly those belonging to the genus Stegomyia. Wells in many places do not seem to be breeding places, but in other places they certainly are. On the whole, artificial breeding places are usually the work of Europeans, and the worker in the Tropics has rarely to go beyond his own grounds to find larvae of several species of mosquitoes. Too little attention has been paid to the breeding places of different species. We know that as great differences occur in the preferential breeding places of different species as of different genera, but little exact work has been done on the subject. CARRIAGE OF MOSQUITOES 269 For exact descriptions of the larvae of one species which might serve as an example the reader is referred to the articles on Anopheles maculipeniiis, by Nuttall and others, in the first volume of the Journal of Hygiene. The pupcE differ less from each other than the larvae, and many insects form pupae that are not unlike those of the CulicidcE. The greatest differences are to be observed in the respiratory tubes. In all the Cullcidce they are simple tubes with one opening. In the Anopheles the opening of the tube is a wide, expanded, trumpet-shaped one ; in the Culex the opening is more of a slit and the termination is little expanded. In Mansonia, according to Dr. Low, the tubes are very long and slightly bent forward. In the different species there are variations in the size of the pupa and in the colour. The majority are, after exposure to light, brown or black, though when first formed they are yellow. A few are green, though most of these become dark before maturity. To hatch out the pupae all that is required is that they should not be disturbed and that they should be kept in clean water. No food is needed. They should be kept in a half light. Carriage of Mosquitoes. — Mosquitoes may be carried in any stage of their existence. As eggs they are not very easy to carry, as those that float are often washed on to the sides of the vessel and there are dried and killed. Eggs like those of Stegomyia, which sink and are not injured by immersion, are easily carried, and it is probably owing to this that these mosquitoes or their larvae are so often found on board ship. The carriage of larvae we have already dealt with. For the development of many species light is a necessity, and consequently such species, including most of the Anophelince, are not carried far by the sea, as most of the fresh water is necessarily in closed casks or other dark receptacles. The adult mosquitoes must be carefully carried as they are easily injured by rough handling or bruising. 270 CARRIAGE OF MOSQUITOES On the whole glass vessels should be avoided because of the hard surface of the glass. Mosquitoes cannot hold on to it. If grass or other substances be placed in the glass vessel water of condensation is often de- posited on the glass and the mosquitoes adhere by the wings to this wet surface and speedily die. If glass vessels, test tubes, &c., are used, the mosquitoes must be carried very carefully, and no water be placed Fig. n;. in the vessel. Jungle mosquitoes will be killed by ex- posure to dry air, and with them the plug of cotton-wool should be kept wet. A light cage covered with mosquito netting is as good an arrangement as any, though at a pinch a small box covered with netting on the open side will work satis- factorily. The box designed by Dr. Sambon and containing four MOSQUITO CAGES 271 compartments, each containing a cylindrical wire cage covered with netting, is an excellent one (fig. 117). It was in such cages that infected mosquitoes were sent from Italy to the. London School of Tropical Medicine for the well-known infection experiments, which resulted in the practical demonstration that mosquitoes infected with the malaria parasite could infect men in a country where there was no other possibility of acquiring an infection. Fig. 118. A simple cage can be made by having a portable wire cage that can be folded flat, and bags of mosquito netting which are pulled over the framework. The open end is then tied in a knot (fig. 118). Adult mosquitoes can be kept in test tubes or wide- necked bottles covered with fine gauze or mosquito netting. A little water should be placed at the bottom and some resting place, such as a piece of stick, blade of grass, or folded card placed above the water. Many mosquitoes will feed readily through the netting, others will not, though they feed readily in a larger space. Mosquitoes thrive better if kept in a larger space. The box slightly modified from Dr. Sambon's cage is \TPrv r-nnvpnifitit for this Duroose. The front is c.nm- 2/2 MOSQUITO CAGES posed of glass in two pieces for convenience in packing, while the ends are of fine wire gauze to allow the entrance of air. The hole in the centre of this gauze is covered by cotton sleeves. These are convenient for the intro- duction of an arm for feeding experiments. Through these holes the hand and test tube can be introduced when we wish to catch a mosquito. The pieces of glass forming the face of the box slide in a groove and can be removed when required (fig. iig)- Fig. 119. Ripe fruit, such as apples, dates and' bananas, serves as food for mosquitoes, but some will not lay eggs unless supplied with blood. As substitutes for fruit, sugar, syrups or jams will serve. Some species, Stegomyia for example, are easy to keep in captivity and can be kept alive for months ; others will die in a few days. Ants of many kinds are very destructive to mosquitoes, particularly to those confined in small spaces. To avoid this the mosquito cage should be placed on legs, each of which rests in a small tin containing kerosene. 273 CHAPTER XV. Fleas, Lice and Bed-bugs. In view of the increasing evidence in favour of the hypothesis that rats and their parasitic insects play an important part in the dissemination of plague, it is con- sidered desirable to include here some notes on fleas that may aid the worker in recognising the more common species. It has long been known that fleas may serve as the intermediate hosts of certain parasites. The cysticercus stage of Dipylidium caninum is passed in the dog-flea, Ctenocephalus serraticeps, as well as in the dog-louse, and healthy rats have been infected , with Trypanosoma lewisi by allowing fleas taken from infected rats to feed upon them. The work of the Indian Plague Commission, as well as observations elsewhere, give ground for thinking that fleas are the carriers of the Bacillus pestis from rats to man. Fleas are usually included in the order Diptera as a sub-order, the Aphaniptera {vide p. 162). They differ from most other dipterous insects not only in the absence of wings, but also in that the three thoracic segments are distinct from one another and that, beyond carrying legs, these segments differ but little from the abdominal segments. The antennae also differ from those found in other Diptera. Capture and Examination of Fleas. — To collect fleas from rats, mice, &c., it is best to confine the animal in a vessel at the bottom of which is a piece of cotton-wool soaked in chloroform. The fleas are killed more quickly 274 FLEAS than the host, and may be picked from among the hairs or from the bottom of the vessel. If it is required to examine the internal structure this method is not very satisfactory as the fleas are frequently found filled with blood, and this obscures details. A preferable method is to capture them alive and keep them in a test-tube until they die of starvation. They may be examined directly, or if it is desired to make permanent preparations they should be rendered MdHMd Lp. Fig. izo. — Mouth-parts of a Flea (Vermipsylla alakurt, i). (After Wagner.) A., Median lancet; if., labial palpi; md., mandibles; mx., maicills ; mx.p., maxillary palpi. transparent and mounted! There are many methods for clearing and mounting small insects. Glycerine is a good clarifying medium but it takes a long time to penetrate. A method that gives good results is to fix in absolute alcohol, leaving them about one day to completely dehydrate, clear in xylol and mount in Canada balsam. The preparations should be examined not only by trans- mitted light but also by direct illumination to note the depressions, the direction of hairs, &c. EXTERNAL ANATOMY 275 External Anatomy. — Mouth-parts. — These comprise a perforating and suctorial tube and two free pieces, the maxillae (fig. 120). The maxillce (mx.) have the form of a triangular pyramid, the apex of which projects downwards. Aris- ing from the base of each maxilla is the maxillary palp (mx.p.). The piercing and suctorial apparatus is formed by the mandibles (md.) and the epipharynx (h.). The labial palps (l.p.) act as sheaths. The mandibles have the form of elongated styles with serrations along their distal two-thirds. Each contains on its mesial surface a salivary groove, which at the base widens out into a trough and towards the tip is nearly closed by the approximation of its edges forming a canal. The epipharynx is a pointed, pricking organ, grooved ventrally. By the approximation of the epipharynx and the two mandibles a channel is formed along which blood is sucked. The epipharynx makes a way through the skin, and the mandibles enlarge and lacerate the hole thus made, and convey into it the salivary secretion, and blood is aspirated from the wound along the channel formed by the epipharynx and mandibles. The antennce are contained in fossae at the back part of the head, behind the eyes when these are present, and are directed obliquely downwards and backwards. It is important not to mistake the maxillary palps for them. The antennae are three-jointed. The three thoracic segments known as pro-, meso-, and meta-thorax are freely movable on one another, and to each segment is attached a pair of legs. Each leg is described as composed of five pieces — coxa, tro- chanter, femur, tibia and tarsus, the latter consisting of five segments, on the last of which (metatarsus) are attached two claws. The hairs on the metatarsus of the hindmost pair of legs are of value in classification. The abdomen is oval in shape and is composed of nine 276 FLEAS — METAMORPHOSIS segments. The ninth segment is the smallest and is known as the pygidium. The males are recognised by their small size and by the presence of the coiled penis extending some distance inside the abdomen. Metamorphosis of Fleas. — As in other Diptera, the metamorphosis in fleas is complete. The eggs are laid at all seasons of the year, but their development is more rapid in summer than in winter. The female does not attach the eggs to the skin or hairs of the host, but allows them to fall anywhere, whether it be on the host, on the Fig. 121. — A, Head; b, thorax (l) prothorax or pronotum, (2) mesothorax or mesonotum, (3) metathorax or metanotum ; c, abdomen. earth, or elsewhere. The ova are small, ovoid or rounded in shape, and usually eight to twelve are laid. The larva is hatched out in four to six days in summer and in nine to twelve days in winter. The larvae (fig. 122, e), are footless maggots, whitish in colour, and have thirteen segments, of which the first is provided with a buccal apparatus and antennae. The buccal apparatus is formed for masticating. They are always found in dry places, dust, sand, or clothing.. At the end of about ten days the larva ceases to feed and becomes immobile and forms about itself a small cocoon, to which dirt, sawdust, &c., adhere. In about eleven DISSECTION OF FLEAS 277 days the larva is transformed into a nymph, which hks three pairs of legs and resembles the perfect insect. These nymphs after a further period of about twelve days are transformed into perfect insects and come out of the cocoon. The whole process is accomplished in about a month in summer and six weeks in winter. Dissection. — This is best carried out in normal saline solution, using needles with very fine points. With a simple lens the dissection is very difficult — a stereoscopic microscope is almost an essential. The flea is traAsfixed through the head and held by the left-hand needle. The point of the right-hand needle is then inserted under the edge of the third or fourth abdo- minal segment and the chitinous covering peeled off. The internal organs float out in the salt solution and may be further separated. Internal Anatomy. — The mouth is situated at the base of the bulb of the ventral portion of the epipharynx and forms the commencement of the alimentary canal. The pharynx extends "from the mouth to the oesopha- geal commissure. It is aspiratory in function. There are two salivary glands, each consisting of two lobes, and they lie on each side of the anterior end of the stomach embedded in the fat body. At the junction of the pharynx and the stomach is an organ to which the name of " gizzard " has been applied. It is suggested that its function is to prevent regurgitation of fluids from the stomach. The stomach consists of a basement menibrane, two oblique layers of muscle fibre and a lining layer of cubical cells. I Classification. — Fleas are divided into three families, Pulicidce, Sarcopsyllidce, and Vermipsyllidce, of which only the first two are of interest to us. Family PULiciD^. — Generally speaking larger than unimpregnated Sarcopsyllidce. Head relatively small, rounded above and frequently armed with combs upon Fig. 122. — a, Sarcopsylla penetrans ; b, PuUx irritans ; c, Ctenocephalus serraticeps, d, Ceralophyllus ; e. Larva of Pulex ; f, Pulex cheopis. CLASSIFICATION 279 the sides, or along its inferior border. Labial palps always consisting of four segments. Thorax longer than in Sarcopsyllidce, in some genera having a comb of spines at the posterior border of the prothorax, sometimes also at that of the metathorax. In others there are combs at the posterior border on one or more of the segments of the abdomen. The females never become fixed in the tissues of the host, nor does the abdomen become greatly distended as in Sarcopsyllidce. Genera of family Pidicidce. A. Eye well developed. Comb along inferior border of head and another along posterior border of prothorax. . . Ctenocephalus. Comb along posterior border of prothorax. No comb on the head Ceratophyllus. No combs Pulex. B. Eyes rudimentary or absent. Nearly always spines on each side of head. Always a comb on posterior border of pro- thorax. a. Comb on posterior border of metathorax and on one or more abdominal seg-. ments. On metatarsus of hindmost pair of legs four pairs of lateral hairs and one pair of accessory hairs Ceratopsylla. 0. No comb or posterior border of meta- thorax or on abdominal segments, (i) Hairs on metatarsus as in Ceratopsylla Ctenopsylla. (2) On metatarsus three pairs of lateral hairs ; accessory hairs well developed Typhlopsylla. (3) On metatarsus four pairs of lateral hairs ; accessory hairs absent Neopsylla. y. Combs on some abdominal segments. Body covered with hairs and small spines Hystrichopsylla. Pulex cheopis. — This flea has been described under various names, P. pallidus, P. murinus, &c. It is the commonest flea found on rats in warmer climates, and 28o SARCOPSYLLIDiE in some localities is the only flea. It has been found in Marseilles, Italy, Sydney, Philippines, India, Sudan, South Africa and South America. It is a non-pectinated flea, resembling the human flea, but differs from it, among other characteristics, in the body being lighter in colour, yellowish instead of brownish, and in the position of the ocular hair, which is situated in front of the eye, while in P. irritans it is situated below the eye. Other rat-fleas are Ceratophyllus fasciatus, Ctenopsylla tnusculi, Ctenocephalus serraticeps, Hystrichopsylla talpce, as well as many less common species. '■ Family Sarcopsyllid^. — Body small, head relatively large, angular or rounded above, never armed with spines. Labial palps unsegmented. Thoracic segments small. Abdomen variable, more or less swollen in the fertilised female. Never any combs on thorax or abdomen. The fertilised female burrows into the skin of the host. This family comprises two genera, Sarcopsylla and Rhynchopsylla. The genus Sarcopsylla contains the important parasite, Sarcopsylla penetrans — the Jigger or Chigoe. It is a small flea, the male measuring about i mm. in length, and the unimpregnated female about the same size. The general colour of the body is brownish. The head relatively to the body is large ; its upper surface slopes obliquely in front, and joins the lower surface at an acute angle. The eye is situated in the front part of the head at the border of the fossa of the antenna. The perforating and suctorial mouth-parts are well developed, but the maxillae are very small quadrilateral structures, only seen with difficulty. The three thoracic segments are very small. The males and unimpregnated females are parasitic on man only occasionally, for the purpose of sucking blood. The impregnated female, however, bores her way into the skin, particularly about the feet, and the abdomen of the insect undergoes great distension following the HEMIPTERA 281 development of the eggs, the head and thorax remaining unchanged. The eggs develop on the soil, and the metamorphosis is similar to that which obtains in other fleas. The Jigger is widely distributed in the Tropics. Origm- ally found only in Central and South America, it was introduced into West Africa about 1872, and in a few years had become disseminated throughout the greater part of Africa. Another member of this genus, S. gallinacea, attacks fowls, particularly about the head, and causes great destruction among them. S. gallinacea does not bury itself in the skin so completely as does S. penetrans, nor does the abdomen become so greatly distended, as the eggs are laid one by one as they mature. Order HEMIPTERA. This order includes insects of very dissimilar charac- teristics. The rostrum is the feature by which they are most easily distinguished. This organ is a modification of the inferior lip ; it is tubular in character, and in a state of repose is folded up under the head and thorax. The rostrum encloses the hairlike penetrating parts. Two groups of parasites, known popularly as lice or pediculi, and bed-bugs, are included in this order. The term lice is loosely used, the " lice " of pigeons, bird-lice, are degenerate Orthoptera. Some Hemiptera, as the aphis and scale insects, are very destructive to vegetable life. Family Pediculid^e. — The body is flattened. The rostrum is provided with barbed hooklets. The antennae are inserted in a kind of sinus in the front of the head, and are composed of three, four or five segments, the number of these being used for the separation of genera. These three thoracic segments are distinctly separated. The abdomen comprises six to nine segments. Legs are terminated by one or two long claws. 282 PEDICULI The eggs are pyriform in shape, fixed by their smaller end to the bases of the hairs of the host : the larger end is provided with an operculum which the young split off in emerging. After the eggs are laid the young are hatched out. They resemble the adults and mature rapidly. There is no metamorphosis. The family comprises several genera, of which we consider only Pediculus and Phthirius. _, r Distinct neck ... Pediculus. .Thorax narrower I Antennas with five sag- ( than abdomen l», j,. .. ^ 1 n ^ j.- ments. Legs with a J '"*" ^''°°'"=" I No distinct neck ^«-»?a;.,/<««.. sing e c a VThorax broader than abdomen ... Phthirius. Fig. 123. — Pediculus vestimenti. Pediculus capitis (Head-louse). — The male is i to 2 mm. in length, the female somewhat larger. The head is triangular in shape, the thorax as broad as the abdomen, and the abdomen has eight segments. The female lays fifty to sixty eggs which hatch out in about seven days. The young are able to reproduce in seventeen to twenty days after birth. Pediculus vestimenti (Body-louse). — Male 2 to 3 mm. PEDICULI 283 in length, females larger. Head somewhat rounded. Antennae longer than in P. capitis. The thorax is narrower than the abdomen. The female lays seventy to eighty eggs, which hatch out in three to eight days. Phthiriusinguinalis {Cv2ih-\ovise). — The genus Phthirius includes a single species. This species is peculiar to man and has a preference for the white races. Male o*8 to i"o mm. in length ; female i to i'5 mm. The head is large and provided with two long antennae. Thorax is broader than the abdomen and shows no trace of separation from it. Fig. 124. — Phthirius inguinalis. The female lays ten to fifteen eggs, which hatch out in six to seven days. An interesting observation made by Murray, that the pediculi of different races differed in colour and even in certain anatomical details, has since been confirmed by many observers. This is regarded as a kind of protective mimicry. From observations on these parasites in the Federated Malay States, we believe that the pediculi of one race, for example the Chinese, do not pass readily, if at all, to another race, for example, the Tamils. Family CiMiciD^. — This family of the Hemiptera be- longs to the division known as Heteroptera, as there is a marked difference between the two pairs of wings. 284 BED-BUGS The family is characterised by the absence of ocelli, by the wings being rudimentary, very short and broad, so that the abdomen is left uncovered. The head is short and broad, and the rostrum is received in ' a groove beneath the head. Tarsi are three-jointed. The genus Cimex comprises at least two species parasitic on man, C. ledularius and C. rotundatus. Considerable interest centres about these, in view of Patton's suggestion that they are the carriers of the parasites of kala-azar, and the older hypotheses that they may convey relapsing fever and leprosy. There is no proof in support of these hypotheses. Fig 125. — Cimex lectularius. Cimex lectularius. — This insect is oval in form, brownish- red in colour, with a much flattened body. It measures 4 to 5 mm. in length and 3 mm. in breadth. Eight abdominal segments. The female lays about fifty whitish eggs, three or four times a year. The complete development takes about eleven months during which time it casts its skin five times. The young larvae are at first pale white. Bed-bugs live in cracks in the walls, under carpets, behind pictures, wail paper, &c., and only come out at night to suck blood which is their only food. They may live from five to ten weeks, or even longer, without food. The average duration of life is three to four months. BED-BUGS 285 Cimex rotundatus. — This species was originally described from the island of Reunion in 1852. Patton has shown that it is distributed throughout India, Burma and Assam. It is rather smaller than C. lectularius, and in general outline less rounded. The main feature, however, is the character of the prothorax, which has well-rounded borders and gives to the anjmal an appearance of rotundity. ' Other species of Cimex described are C. ciliatus from Kasan, Russia, which is smaller than the common bed- bug, yellowish-red in colour, and thickly covered with hairs, C. columbarius, C. hirundinus, and C. inodorus, which have been found attacking birds. 286 CHAPTER XVI. Arachnoidea — Ticks, Mites, Porocephalus. CRU STACEA — C YCLOPS . The Arachnoidea are a class of the Arthopoda readily distinguished from insects, as they have four pairs of legs in the adult stage. The head and thorax are usually fused into a compact mass, the cephalothorax. The abdomen is generally without appendages; it may be segmented or unsegmented; it is generally distinct from, but may be fused to, the cephalothorax. Compound eyes such as occur in insects are never present — the eyes when present are always simple. Respiration may be by means of tracheae or cutaneous. The genital orifice is in the middle or in the anterior half of the ventral surface and is dis- tinct from the alimentary canal. The sexes are distinct. In most cases the newly hatched young are essentially like the adults. The class Arachnoidea is divided into a number of orders, including Scorpionidce (scorpions), Pseudoscorpion- idce (book scorpions), Pedipalpi (whip-scorpions), Ara- neidce (spiders), Acarina (mites and ticks), and certain aberrant orders, as LinguatuUda or Pentasiomiidce. Order ACARINA (Mites and Ticks.) — These are small Arachnoids, usually parasitic. They occur in earth, in fresh or salt water, or on animals or plants. They feed on the organisms they infest or upon organic matter. The abdomen and cephalothorax form a fused mass. The mouth-parts are formed for biting or for puncturing and sucking, according to the mode of life. Respiration may be cutaneous, but in most there are tracheae with two TICKS 28: stigmata. Many of the young have only three pairs ol legs when hatched, but after moulting have four pairs. The Acarina which cause disease or act as carriers ol disease are divided into families as follows : — I. Non- vermi- form acarina /{a) Legs inserted Legs with Trachea open- directly into in- six joints ing in the tegument posterior part of body Chelicerffi with hooklets Chelicerae di- dactylous or styliform {i) Legs articulated on distinct epimerse ' Tracheae open- Chelicer8e styli- ing in ante- form or with rior part of hooklets; body palps free 7xc Ga Tn ,No tracheae II. Vermiform acarina Legs articulated Legs with No trachese on distinct epi- three merae joints IXODID^. Chelicers di- Sari d a c ty 1 o u s ; Palps cylin- drical and adherent to inferior lip Cheliceise styli- De form ; palps with hooklets The family Ixodidce is composed of comparatively large Acarines with leathery skins. They have flattened bodies, but after sucking blood become much distended. The mouth-parts, which are characteristic, comprise a median penetrating organism, the hypostome, armed with recurved teeth, two rod-like organs surrounded at their bases by a sheath, the chelicerce, each of which is terminated by a process bearing large hooked teeth. On either side of the chelicerae and hypostome lies a four-jointed palp, which in some genera is closely applied to the piercing organs and appears to act as a sheath for them, but in other genera is quite free (fig. 129). Each leg consists of six main segments known respec- tively as coxa, trochanter, femur, patella, tibia and tarsus. The trochanter may have spines or teeth. The tarsus bears on its terminal segment two claws (fig. 126). There are two sub-families, Ixodince and Argasince. 288 TICKS In the sub-family Ixodince the skin of the dorsal surface is thickened to form a hard, leathery, chitinous plate, the scutum or dorsal shield, which in the male covers almost the whole of the dorsum, while in the female it covers only the anterior portion of the dorsum. This arrange- ment serves to distinguish' males from females. In the sub-family Argasince these shields are not present. Fig. ia6. Eyes are not always present. They consist of a simple lens only and may be globular or flat. In the Ixodince they are situated at the margins of the dorsal shield ; in the Argasince on a ridge above the coxae. The stigmata or openings of the tracheal system are situated behind the level of the fourth pair of legs in .fVSji Fig. 127. — Ixodina (Female), a, Dorsal aspect, showing shield ; i, ventral aspect. 19 290 EXAMINATION OF TICKS Ixodince and between the third and fourth pair of legs m the Argasince. The genital orifice is situated in the middle line on the ventral surface, a short distance behind the rostrum at the level of the second and third coxae. In the middle line, behind the level of the last pair of legs, is the valvular anus. On either side of the anus in certain genera are situated thickenings of the cuticle known as anal plates : the number, shape and position of these are of value in classification. Examination of Ticks. — Ticks can be examined living or di-y, and the main points clearly made out with the aid of a hand lens. The dorsal and ventral aspects must be examined in turn. The points to observe on the ventral surface are along the median line, the rostrum, with the palps on each side ; the opening of the genital organs in the anterior half of the body and the anal opening in the posterior half, and any deep furrow in front or behind this anus, anal furrow. At the sides the position and any special markings on the coxae should be noted ; the respiratory area, stigma, anterior or pos- terior to the fourth coxae and its shape, and the presence or absence of any " shields " or chitinous plates on each side of the anal, adanal shields. On the dorsal aspect the presence or absence of a dorsal shield should be noticed, and its extent as to whether it covers the whole of the back or the anterior part only. Any markings, bosses or protuberances, and how far these form a continuous pattern to the edge of the dorsum, or vary at the margins. Eyes should be looked for. The details in the structure of the mouth-parts are best seen in specimens rendered translucent by treatment with alkalies. In smaller ticks, if not distended with blood or eggs, boiling for a few minutes in a 10 per cent, solution of caustic soda will suffice, but it is better to leave for a longer time in the solution. Many ticks contain chro- matin and are not readily decolourised. After the treat- ment with caustic soda they should be well washed, and Fig. 128. — Ixodiiia (Males), a, Dorsal aspect, showing dorsal shield ; b, ventral aspei in species with adanal plates ; c, ventral aspect in species without adanal plates. 292 ANATOMY OF TICKS may be examined mounted in glycerine or dehydrated with alcohol and oil of cloves and mounted in Canada balsam. Dissection of Ticks. — Christophers recommends the fol- lowing method : Holding the tick between finger and thumb, with a pair of sharp scissors snip fine slices from the edges. Gently wash in normal saHne solution and place in a small dish containing the same solution. By using one pair of forceps to seize the ventral flap and another the dorsal flap at their posterior ends, the latter may be dragged forward over the head, leaving the viscera attached in situ to the ventral surface. The chief anatomical features can be made out by displacing the larger organs and removing with fine forceps the tissues which hold these in position. Internal Anatomy of Ticks. — The chitinous pharyngeal pump, which opens into the mouth, leads posteriorly to a narrow, straight oesophagus. The cesophagus, after perforating the central nerve ganglion, enters the enor- mous saccular portion of the alimentary canal which, with its diverticula, forms the great bulk of the body contents. Posteriorly an extremely fine canal joints the central saccular gut with the rectum. Opening into the rectum on either side is a Malpighian tubule. These latter are two fine white or transparent tubules of great length which, after a complicated course among the viscera,, end blindly m the anterior part of the body. The rectum has several diverticula and terminates in the anus. The salivary glands, two in number, lie over the bases of the first two pairs of legs. There is a central duct which arises near the free extremity, and passing through the whole length of the gland becomes the salivary duct. The salivary ducts open into the mouth. In the female the single ovary lies upon the diverticula of the alimen- tary canal in the posterior portion of the body. Leading from it on either side are the long coiled oviducts. In front of the ovary is the spermatheca. In the male, in the LIFE-HISTORY OF TICKS 293 same position as the ovary in the female, is a thin delicate tubule, the testis. Life-history. — The ova of ticks are laid in recesses in the soil : in the Ixodiiice the number of these is 2,000 to 4,000, in the Argasince a few hundreds only. After a variable period the six-legged larva is hatched out. The larvae attach themselves at the first opportunity to some vertebrate, from which they suck blood. In some ticks the larva, having gorged itself with blood, drops off upon the ground, and after a few days casts its skin and emerges as the eight-legged nymph, which differs from the adult form mainly in the absence of a genital opening. The nymph in turn attacks a fresh host, and having fed, drops off, and after moulting becomes the adult tick. Hcemaphysalis leachi undergoes its development in this way. In other ticks, such as Megaropus annnlatus, the metamorphosis from larva to nymph takes place on the host. The nymph may or may not leave the host before reaching the adult stage. In Ornithodoros moubata and 0. savignyi the larvae do not suck blood. Ticks as Carriers of Disease. — Ticks are known to be carriers of the several species of haematozoal parasites be- longing to the genera Piroplasma and Spirochceta. In man a spirochaete (S. duttoni) which is the causative agent in African relapsing fever is carried by a tick, Ornithodoros moubata, and an analagous disease of fowls, also due to a spirochete, is transmitted by Argas niiniatus. In mammals a number of diseases conveni- ently grouped under the name Piroplasmoses are carried by ticks belonging to the sub-family Ixodince. It is important to note the stage in its development at which a tick may transmit parasites, as this differs accord- ing to the species of parasite and the species of tick con- cerned. For example, larvas of Megaropus annulatiis from an infected mother are able to transmit Texas fever, while in the case of Hcemophysalis leachi, one of the carriers of canine piroplasmosis, the larvae and nymphs are not infective, and only the adult can convey the dis- 294 CLASSIFICATION OF TICKS ease ; again, in the case of A mblyomma hebrcerum, which conveys the parasite of heartwater in sheep and goats, the nymph is infective if fed as a larva, and the adult if fed as a nymph. Systematic Classification of Ticks. Rostrum on ventral surface of body not visible from above in the adults. No dorsal shields or adanal plates. Stigma in front of fourth coxa. Palps fine Sub-family Argasina. Fig, 129. — Mouth-patts of OrnithoJoros. Rostrum terminal. Dorsal shields present. Adanal plates in males of some genera only. Stigma posterior to fourth coxa. Palps grooved on internal aspect and usually closely applied to rostrum Sub-family Ixodina. Sub-family Argasina. Body with sharp edges. No eyes. No deep furrows on ventral surface. Skin wrinkled. Pattern of marking on the edge differs from that on the remainder of the dorsum Genus Argas. Body with thick edges. Eyes present in some species. Often deep furrows on ventral sur- face. Skin covered with bosses and the pattern of these is continued to the edge... Genus Omithodoros. IXODIN^ 295 Sub-family Ixodina. Palps long, the second joint much longer than broad Group I. Ixodina. Palps short, the second joint much broader than long Group II. Rhipicephala. Fig. l30.^Mouth-parts of Ixodes. Fig. 131. — Mouth-parts of Rhipicephalus. Group I. IxaiUncB. 1. Anal furrow in front of anus. No eyes Genus Ixodes. 2. Anal furrow behind anus, often continued laterally to genital furrow (fig. 127). (i) Anal plates present in $. Eyes present Genus Hyalomma. 296 IXODIN^-ORNITHODOROS (2) No anal plates in ^ . (a) With eyes Genus Amd/yomma. {d) Without eyes Genus Aponomma. Group II. Rhipicephalce. 1. No eyes present. No adanal plates in $ . Second segment of palp has a well-marked lateral projection... Genus Hcemaphysalis. 2. Eyes present. {a) No adanal plates in $ . Base of rostrum quadrilateral Genus Dermacentor. {b) With adanal plates in ^ . Base of rostrum hexagonal, with well-marked lateral angle. (i) Two adanal shields, free or united in front, with or without accessory shields. No pre-anal furrow. Stig- mata circular or oval (fig. 128, b.) Genus Megaropus. (2) Two adanal shields, free. Pre-anal furrow present. Stigmata comma- shaped (fig. 128, <5.) Genus Rhipicephalus. Omithodoros moubata. — Length about 8 mm., width 6 to 7 mm. General shape of the body ovoid, somewhat wider behind than in front ; yellowish-brown in colour when young, greenish-brown when adult. Integument studded with small tubercles. On the dorsal surface three pairs of grooves running obliquely downwards and inwards towards posterior end. Above the bases of the legs a longitudinal supra- coxal groove. On the ventral surface there is a deep pre-anal groove joining the supra- cbxal grooves laterally ; behind the anus three pairs of longitudinal grooves. There are no eyes present. Stigma semilunar in front above the supra-coxal groove. Fourth leg one and a half times as long as the first ; tibiae and tarsi of first three pairs with three teeth on the upper side. This species is widely distributed in Central Africa from east to west. Another species, 0. savignyi, in which eyes ■ are present, has been found in SomaHland, German East Africa, the Congo and India. (Fig. T32, 0. savignyi, dorsal and ventral surface.) Fig. 132. — Orniihodoros savignyi. a. Ventral aspect ; i, dorsal aspect ; c, lateral aspect between second and third pair of legs. 298 DEMODEX Family Deinodicidce. — Body small, vermiform, com- prising an anterior part provided with legs and a posterior part showing transverse striation. Mandibles styliform, palps consisting of three segments, the last of which has a curved booklet. No eyes. Four pairs of legs. No stigmata. Larvae have three pairs of rudi- mentary legs or may be footless. These animals are parasites of the hair folUcles and sebaceous glands of the skin of mammals. Fig. 133. — Demodex follicularum. Demodex follicularum (fig. 133) is a common pai^asite in man, and may cause inflammation by obstructing excretory gland ducts. The male is about 3 mm. in length and the female 4 mm. in length. Order LiNGUATUHD^E. — Probably degenerate Arach- noidea. They are wormlike in form and externally annulated ; there is no distinction between head, thorax, and abdomen. No oral appendages are present, but there are two pairs of movable hooks near to the mouth, which LINGUATULID^ 299 are regarded as remains of the antennae and palpi. Alimentary canal simple, no Malpighian tubes. Thei-e are no sense organs or tracheae. The sexes are distinct ; the males smaller than the females. Adults live in the nasal cavity, frontal sinus, or lungs of the dog, wolf, and other animals ; occasionally they are found in man. The females lay eggs, which if taken up by an inter- mediate host give rise to embryos. These embryos pass from the intestine to the liver or lung where they encyst, moult, and pass through a larval stage, in which the characters of the adult are developed. Finally they reach the nasal cavities of the same or a new host and become sexually mature. Porocephalus armillatus. — The larval form originally known as Pentastomum constrictum is probably a Poro- cephalus. It has been foimd on several occasions encysted in the livers of negroes in Africa. The number of rings is never more than twenty-two, which corresponds to the number of rings in the larval forms of Porocephalus armillatus. According to Sambon the adul't forms of this group occur in the lungs of the Royal Python and in those of the Nose-horned Viper. Class Crustacea. Order Copepoda. — Elongated crus- taceans, usually with distinct segments. No dorsal shell. Five pairs of biramose thoracic appendages, the last of which may be rudimentary. Abdomen without limbs. The females carry the eggs in external ovi-sacs (fig. 134). To this order belongs the freshwater Cyclops, a tropical species of this genus is the intermediate host for Filaria medinensis the well-known Guinea-worm. 300 CRUSTACEA Fig. 134. 30I CHAPTER XVII. Pigment Deposits and Degenerations in Tissues. The pigment deposits are in the main those derived from altered haemoglobin. Of these the most important is melanin, the residue from the digestion of the red corpuscles by the parasites of malaria. This pigment is taken up by the leucocytes and other phagocytic cells and deposited in various parts of the body. As seen in the interior of a parasite it is at first finely divided and varies in colour according to the species of parasite, later it may be aggregated in each parasite into a mass. In the large masses which are found in the tissues it is black with a slight greenish tinge, it is insoluble in acids, in alcohol and in ether, but is readily dissolved by alkalies. It is very stable and is not destroyed by putrefaction. In solution in alkalies it has a distinctly greenish tinge, but shows no charac- teristic bands when examined with the spectroscope. From this solution it can be precipitated by the addition of acids, and by repeated solution in alkaline fluids and precipitation with acids, can be isolated in an impure condition. Analysis shows that it is very rich in iron, more so than haemoglobin or any of the other haemoglobin derivatives. The iron is in firm organic combination, and does not give the inorganic iron reactions. After frequent precipitations it becomes brown in colour, and the same brownish tinge can be observed in pigment left in certain organs as a result of old malarial invasion. 302 MELANIN Melanm in an acute attack of malaria is found (a) in parasites, (6) in leucocytes, and (c) in certain cells of the connective tissue type in the liver, in the parenchy- matous cells in the spleen, and occasionally in the nuclei of the endothelium of the capillaries in various parts of the body, such as the brain, liver, suprarenals, &c. In an invasion of short duration the only pigment found is in small granules, often no larger than those set free after the breaking up of a sporulating body. At this stage the cells containing the pigment stain normally and do not differ in appearance from other cells of the same type which do not contain pigment. When death occurs some little time after the termina- tion of an attack of malaria the distribution of tjie melanin deposits is much more limited. It will not be found in the red corpuscles as there are no parasites, nor in the leucocytes or endothelial cells, but will be restricted to the connective tissue cells in the liver and the paren- chyma of the spleen. The pigment itself is now aggre- gated into larger masses, though these may be seen to be composed of separate granules. The cells stain faintly with ordinary stains and often appear to be shrunken or distorted. If examined still later the pigment will only be found in blocks or masses, and the cells containing these blocks will not take basic stains, and appear merely as an outline round the pigment masses, some of which appear to be free. The change to a brownish colour, particularly at the edges of such masses, is sometimes to be noted. Later, even months or years after the malarial attack' pigment may be found. If present it will be imbedded in the fibrous stroma and no trace of the cells will be seen. At this stage the spleen is usually the only organ in which the pigment will be found. A consideration of these changes will show that the date of a malarial invasion or invasions can be inferred from the melanin deposits in the organs if the state of division of the pigment, the staining reactions of the cells HEMOSIDERIN ' 303 containing the pigment, and the situation of the pigment be observed. It cannot be concluded because no pigment is present that there has not been antecedent malaria. We have proof that pigment is rapidly removed from all organs except the liver and spleen (and occasionally the lymphatic glands), and also that it may be removed from either the liver or spleen. In all recent attacks both oi-gans contain pigment ; after a longer interval it may be present in the spleen only, or rarely in the liver only. In the older cases when malarial infections have not occurred for a prolonged period the amount of melanin found is usually small. In some cases, even a few weeks after an attack of malaria, the amount of melanin is so small that careful search is required to reveal it. It seems probable that as long as the pigment is contained in living cells it is fairly readily removed. In many cases there is evidence that the pigment (melanin) deposited is the result of several distinct attacks, as in the same specimen finely divided pigment in cells which stain normally, coarse pigment in cells that stain poorly, blocks of pigment with no traces of a cbntaining cell, and pigment in between strands of fibrous tissue, can all be seen. Melanin is the only pigment which is characteristic of malaria. Another pigmentary deposit of a bright yellow colour is often found in the organs in cases of malaria, but this is also found in pernicious anaemia, in the anaemia of ankylostomiasis, and in other cases where haemolysis or blood (destruction has taken place, such as trypanoso- miasis and kala-azar. This yellow pigment differs from melanin not only in colour but in that it is insoluble in alkali as well as in acid. It appears to be slightly soluble in alcohol. Whether or not it contains iron is difficult to ascertain, as it ia frequently associated with other substances con- taining iron in inorganic combination. When found 304 ' IRON DEPOSITS alone it usually does not give the reactions for inorganic iron. This yellow pigment is found in the true hepatic cells, in the secreting cells of the kidney, particularly in the first part of the convoluted tubules, and in the spleen. It is evidence of blood destruction from any cause, whether acute, as in blackwater fever, or chronic, as in pernicious anaemia or ankylostomiasis. Both these pigment deposits can be observed without cutting sections by making "squash" preparations of tissues of the organs, but the arrangement is better shown in sections. Merely to detect the pigment no stain is needed, but to show the character of the cells containing the pigment it is well to stain lightly. Haematoxylin gives good results, but a better stain is carmine, as both the melanin and the yellow pigment stand out better against the red background. Thionin should not be used as it has an affinity for these pigments or the protoplasm surround- ing them. In many cases granules that contain iron in a condi- tion to react to the usual tests for inorganic iron are associated with the yellow pigment. Ammonia sulphide is sometimes used as the test for the demonstration of inorganic iron, but has the disadvantage that the brown sulphide of iron deposited can be confused with m'alarial pigment. A better reagent is ferrocyanide of potassium in an acid solution, as the blue ferrocyanide of iron is characteristic and causes no confusion. The section should be first treated with a 2^ per cent, aqueous solution of potassium ferrocyanide for five minutes and then with a i per cent, solution of hydro- chloric acid in glycerine, or with acid alcohol. The acid glycerine should be slightly warmed, and must be left on till the blue colour is quite distinct. If the blue colour only shows faintly the specimen can be replaced in the ferrocyanide solution and again treated with acid glyce- rine or acid alcohol. The specimen can then be washed IRON DEPOSITS 305 in water, dehydrated in alcohol, cleared in xylol, and mounted in balsam. It is important that the sections should not be touched with iron after they are cut, so that they must not be lifted with a needle, as in any place touched with iron there may be a deposit of the blue ferrocyanide of iron. Loosely combined iron will be shown blue, whilst the melanin in which the iron is in firm combination will remain black. The yellow pigment may be in part turned blue or may be unaltered. The outlines of the cells can generally be seen and counter-staining is not necessary, but weak carmine solutions can be used if it is desired. The iron may be diffused throughout the cells or may be found in granules either alone or mixed with yellow pigment. The relationship of these ferruginous granules to the yellow pigment is not definitely known. In the most acute haemolytic processes, such as in blackwater fever, both are present and the iron-bearing granules are the most numerous. In the most chronic forms, such as some cases of ankylostomiasis, yellow pigment alone will be found. The balance of evidence is in favour of the view that ferruginous granules are evidence of active and recent haemolysis, whilst the yellow pigment is a more perma- nent substance, and though formed as a result of acute haemolysis is, when in considerable amount, evidence rather of a prolonged or chronic haemolysis. Some authorities hold other views, and consider that the yellow pigment when old gives the iron reaction. The important point is that both these substances are proof of blood destruction, and are not evidence of malaria, although often found in cases of malaria. They are evidence of the general blood destruction that may be caused by the parasites of malaria as well as by other organisms. In the vicinity of certain skin lesions there may be 20 306 PIGMENT considerable disturbance in the normal arrangement of the pigment, so that instead of being deposited only in the deeper layers of the epidermis it is scattered not only in th^ superficial layers of the epidermis, but also in the subcuticular connective tissues. The disturbance of the arrangement of pigment is most conspicuous in growths of the granulomatous group, including lichen hypertrophicus. Pigment is normally present in the skin, and it is com- mon to find pigment in the mucous membranes, particu- larly of the mouth in the coloured races. Such pigment is generally found in patches in the mucous membrane of the tongue, cheeks, or gums. It has no connection with malaria or other disease, but is more conspicuous in cases of advanced anaemia, as the pigmented patches then stand out more markedly against the general white background. The normal pigmentation of the pia mater has been already mentioned. It is extravascular, and is not dis- solved by alkalies. The pigment in all these cases is much less soluble in alkaline solutions than the melanin of malaria. Pigmen- tation of the skin as a result of Addison's disease is also well known. In melanotic sarcoma, black or brown pigment is also deposited in the growth. Degeneration. — Cells exposed to various 'influences undergo degenerative processes. Death or necrosis of cells may take place, and in such cases the cell ceases to stain normally, so that instead of taking up basic stains it stains with acid stains, or feebly with both acid and basic stains. The nuclei break up and lose their characteristic staining reactions, and the whole cell may disintegrate and be converted into granular debris, or caseation may take place in which a mass of cells is replaced by granular fatty material. The mass may become calcified. Where the morbid influences are insufficient to cause DEGENERATION 307 cellular death, changes occur in the protoplasm. Of these the more important are : (i) " Cloudy swelling," in which the protoplasm of the cell becomes swollen and the aspect of the cell changed so that its contents become obscured and very finely granular. This change is best seen in fresh, unfixed cells and is shown in stained speci- mens by an irregularity in the staining. This change occurs in the early stages of inflammatory action and may be general in any prolonged pyrexia. (2) Fatty degeneration may affect any cells, but more especially muscular fibres and the glandular cells of the liver, kidney, intestinal mucosa, &c. In this latter form droplets of fat are found in the interior of the cells : these at first are small, but in advanced cases the whole contents of the cell appear to be replaced by fat and the nucleus is squeezed to one side. With fresh specimens the high refractive index of the fat renders the diagnosis easy. In specimens passed through alcohol, &c., the fat is dissolved out and the condition is then recognised by the meshwork of the protoplasm having clear, round, unstained spaces which were previously occupied by the fat globules. Special methods show this form of degeneration more clearly. In specimens hardened in any of the osmic acid fixatives, such as Fleming's solution, or cut fresh and treated with weak osmic acid, the fat will be stained a deep and intense black. Such sections can be counter- stained with safranin. Soudan III. and Scharlach R. are also good stains for fat. Fresh tissues or tissues hardened in formalin must be used. The sections are treated with a saturated alcoholic solution of the stains (80 per cent, alcohol) for fifteen minutes, rapidly washed in 50 per cenf. alcohol and washed in distilled water. They can be counter- stained with haematoxylin and mounted in any glycerine medium. The fat will be stained a deep red. A rough estimate of the amount and extent of the fatty degenera- tion may be made from such a section, but the main 3o8 FATTY DEGENERATION advantage of the method is to show the distribution of the degeneration and the class of cells mainly involved. A promising method for the estimation of the extent of this degenerative process is the determination of the specific gravity of the organs. In some cases the fat is in sufficient amount to cause the entire liver to float in water, but more commonly it is short of this. To determine the specific gravity a large portion of an organ is weighed and the volume of this portion deter- mined. This volume can be ascertained in the course of an ordinary post-mortem examination by the use of a vessel with an open tube fixed at the side. The vessel is filled with water till the water escapes from the tube. When the water has ceased to escape a receiver is placed under the tube and the weighed portion of the organ is placed in the vessel. Water will again escape from the tube, is collected in the receiver and measured. The volume of this water is the same as that of the organ placed in the vessel, as it is the amount displaced by it. We now know the volume of a given weight of the organ and therefore its specific gravity. This method is sufficiently exact for ordinary purposes if a sufficiently large piece of the organ is taken, but for comparative purposes more information is required than we at pre- sent possess as to the normal variations in the specific gravities of organs. Fatty degeneration is an important factor in many tropical diseases. It is marked in yellow fever almost as much as in poisoning by phosphorus. In the anaemia of ankylostomiasis if is constant and pronounced, and as it affects extensively the intestinal mucosa, it is, in the more chronic cases, largely responsible for the impair- ment of the digestive processes in some of these cases. It also occurs in the liver and kidneys in this disease, and as there is also a deposit of hsemosiderin these organs often appear to be of a dusky chrome-yellow • AMYLOID DEGENERATION 309 colour. The occurrence of this degeneration in the cardiac muscles is more serious, as cardiac failure, either acute or chronic, frequently is due to this condition. Amyloid degeneration is best shown in fresh sections. Macrosopically it can. usually be determined by treat- ing a cut surface of an organ with tincture of iodine ; a deep brown colour is produced in such portions as contain this amyloid material. Sections can be similarly treated and mounted in glycerine media. Methyl violet stains amyloid material a deep red, standing out clearly from the surrounding violet. Amyloid degeneration is not common in tropical diseases, with the exception of leprosy. In that disease, even when there has been no extensive suppuration, amyloid degeneration is sometimes found. Fibrous Degeneration. — As a result of degenerative changes in many parts, and particularly in the nervous system, the nerve elements are replaced by fibrous tissues. These are well seen in spinal diseases in which degenera- tion of nerve tracts is followed by the formation of fibrous tissue in the tracts occupied by the degenerated nerves. This fibrous tissue stains with ordinary basic stains and is well shown by carmine. This change is sclerosis, but is the final result of the degenerative changes. The early nerve degenerations require special and com- plicated methods and could not be satisfactorily studied without special knowledge and appliances. The simplest method for demonstration of early nerve degeneration is that of Marchi. Small pieces of tissue are hardened in Mijller's fluid for one week, taking care to avoid mechanical injury. The tissues are then trans- ferred to freshly prepared Marchi's fluid (Miiller's fluid two parts and i per cent, aqueous solution of osmic acid one part), in which they remain for one week at about 37° C. Brain tissues require a longer time. The tissues are then washed for twenty-four hours in running water, hardened in increasing strength of alcohol, ira- 3IO NERVE DEGENERATION bedded and cut. Sections are then dehydrated, cleaned and mounted in balsam. Nerves are best teased out and then mounted. Degenerated nerve tissue (fat) is stained black ; all else brownish-grey. The earliest degeneration is shown by a flecking with black about the internodes ; later fat drop- lets staining black more or less completely replace the white substance of Schwann. 3" CHAPTER XVIII. Parasites in the Tissues. Bacteria, protozoa, trematoda, nematoda, and their eggs and larvaj, may be found in the tissues in various diseased conditions in man and animals in the tissues of various parts of the body. Bacteria are considered separately. Protozoa. — The common parasites are coccidia and sarcosporidia. They occur rarely in man, and are probably in those cases accidental parasites, but in other mammals are very common. Coccidia are sporozoa which are encysted, and by their continuous asexual multiplication form visible white masses not unlike tubercles. The rabbit is very commonly infected, and the large masses are found in the liver, though the intestinal mucosa may also be infected. The young coccidia enter hepatic cells or those of the bile duct and speedily destroy the cell. They may develop asexually so that a cyst is formed, in which the protoplasm divides into a number of young coccidia, which, on the rupture of the cyst, enter neighbouring cells and in turn multiply. Massive tumours composed of these encysted sporozoa are thus found. Some of the young coccidia develop into sexual forms, male and female. In the male forms the cell protoplasm and nucleus divide, so that a number of motile bodies, microgametes, are formed. When the cyst ruptures, these microgametes enter a female and fertilise it. The female forms, macro- 312 COCCIDIA gametes, do not divide till the microgametes have entered and fertilised them. After this fertilisation, which takes place in the liver, the cyst wall of the fertilised macro- gametes becomes impervious, and ultimately this cyst, now known as the oocyst, becomes detached and passed with the bile into the faeces of the host, and is passed externally. Lying on the ground the cell contents of this oocyst or fertilised macrogamete divide so that eight spores are formed. When swallowed by a suitable host the cyst wall is dissolved off, and the spores enter cells and become young coccidia. The different coccidia which have occurred in man are : — Coccidium mniculi (C. qviforme), in which the oocysts measure -033 to "049 mm. long by '015 to '028 mm. wide. This parasite has been found in the liver in man on three or four occasions. Coccidium hominis, in which the oocysts measure •024 to -035 mm. by -012 to -020 mm. This is a parasite of the intestinal epithelium of rabbits in which it causes death from severe diarrhoea. It has been described in the intestine of man on a few occasions. Some authori- ties hold that it is identical with C. cuniculi. Coccidium. bigeminum, in which the oocysts measure ■012 to -015 mm. by -007 to -010 mm. In this form the oocyst divides into two parts, each of which encysts and forms sporoblasts. A small number of cases are described in man. Demonstrations. — The demonstration in tissues of the coccidia is not difficult, as even without staining the shape and general appearance can be readily made out. They are very liable to be mistaken for eggs of trema- todes. To stain the cell contents is a difficult matter. The wall of the organism is not readily penetrated by stains, and when the stain does penetrate the contents are apt to be so deeply stained that details cannot be made out. Good results can be obtained by staining with iron alum. SARCOSPORIDIA 313 The method is as follows. The solutions required are : — A. 2'5 per cent, solution of iron alum. B. Hsematoxylin crystals I gramme. Absolute alcohol 10 cc. Distilled water ... .'. go cc. This solution should be kept for one month to "ripen." Then add water 100 cc. Sections are first placed in the iron alum solution (A) for six to twelve hours, washed in water for one minute, then left in the haematoxylin solution (B) for twenty- four to thirty-six hours. Afterwards they are washed and differentiated in iron alum solution until the section becomes deep blue, and the nuclear structures stand out sharply. This stage is best controlled by watching the process under a low power. When completed, wash in running water for fifteen minutes. Counterstain very lightly with eosin if desired. Dehydrate and mount as usual. Sarcosporidia. — In the muscles of some animals, such as cattle, sheep, pigs, rats, &c., elongated bodies are found which are often visible with the naked eye. On micro- scopic examination these " Hielscher's tubes" are seen to be filled with retractile bodies, which are usually curved and sausage-shaped, but may be round. Nothing is known of the extra-corporeal life-history or sexual development of these bodies. Similar bodies have been found in man. Each of these bodies is a single in- dividual, only a part of the protoplasm is converted into spores, the remainder continues to live and form spores. This continuous formation of spores without the destruction of the original cell leads to the formation of these large masses. This peculiarity in the asexual development, separates the sarcosporidia and the allied forms which occur in fishes, Myxosporidia, from the other sporozoa, and they are known as Telosporidia ; whilst the parasites in which the whole protoplasm divides and the parental cell is destroyed in the pro- 314 LEISHMAN-DONOVAN BODIES cess are known as Neosporidia. The sarcosporidia stain readily with basic stains. Little attention has been paid to these bodies. The separate organisms are invisible to the naked eye, but as they usually occur in masses and produce some colour change, appearing as light streaks or nodules; such streaks should be looked for and scrapings of them examined microscopically. The nature of the bodies can be demonstrated in sections or, better, by isolating one of the bodies and rupturing it. A large mass of spores are set free and can be readily stained after drying by Leishman's stain or by Giemsa's stain. They are oval, often sausage-shaped bodies, with a nucleus with diffuse chromatin staining. Often detached granules staining with the polychrome red are present in the protoplasm. At one end is a clear space which may be unstained or very deeply stained, and is known as the polar capsule. The few species of the sarcosporidia known are in- cluded in the genus Sarcocystis. The most important of the protozoa found in the tissues in man are the Leishman-Donovan bodies, now usually believed to be the resting stage of a flagellate. These bodies are found in the spleen, liver, mesenteric glands, submucosa and in the lungs, and are present in enormous numbers. They are contained in endothelial cells in the macrophages and imbedded in a hyaline matrix, possibly the remnant of a broken-down tissue cell between the cells of the organ or tissue. They are small, round, oval or oat-shaped bodies, characterised by having two unequal chromatin masses, one large and oval, staining moderately well with the red polychromed methylene blue, and the other smaller, more compact, usually rod-shaped, and staining very deeply with chromatin stains. They may be readily seen in smears made from an infected organ and can be recognised in sections. During life they may be obtained by puncture of the spleen or liver. They are more easily LEISHMAN-DONOVAN BODIES 315 obtained from the spleen, but there is a certain amount of risk in puncturing that organ. They are not so numerous in fluid drawn from the Hver, but the risk in puncturing that prgan is sHght, and therefore it is on the results of liver puncture that the diagnosis should be made. The puncture must be made with sti-ict antiseptic precautions, and there must be no fluid in the syringe used, or the bodies will be broken up. Aspiration must be slow and gradual, or blood will be sucked up in too great a quantity. In the peripheral blood at some stages of the disease the bodies may be found in the leucocytes. Leishman's stain is an excellent one for smears of the organs, or smears of the fluid obtained by puncturing the liver, or to demonstrate the parasites in the leucocytes ; dilute carbol fuchsin also gives satisfactory results. In sections, carbol thionin or haemalum used as described for malaria parasites in sections of the blood- vessels gives good results. Van Gieson's stain brings them out rather better. In this method, after overstain- ing ten minutes in the haemalum solution and "blueing" the section is covered with a saturated solution of picric acid to which i per cent, of acid fuchsin has been added. The bodies appear very small in section and the two chromatin masses are close to each other, as in the hardening process there is great shrinking of the parasites. The disease caused by these parasites is that known as kala-azar. Similar bodies can be obtained by scraping the floor of certain ulcerated surfaces on the skin. These ulcers are the Oriental sores or Delhi boils. Probably they are a different species, as the topographical distribu- tion of the two diseases is different. The two unequal chromatin masses suggested to Leish- man the similar appearances found in degenerate try^ panosomes. Rogers and others have shown that in a medium containing 4 per cent, citrate of soda changes occur in 3l6 WORMS the Leish man-Donovan bodies and that ultimately they develop flagella. It is essential that the temperature of the culture should not exceed 25° C. Rogers states that by adding a little citric acid to his cultures, development occurs more rapidly and certainly; this addition is not however, an essential. Trematodes may be present in various tissues. Some, such as the schistosoma, are found in blood-vessels other trematodes may be found in bile ducts or in the pul- monary alveoli. Filaria in man and many animals are found in con- nective tissue, or in the muscles, or in the peritoneal cavity. Others, as in some cattle, are found beneath the endothelium of large vessels, such as the aorta, and others, again, partly beneath the endothelium and partly free in the lumen of the vessel. Some nematodes penetrate the intestinal wall, as the Trichinella spiralis and Strongyloides intestinales and others are encysted in the submucosa either commonly or, as in the case of the Anchylostoma duodenale, exceptionally. Eggs are not uncommon in the tissues. Those of Schistosoma hcematobium are commonly present in the submucosa of the bladder or rectum, and of the S. japonicum in the submucosa of the small and large intestines. Eggs of these worms may be found in other parts and those of S. japonicum appear to be constantly present in the liver and lymphatic glands. Embryos are frequently found in blood-vessels and exceptionally are extravascular, but in the natural life- history of Trichinella spiralis the larvae are encysted in muscles of various parts of the body and in the dia- phragm and intestinal walls. Nematode embryos, such as those of the Anchylostoma duodenale, penetrate the skin and can be found in the skin, subcutaneous tissues, lungs and various parts of the body. Eggs and larvae can be readily stained in section with any basic stain — hasmatoxylin and carmine perhaps give the best results. They can be detected unstained. WORMS 317 Larvae of Trichinella spiralis can be well seen in teased- out specimens or in the intestinal walls of small animals, such as rats, spread out between two slides. If hardened whilst so spread out in alcohol, the intestine can then be rapidly treated with sodium hydrate solution till the intestinal wall is rendered transparent. The worms are then more readily seen. 3i8 CHAPTER XIX. Fmces. The examination of fseces is of the greatest import- ance, as most of the intestinal entozoa deposit their eggs whilst in the intestinal canal ; others inhabiting the liver deposit their eggs in the biliary'ducts, while others, again, pass their eggs or embryos through the tissues into alimentary canal. The eggs of all these are passed with the faeces. In other cases the parasites themselves may be passed, or in the case of tape-worms the mature seg- ments or proglottides only. In dysentery, cholera, &c., the organisms found in these diseases are present and can be isolated from the stools. Macroscopic examination of the stools is very necessary and much information can be gained. The stools can be examined as passed in any vessel, but are more con- veniently examined if passed into transparent glass vessels ; these can be covered with a larger glass cover fitting over the lower vessel like an enlarged Petri dish., The points to observe are : — (i) The presence or absence of blood, mucus, muco- pus or pus, and the arrangement relative to the stool of such a discharge. (2) The colour of the stool and its consistence. (3) The presence or absence of evidence of gaseous fermentation. ^ (4) The odour. (5) The reaction, determined as soon as possible after the stool is passed. MUCUS IN F^CES 319 (6) The bulk of tlie stools. (7) Any visible signs of animal parasites, such as the worms themselves or the proglottides or segments of tape-worms. (i) Muois alone, or streaked or mixed with the blood, usually indicates inflammatory action in the lower bowel, not necessarily dysenteric. It may be caused by anything that sets up such inflammation, such as bilharzia, ulcer- ated haemorrhoids, or chronic ulcerations of various kinds of the rectum. These latter include malignant growths, granulomatous growths, and the ulceration left as a sequela of dysentery. Rarely mucus derived from the small intestines is passed with the fasces. Such mucus is recognised easily as it is stained yellow with bile. Clear mucus, whether streaked with bright blood or not, without any admixture of faecal matter, is met with in early or acute dysenteric attacks. Turbid or purulent mucus, sometimes in large quantities and passed either without any stool or with solid formed motions, is more indicative of a chronic ulceration of the rectum, from whatever cause. Sometimes the mucus is passed in large masses and condensed, and may include much debris and numerous epithelial cells. In the condition known as membranous colitis, complete casts, several inches in length, of the rectum may be passed. These are usually twisted up when passed, and may be mistaken for worms. They can sometimes be floated out in water, and in any case the microscopic structure should render any mistake impossible. With ulceration limited to ihe rectum, stools are often coated with mucus. The more intimately the mucus and blood are mixed with the faeces the higher up are the lesions from which the mucus or blood is derived. In some lesions the mucus is so intimately mixed with the fluid fasces that it is difficult to discern, but tilting the vessel from side to side will often indicate its pre- sence by the manner in which the stool flows. In some 320 BLOOD IN F^CES cases it is better shown by adding water to the faeces, when the flakes or masses of mucus can be more readily seen, especially if the diluted faeces are poured from one vessel to another. Amoeba are killed or have their motiHty destroyed by this addition of water, and there- fore this method should only be adopted when the masses cannot be seen on inspection. Blood may be passed, bright red or in clots, in large quantities. This is no proof that it is passed from the rectum, as if in sufficient quantity and not mixed with the faecal contents of the intestine it need undergo very little change in passing through the large intestine. Such blood is occasionally passed in ankylostomiasis. If intimately mixed with the faeces it may have lost com- pletely the red colour and appear black and tarry — melaena. When in vei-y small amount altered blood can be recognised by Weber's test. The fat is first extracted with ether and the stools are then rubbed up with water and a third part of acetic acid added. They are now shaken up with ether and an ethereal solution of acid haematin is obtained if altered blood is present {vide table of spectra). In other- cases, though still red, the stool has a duller colour, more like anchovy sauce. Such stools are passed in some cases of dysentery where the small intestines are implicated, and may also be passed in cases of extensive enteritis secondary to malaria. Microscopic as well as macroscopic examination of the mucus and blood, as well as of the stool, should be made. (2) The colour of the stool is much modified by the diet, milk especially causing pale stools. Articles of diet taken by a patient have a marked effect, and amongst abnormal articles that may be met with are earths of various kinds, coal-dust, &c., which to the inexperienced may cause much confusion. The pipe-clay stools of obstructive jaundice may be simulated by those of some earth-eaters, and the black stool of the coal-dust eater has BILE ACIDS AND PIGMENTS 321 been mistaken for melsena. The dark blue stools passed by patients taking methylene blue are easily recognised. Bile pigments and acids ai-e not usually pi'csent in normal faeces, as they are absorbed in the small intestine or broken down in the large intestine. They may be present in diseased conditions or when the food is passed too rapidly through the intestinal canal. Bile acids may be recognised by Pettenkoffer's reaction. A small portion of the faeces is mixed with a little sugar and placed on a white porcelain dish. A little sulphuric acid is allowed to come in contact, and if bile acids are present a crimson colour appears. Bile pigment may occur either as Bilirubin or as Biliverdin. {a) Schmidt's Reaction. — A saturated solution of per- chloride of mercury is added to the faeces, and in the presence of bile pigments a bright green colour is produced. (6) Gmelin's Reaction.- — A drop of yellow nitric acid (i.e., containing nitrous acid) is brought into contact with the faeces, and in the presence of bile pigment there is a play of colours, one of which must be green. This test is more decisive with a watery extract of the faeces. (c) Huppert's Test. — The faeces are mixed with slaked lime suspended in water, and the precipitate is filtered and washed. An extract of the dried precipitate is made with hot alcohol and a liltle sulphuric acid. If bile pigments are present this extract is green. The consistence of the stool is of great importance, and it will be found that " looseness " of stools is of more importance in tropical practice than in England. In ulceration of the cscum and upper part of the colon, eveh when this is acute and extensive, there need be neither visible mucus nor blood, nor even tenesmus. " Tropical diarrhoea " is frequently shown at post-mortem examinations to be dysenteric. It is very fatal. On the other hand, mucus and blood may be passed with formed 21 322 ODOUR AND REACTION OF F.4!;CES or even hard stools when there are a few chronic ulcers high up in the large intestine. (3) In some forms of tropical diarrhoea, particularly that form known in the East as sprue, T:he stools passed are full of air-bubbles and are undergoing active gaseous fermentation. (4) The odour varies so greatly with the diet that it is of minor importance. In the i-aces subsisting mainly on a scanty vegetable diet the odour is singularly slight. The smell is mainly due to indol and skatol. In cases of dysentery associated with formation of sloughs the ordinary faecal odour is replaced by the peculiar pene- trating smell associated with that condition. Excessive decomposition of the stools may cause an increase in the intensity of the normal smell, or if the diet is mainly of carbohydrate food-stuff no increase but even a dimunition. Variations in the odour indicate changes in the decom- position of the contents of the intestine, often from variations in the food, but sometimes from variations in the "flora" of the intestinal contents, rarely from struc- tural lesions of the intestinal wall. The result of the administration of intestinal anti- septics is more often a diminution in the putrefactive changes in the contents of the bowel than any real improvement in the diseased condition of the intestinal wall. (5) The normal reaction of the faeces as determined by litmus is nearly neutral, when fasting it is acid, with a milk diet faintly alkaline. It is usually acid to phenolph- thalein. In many cases of diarrhoea and dysentery this is replaced by a decidedly alkaline reaction. To determine the reaction the faeces must be examined as soon as they are passed, as a change rapidly occurs in most fasces, particularly when fluid, rendering them alkaline. Solid motions must be rubbed up with water in a mortar. (6) Bulk of Faces. — The amount of fasces passed by a European on the average is about 130 grammes per diem. BULK OF EXCRETA 323 On a meat diet it is about half this. In vegetarians it is much greater. The amount passed varies with the amount of food taken, and inversely as the amount digested. The excretions from the intestinal v^rall form a proportion of the faeces, as during prolonged fasts 22 grammes may still be passed. Most native races con- sume a large amount of crude carbohydrates in bulky vegetables. Much of this is indigestible and is therefore passed with the faeces. The average weight of the excreta in native races is greater than in Europeans, and in India is about 233 grammes. These amounts are of importance in estimating the amount of excrementitious matter that has to be disposed of in a community. It is usually estimated per 1,000 of the population, and is given for a European community as half a ton, and for a native community as two-thirds of a ton. The bulk of the excreta is variously affected in disease. Discharges from the intestinal walls, usually watery in character, may form an important part or even, as in cholera, nearly the whole of the excreta. In other cases where there is an extensive ulcerated svirface muco-pus may be discharged in quantity and no faeces at all. Abscesses such as hepatic abscesses may open into the intestine, and then there may be a profuse discharge of pus. Water is generally absorbed in the small intestines, but in many conditions where the intestines are irritated, food and even water are hurried so quickly through the alimentary canal that little absorption takes place. In some diseases, as in pneumonia, and towards the crisis in relapsing fever, there is a great tendency to the occurrence of frequent large watery motions. In dysentery, as a rule, though the motions are frequent, the amount of faeces passed is small, and in the acute and early stages no f^ces at all may be passed, though mucus or blood, or both, are passed in quantity. In most general diseases, as in malaria, digestion and absorption are fairly active, and the motions then are 324 ANALYSIS OF F^CES constipated, but the amount passed is not more dimin- ished than might be anticipated from the diminution in the amount of food taken. In the disease known as sprue or psilosis the motions are usually bulky. Digestion and absorption are both imperfect. As the guiding principle in this disease is to give as complete rest to the alimentary canal as is con- sistent with a sufficient supply of nutriment to the tissues, it is of importance to know what forms of food are not digested and which are. In other diseases; as in obstruction of the pancreatic duct, it is also important to know which of the digestive juices is wanting, as it may be possible to supply the deficiency or so modify the diet that little or no call for this agent is necessary. An analysis of the feeces will show which of the important food elements have undergone little or no change, provided that the amount, and composition of food taken from which the faeces is derived is accurately known. Allowance has to be made for excretions from the intestinal wall, and for the secretions from the liver, pancreas, &c., which are poured into the intestine in variable amounts. The investigation is difficult, as the amoimt of the main ingredients excreted in this way may be consider- able, and is affected by the food taken. The amount of fats excreted, for instance, is greater when fat-free food is taken than in the same individual when he is fasting. For any investigation food of known composition and amount must be taken. The simplest diet is a milk diet. Some inert, easily recognised substance, such as charcoal or carmine, should be taken at the commence- ment of the experiment. The first fasces containing this substance, usually twenty-four hours after the administra- tion, but in cases of diarrhoea four hotirs, or even less, must be saved, as well as all subsequent excreta. A period of three days or five days should be taken, and at the ANALYSIS OF F^CES 325 completion of the experiment a second dose of charcoal given. As soon as the charcoal appears in the faeces the experiment is over. The faeces passed during the period must be all collected, and should be analysed : (i) For water ; (2) for nitrogen ; (3) for fats ; (4) for carbohydrates. (i) The water can be determined by weighing before and after drying, but as volatile substances are present this must be conducted over sulphuric acid in a drying chamber not over 60° C. The average amount of water is 75 per cent. (2) Nitrogen can be determined by taking a weighed portion of the faeces, to which is added 15 or 20 cc. of ^ sulphuric acid to prevent loss of ammonia. This mixture is then dried in a water-bath till fairly hard, and the desiccation completed in a drying chamber at 10° C. over sulphuric acid. A gramme of this dried powder is then mixed with 25 cc. of sti"ong sulphuric acid and i gramme of sodium pyro- phosphate, and this is allowed to stand for some hours, and then cautiously boiled. The nitrogen will all now be in the form of ammonium sulphate. This can be estimated after allowing to cool by adding 600 cc. of water and sodium hydrate solution till strongly alkaline. Some granulated zinc to prevent bumping should also be added. The mixture is then distilled and the distillate allowed to pass into a measured amount of ^ sulphuric acid. The ammonia will neutralise a certain amount of this, so that by subsequent titration the amount of ammonia that has distilled over can be estimated. Undigested proteids passed unchanged form a very small part of the nitrogen. (3) Fats are usually determined as "total fats" consist- ing of> fats, soaps and fatty acids. The fffices should be thoroughly dried over sulphuric acid and treated with i per cen^. hydrochloric acid to split up the soaps. 326 PARASITES Ether is then added and the ethereal extract dried is re-dissolved in water-free ether. The residue left after the evaporation of the ether is considered as total fats. In any question of the digestion of fats it must be remembered that fats differ greatly in digestibility, and that a healthy person will pass unchanged about 8 per cent, of mutton suet, which melts at 52° C, whilst he will only pass 2-5 per cent, of pork fat, which melts at 30° C. A healthy adult on a pure milk diet will digest about 95 per. cent. (4) Sugar and carbohydrates are usually digested com- pletely unless enclosed in an impervious capsule, as in some vegetable foods. The amount of gas formed from such faeces, kept at blood-heat for twenty-four hours, will give a fair indication of the amount of carbohydrates present. Parasites. (7) Parasites of various kinds may be seen by direct examination, but more often it is necessary to strain the stools. This is best done by placing the stool on a muslin or strong fine wire gauze strainer, and adding water and stirring well. By repeating this process all the smaller particles of the faeces will be carried through the muslin, and only the coarser particles and any entozoa present will be left on the strainer. Some of the smaller entozoa may be carried through the strainer. The fluids that have passed through can be strained again (fig. 135), or passed through a muslin bag. This is conveniently done with a bucket big enough to hold the sieve. The bucket is filled with water so that it covers the wire gauze. The faeces are then placed on the gauze and stirred well. From time to time the sieve is lifted so that the stirred faeces pass into the bucket. When the water becomes turbid the strainer is placed in a second bucket filled with clear water and the process MICROSCOPIC EXAMINATION 327 repeated. On examining the strainer tiie greater number of the worms will now be readily seen. After standing a little the superjacent fluid in both buckets should be poured off and the deposit again passed through the sieve. More worms will be found. Entozoa are damaged a little by this proceeding. Where they are required for detailed examination they should be picked out of the undiluted faeces. Fig. 13s — Wire Gauze Strainer. Microscopic Examination. — The most important objects of this are (i) the detection of ova of parasites, (2) the detection of animal micro-parasites, and (3) the investi- gation of the bacteria present. Ova are readily seen with a low power, two-third inch objective, but for their identification at least half an inch or, better, quarter of an inch objectives are requisite. The preparation of the stool is very simple. A small particle of the faeces is placed on a slide ; it can be con- veniently taken up with a splinter of wood such as a match stick. If not too hard it should then be com- pressed by a cover-glass into a thin layer ; if too hard for this it can be mixed with a little water. If the stool be 328 EGGS watery it should be allowed ■ to stand, and with a pipette some of the fluid taken from the bottom, as the eggs are heavier than the fluid stool and sink to the bottom of the vessel containing it. The eggs that may be met are those oi. the A scat is lunibricoides, Tricocephaliis dispar, Ankylos- tomum duodenale, Oxyiiris vcrmicularis, several species of tape-worms, several species of Fasciolidce, those of Schis- tosomum hcematobium with the lateral spine sometimes, and those of S. japonicum, and the embryos of Strongy- loides intestinalis. The attached diagram shows the appearance of the more important of the ova. The eggs of Ascaris lumbricoides (round-worm) are enclosed in a thick, clear capsule usually coated with an albuminoid covering stained yellow or brown by the fzecal colouring matter. The protoplasmic contents are granular and do not as a rule completely fill the inner capsule (fig. 136, a). If too much pressure has been used the albuminoid covering may have been ruptured and the egg is seen surrounded only by its thick transparent capsule. These Ascaris eggs can be readily distinguished from eggs with thin capsules, such as those of A. duodenale, not only by the thickness of the capsule, but by its more spherical shape, and by the granular and unsegmented character of the egg contents. Unfertilised eggs are larger, more oval, and the egg contents contain numerous refractile globules, which should not be confused with segmen- tation. The eggs of Trichocephalus dispar (whip-worms) are easily distinguished, as they are small oval eggs contained in a thick, deeply stained outer capsule which has an opening at each end. Inside this capsule is a thinner, unstained capsule, and the egg contents are granular. In many instances the openings in the outer capsule are seen to be plugged by mucus (fig. 136, b). The ovum of Ankylostomum duodenale is enclosed in a single, thin, transparent, unstained capsule. At the time the egg is passed segmentation usually into about four segments has taken place (fig. 136, c^, d), but if the stool Fig. 136.— a, Ascaris lumbricoides ; b, Trichocephalus dispar ; c, Oxyuris 'oermiculafis ; c\ c^, Ankylostomum duodenale ; d. Oncosphere of Cestode ; «,/,,?'j various Fasciolida ; //, Sckistosomum (?) hamaiobium (from feces); i, Sckistosomum hamatobium (from urine). 330 EGGS be kept a large number of segments will be present according to the time and temperature, and in twenty- four to forty-eight hours a fairly well-formed embryo will be found in many of the egg capsules, and the egg then closely resembles that of Oxyuris vermicularis. The eggs of the closely related Necator americanus are slightly larger but otherwise similar to those of the Ankylostome. Oxyuris vermicularis (thread-worm) has an egg that in size and general appearance is not unlike the anky- lostome but is usually flattened at one side. At the time the stool is passed this egg contains a well-formed embryo (fig. 136, c). The " eggs," or rather oncospheres, of the different species of tape-worms may only present slight differences from each other, but they are readily distinguished from all other ova by the radial striation of the thick capsule and the presence of a differentiation in the contents into an embryo ; the booklets of this embryo can usually be made out (fig. 136, d). These oncospheres consist of the embryo and embryonic capsule only, the outer part of' the egg has usually disappeared. The eggs of the various Fasciolidce (flukes) can be recognised by the presence of an operculum or lid (fig. 136, e, /, g,), and distinguished from each other by their size. The eggs are usually yellow or brown of the Trematodes found in man. Schistosomum hcematobium has ^a highly characteristic egg, as it is armed with a sharp spike. In eggs passed with the faeces, with which they may be mixed, or con- tained only in mucus on the surface of the stool, this spike is at one side, (fig. 136, h), in urine the spine is terminal (fig. 136, i), and by some it is believed that the eggs with a lateral spine are from different species of Schistosoma. If water be added to the fceces it will be seen that the egg contains a ciliated embryo which soon becomes active and bursts through the egg capsule. The free-swimming embryo remains alive in water for some days, but undergoes little further change. An intermediate EGGS AND WORMS 331 host, perhaps a fresh-water mollusc, is probably necessary for its further development. Schistosomum japonicuni. — -The eggs are passed into the intestinal canal higher up than those of S. hcematobium. They have no spike, no operculum, and are about the same size as the eggs of ankylostomes. They contain a formed miracidium when passed. Measurements of Ova. T. saginata ... 0-03 1 to 0-04 mm. by 0-02 to 0-03 mm. T. solium ... (Spherical) 0*03 mm. H. nana a» 0-4 „ D.latus ... o'o68 to 0'07i mm. by 0*045 """• (operculated), F. hepatica ... 0-13 by o-oS mm. F.buski ... 0-125 ., 077 ., D. lancealmu ... ... 0-04 „ 0-03 „ 0. sinensis ... 0-027 ,, o-oi6 ,, C. heterophyes ... ... 0-03 .. 0-017 .. [Paragonimus westermani o'o8 to o-i mm. by 0*052 to 0-075 "im.] G. hominis ... o-is by 0-07 mm. A. watsoni ... 0-12 .. 0-075 >. S. hcematobium ... ... o-o8 .. 0-03 „ S. japonicum ... o-o6 to 0-09 ,, by 0-03 to 0-05 ram. A. lumbricoides ... ... 0-05 .. 0'07 „ „ 0-04 ,, 0-05 „ 0. vermicularis . . . ... 0-05 by 0-016 ,, to 0-024 «"". A.duodenale ... 0056 to o-o6l ,, by 0-034 to 0-038 mm. N. americanus ... ... 0-064 „ 0-072 „ „ 0-036 mm. T. dispar ... 0-05 .. 0'054 ., » 0-023 ,. The embryo of the Strongyloides intestinalis is fre- quently passed with the stools. The embryos of the Trichina spiralis are very rarely passed in the stools as they normally penetrate the intestinal walls and pass into the surrounding tissues. In the fceces, thread-worms, segments of tape-worms, and occasionally round-worms are passed naturally. After the administration of powerful anthelmintics the whole tape-worm, round-worms, ankylostomes, flukes, and whip-worms may be passed. Some species are never found under any circumstances in the faeces. OJ- CHAPTER XX. Intestinal Parasites. The worms met with in the human intestine and its appendages belong to the following classes : — A. Cestoda. — These flattened worms have a segmented body, no digestive tube, and are hermaphroditic. B. Trematoda. — In these the digestive tube is incom- plete ; there is no anus and the body is not segmented. They are hermaphroditic except the Schistosoma. C. Nematoda. — These usually have a complete diges- tive tube. They are cylindrical worms and they are not hermaphroditic. A. Cestodes. The human Cestodes are : Tcenia solium, T. saginaia, T. confusa, T. africana, Dipylidiuiu caninum, Hymeno- lepis murina (T. nana) and H. diminnfa, Davainea madagascariensis, Rothriocephalus latns, Diplogonoporns grandis. Cestodes or Tape-worms. — The embiyonic or cystic forms of the Taniia echinococcus may be found in the liver, muscles of man, &c. The definitive host is the dog. These cysts, the hydatid cysts, can hardly be mistaken for non-parasitic cysts ; they can be readily distinguished if there is any doubt by the laminated cyst wall and the presence of booklets in the cyst or discharges. In the case of the echinococcus man is the intermediate host. A larval form of Bothriocephalus (B. mansoni) has been found in the connective tissues of men in Japan, and similar larval forms have been obtained from an TAPE-WORMS 333 aboriginal of British Guiana and in Central Africa. These larvae may be the larval form of a Dibothriocephalus, but it is not certain. They are classed as a separate genus, Sparganum (Stiles). The greater number of the tape-worms found in man attain sexual maturity in him. Man is therefore the definitive host of these worms. The general structure of tape-worms should be known, and the differences indicated in the tabular statement of the well-known human tape-worms will then be understood. Tape-worms consist of a head or fixed portion attached by hooks or suckers, or both, to the intestinal wall. This "head" is called the scolex. From this scolex growth takes place continuously in one direction ; at first as a narrow neck which is not segmented, but which rapidly becomes segmented, and as growth continues each seg- ment increases in size and becomes sexually mature. Each segment is known as a proglottis, and together these form the strobila. When sexually mature, the eggs are fertilised, and finally the genital organs atrophy and the proglottis is reduced to a muscular sac distended by a uterus filled with fertilised eggs. These proglottides become detached and ai-e passed in the stool. Each proglottis is motile and may live for some time after it has been passed in the stool. It creeps about discharging its eggs. These eggs are taken up by the intermediate host, another mammal, a fish or even an insect, and develop in that animal into the cystic or larval stage. In the case of some of the tape-worms, as in Bothrio- cephalus, a ciliated embryo is formed which swims freely in water, and in its intermediate host does not form a cyst but an elongated, worm-like larva known as a "plerocercoid" lai^va. If taken, with food or otherwise, into the intestinal tract of man, the cyst is set free and the head becomes the scolex of the mature tape-worm. This scolex fixes 334 STRUCTURE OF TAPE-WORMS The tape-worm derives its nutriment by osmosis from the intestinal tract. There is no intestine and no trace of one. There are water vascular tubes, the water vascular system, running the whole length of the worm. With this exception, and the nervous system, each seg- ment or proglottis is a distinct individual jointed on to its predecessor and successor. The points in the structure of a proglottis are best observed in a half-grown proglottis, as earlier the organs are not fully developed and the last segments are merely muscular egg-sacs with atrophied organs. For permanent specimens the method to be adopted is as follows : Stain for twenty-four hours with very weak borax carmine ; soak in glycerine for some months. Compress between two slides clamped together and place in methylated spirit. When partially hardened the pressure can be relaxed and the specimen dehydrated in alcohol. Clear with oil of cloves and mount in balsam. Pressure should be applied to the cover-glass till the balsam has hardened. The proglottis is covered with a transparent cuticle and has a powerful muscular wall with longitudinal and transverse or circular bands. In the interior of the seg- ment are the organs of generation, male and female, as each segment is hermaphroditic. The ari^angement of these organs varies greatly in different species, but they conform to a common type. The space between the organs is occupied by paren- chymatous tissue in which are often included highly refractile calcareous masses which much not be mistaken for eggs. The male genital organs consist of a number of small testes. Minute vasa efferentia unite about the centre of the segment into a common vas deferens, this terminates in the copulatory organ or cirrhus, opening with the vagina into a genital cloaca. The female genital organs consist of the vagina leading as a straight tube from the genital cloaca into an enlarge- STRUCTURE OF TAPE-WORMS 335 merit, the receptaculum seminis ; from this the tube is continued to the shell gland, and near it the ovarian tube, or tubes, if, as is usual, the ovary is paired, open. There is a diverticulum running longitudinally in the centre of the proglottis, Mrhich at first is simple but later w.v.s. Fig. 137. — a, Testes ; d, vasa efferentia ; : Q ^, 'I b2'>% 60 -S tSlV VJl tiu V jz ,;r 1* Is g m. Ion matur doubl ir widt im.lon matur trebl ir widt iS BO 1- sa s S- u SoiS^ I'oSS ; o _ t^— 13 o c ■" O c +- 3 ^^ C in ri « jc S>»5 t^ji U**5 ^ en N 2 M W ° c Sort •^ *-• c O rt 9 o*c OJ o*E b .2 0, : 4) o cue osas o£ US ■a g '^l ro oo m u O- M< ^ ^- e e n .a o a-° ^l s a iri s c !:?:=: -a 2 !^ in A 4^ ed >-• ^ >* J3 : ever more slightly bi than long, hooks with -§-§ E 1° O w o It S a V o 3 J ^ s s M c ■g 1.^ rt ■s — i bo s C > > 2i c o •« V umerous tides, several i tres Ion s |4 .1 c o VI V c o 1) s 1 T3 o 1 Z ■ H C s "rt V ■So c ci5 V 3 1 >< CO ■a go 1 S s « 'Ei a 3 o ■o'S ea ■s ■a CO s ^ s rt OJ a as ■g 3 ~~^->— "" II 0) ..^ ^ lU m j. ■■s 3 C o o o s h CLASSIFICATION OF FLUKES 339 are found in the intestinal tract, so that ordinarily the eggs, and after the administration of powerful anthel- mintics, like thymol, the adults also of these are passed by the rectum. The Trematodes include the Fasciolidce, which are flattened bodies, of oval shape with pointed ends ; from the peculiarity of their shape they are popularly known as "flukes." They are hermaphroditic, non-segmented, and possess an incomplete intestine. They are armed with two suckers placed near each other in most of the genera. One of these, the oral sucker, surrounds the mouth, the other, the ventral sucker, or acetabulum, is on the ventral surface. Fig. 139. In the Paramphistomidce the suckers are at opposite ends of the body, and they differ in shape from the other Trematodes. The intestinal system of the Trematodes consists of a short muscular pharynx leading from the .anterior sucker longitudinally. .The oesophagus terminates by bifurcating into the two caeca which pass round the body towards the posterior extremity of the worm. These caeca end blindly, but are often sacculated or have diverticula. The genital organs are complicated and the arrangement varies. In Schistosomid/x the male and female are distinct, and the female lives in an incomplete canal, the gynaecophoric canal in the male. In the other 340 STRUCTURE OK FLUKES The female organs consist of a convoluted uterus open- ing externally near the second or veintral sucker in the Fasciolidce. This convoluted uterus leads to a dilatation surrounded by the " shell gland," and into this the ovarian Fig. 140. — a. Anterior sucker ; ^, caecum ; c, posterior sucker or acetabulum ; d, opening of uterus ; e, yolk glands ; /, vitelline ducts ; g, ootype ; h, ovary ; i, compound testicles ; /, vesicula seminalis ; k, penis. tube from the single ovary opens, and also the opening from the spermatheca. The common vitelline duct formed by the- junction of the two vitelline ducts which receive the yolk from the numerous yolk glands distributed along the edges of the animals opens with it. There is a canal STRUCTURE OF FLUKES 341 leading from the ventral surface to the oviduct known as the canal of Laurer, which may serve for the entrance of spermatozoa. There are two compound testicles which lie one in front of the other. The ducts, vasa deferentia, from these pass forwards and open into a dilatation, the vesicula seminalis, the duct from which leads to the penis, which opens externally close to the female genital opening (fig. 140). The details of the arrangement vary greatly. Fertilisa- tion is probably by a different worm. The fertilised eggs are passed with the fasces, sputum, urine, &c., of definitive the host. The structure of Fasciolidce is best shown as in the case of the Cestodes by prolonged immersion in glycerine and then passing through alcohol and oil of cloves, after staining with weak borax carmine for some days. In one division of the Trematoda, the Monogenia, the eggs develop into a condition suitable for the invasion of their definitive host without the intervention of an inter- mediate host, and there is no alternation of generations or asexual multiplication. Frorn one egg, therefore, one sexually mature form is developed. The eggs of the other and more important division, Digenia, require for their development an intermediate host, and there is alternation of generations, asexual multiplication, and from one egg several sexual forms may ultimately develop. The full life-history of the human Trematodes is not known. Of those in lower animals that in Fasciola hepatica has been thoroughly worked out. A ciliated embryo is formed in the egg and escapes, the miracidium. It then passes into a fresh-water snail. In the snail it becomes hollowed out, forming a sporocyst. Buds form in the interior of this cyst and secondary larvae, redice, are formed. These escape into the tissues of the snail, and by a further process of internal budding form tertiary larvae or 342 CLASSIFICATION OF FLUKES host are taken up with grass -by their definitive host, the sheep. They pass up the bile ducts into the liver of this animal and there develop into the sexually mature form. The Trematodes found in man belong to four families : — (i) The MonostomidcE, which have only one sucker, are represented by the Monostoma lentis, found in the super- ficial layer of the crystalline lens on one occasion only. . (2) The FasciolidcE have two suckers, one terminal and the other ventral. Of this family, representatives of six genera are found in man. In four of these the ventral sucker is near the oral sucker. Dicroccelium, in which the testicles are in front of the female genital organs ; Fasciola, Fasciolopsis, and Opisthorchis, in which the testicles are behind the female genital organs, and in these the ventral sucker is in the anterior part of the ventral surface, not far away from the oral sucker. These three genera are separated from each other by the shape of the anterior extremity, which is conical in the Fasciola; by the character of the intestinal caeca, which are much branched in Fasciola, unbranched but sinuous in Fascio- lopsis, and'unbranched but nearly straight in Opisthorchis. The Fasciolopsis is further distinguished by the great size and depth of the acetabulum or ventral sucker, and by the conspicuous and long cirrhus. In Fasciola and Opis- thorchis, as well as in Dicroccelium, the ventral suckers are about the same size as the oral suckers. In the other two genera the ventral is nearly in the middle of the ventral surface. Cotylogonimus, in which the genital opening is behind the posterior sucker, which is veiy large, much larger than the oral sucker ; and Paragonimus, in which the genital opening may be median, or right or left of the middle line. The ventral sucker is about the same size as the oral sucker. The Paramphistomidce have two suckers, both terminal, one at the one end and the other at the other end of the animal (fig. 139). They include two genera, species of which are parasitic in man — Amphistomum, or Paraphis- tomiim, and Gastrodiscus. These genera are distinguished a •6 13 'U 3 t-H .s ° J3 .£3 s ■s.= ■E rt .2 -3 ri 1 — . 5- .2 -a c •c V 'in .2 ■a a 1 la U 1 a u B B « >, Ug ■S « o " "O ha B V a, a CO o O B C B N W « ►3 en ■5 a >. ^ a* u ■3 s o 3 "^ « O tJ k< % en O ^ V lu ^ cd cH s -^ tu -— s 15 Sis > V ta O 4J 110^ B a ^ V u " s O .• " VI •I te s " t^a ^ 5 u ■§ ca bi ii-ji g s ^4 § S s •3 B ^ s « Is 344 NEMATODES by their exfernal appearance, as in the Gastrodiscus the anterior extremity is conical and appears to arise as a projection from the dorsal surface of a flat, rounded mass which contains the organs of reproduction. The human species is Gastrodiscus hominis. In the Amphistomum or Paramphistomum the shape is more or less conical, and there is no marked division between the anterior and posterior part of the body. The human representative is Amphistomum watsoni. Shipley considers this to be a Cladorchis, as there are pharyngeal pouches which are not present in the Paramphistoma. In both Paraphistomum and Gastrodiscus the opening of the genital pore is in the middle of the body and not near the acetabulum. Schistosomidce have two suckers, but the male and female organs are in separate animals. Nematodes. The Nematodes found in the human intestine are the Ascaris lumbricoides, rarely Ascaris mystax, Oxyuris vermi- cularis, Ankylostomum duodenale, Necator americanus, Strongylus subtilis, Trichocephalus dispar. Trichina spiralis, Strongyloides intestinalis {Anguillula intestinale). The larger worms are readily found, but some of the smaller ones are only to be seen by very careful inspec- tion. The Nematodes are liable to shrink, rupture, or otherwise become distorted, and are not very easy to stain. As a general rule it is best to examine them un- stained, and describe them as they appear in the fresh condition, mounted in normal saline solution. For per- manent specimens fixation in 70 per cent, alcohol just on the boil gives good results. The 70 per cent, aqueous solution of alcohol should be heated till bubbles begin to form, and the living worms dropped into it. The lamp should be put out, and the worms left in the fluid. After this treatment they may be mounted in glycerine jelly without shrinking. Ascaris lumbricoides. — These are large round-worms. ASCARIDES AND OXYURIDiE 345 The males are 15 to 17 cm. in length and 2 or 3 mm. in breadth. The female is rather larger, 20 to 25 cm. long and 5-5 mm. in breadth. These worms are found in any part of the intestinal tract and occasionally pass through the common duct into the gall-bladder or even the biliary ducts. They have been found in hepatic abscesses. They may be numerous in the intestine. Rarely Ascaris mystax, a smaller ascaris, with lateral alar cuticular appendages on cephalic end of the body, is found in man. Fig. 141. — a, Male ; b, female. Fig. 142. — a, Male ; b, female. Oxyuris verniicularis is a small cylindrical worm which tapers towards the tail to a sharp point. The male is 3 to 5 mm. in length and at the tail is coiled up in a spiral. The female is 9 to 12 mm. in length. This worm is found in the whole length of the large intestine and rectum and may escape through the anus (fig. 141, a, h). The males are found higher up in the alimentary canal than the 346 TRICHOCEPHALI AND ANKYLOSTOMA Trichocephalus dispar (Whip-worm). The characteristic of this worm is a long, thin, anterior portion somewhat resembHng the lash of a whip. The male is 35 to 45 mm. in length, and the female 35 to 50 mm. These worms are found commonly in the caecum and also in the ascending and trapsverse colon. They are very rarely found in the ileum (fig. 142). The Ankylostomum duod^nale is of the greatest import- ance. These worms are found in the small intestine and may be very numerous. Both males and females are found. They fix themselves to the intestinal wall and live on epithelium, debris, &c., but not blood. The female adult worms are 7 to 15 mm. in length and "8 mm. in breadth. They have a mouth surrounded by a power- ful armature consisting of two pairs of curved teeth on the posterior wall of the opening and of two triangular plates terminating in sharp points anterior to the mouth. The intestine is nearly straight and commences as a powerful oesophagus. The anus is subterminal in the female. The genital opening is posterior to the middle of the body. The males are rather smaller in length, 6 to 11 mm., and "5 mm. in breadth ; they have similar mouth-parts. The caudal extremity is expanded into a membranous fold of the integument divided into four unequal lobes, of which the lateral ones on each side are the largest. There are two equal spicules which can be protruded through the cloaca (fig. 143, m). The dorsal lobe is of importance as it differs in the character of the supporting rib from that found in Necator ' americanus. In the ankylostome it commences as a single rib which only bifurcates in the peripheral third and finally divides into three minute divisions (fig. 144, a). A'', americanus resembles the ankylostome in its habits and general characters, but differs in that the two pairs of curved teeth are i-eplaced by sharp chitinous thickenings, and that in the female the genital pore is anterior to the middle line, whilst in the dorsal flap of the caudal bursa of the male the supporting rib bifurcates Fig. 143.^ — m, f, Male and female Ankylostomes ; a, head oi A. duodenale; 348 ANKYLOSTOMUM AND NEGATOR near the base, and each of the divisions divides into two instead of into three as in the ankylostome. It appears to be the indigenous species in Africa and in some parts of Asia. A B Fig. 144.— a, a. Head and tail of male A. duodenale ; d, i, head and tail of male JV. americanus. The ankylostome is supposed to gain access to the body by the mouth, but it has "been shown to be capable STRONGYLOIDES INTESTINALIS 349 in its embryonic form of penetrating the skin, and some experiments seem to show the possibility of these em- bryonic forms obtaining access to the intestine after penetration of the skin. The ankylostome eggs hatch quickly, within forty-' eight hours, and the embryos rapidly increase in size. If kept in the fasces they soon die, but if allowed to escape into the earth they undergo further development, but do not become sexually mature, or reproduce outside the body. In their final stages they are enclosed in their old larval skin as a sheath and are inactive for' long periods. Strongyloides intestinalis, Anguillula intestinalis, or Rhabdonema intestinale. — This is a small worm only I mm. long and 50 jjl in breadth. It is found in the small intestine and the male is not known. Only a small number of eggs are formed, four or five as a rule. The embryos hatch out either whilst still in the adult or when discharged into the intestine. The embryos outside the body are capable of full development to sexual maturity and reproduction. These free-living sexual forms differ in all respects from the parasitic sexual forms. They are shorter and broader, the oesophagus has a double dilata- tion, and the embryos are more numerous. The free- living forms must alternate with a parasitic generation for the continued existence of the species. Trichina spiralis. — The adult forms are found only in the intestines and intestinal walls. of man, pigs, rats, &c. They are found only for a period of a few weeks after eating flesh in which encysted embryos were present. The cysts are dissolved in the stomach, the embryos are set free, and in the walls of the human small intestine pass through several metamorphoses and become sexually mature in a few days. The mature worms are just visible with the naked eye. The male measures i to 5 mm. in length and '04 mm. in breadth. There is a straight intestine with a powerful oesophagus. The anus opens into a terminal cloaca into — !:§ C|^ -^ Q ^ ^ ■ c^ .^ E^ — S fi. r .<3 •*» K E^ s ^^ "t i" ,5; s t>3 <^ ^■s :§ S 1- Q s - *- a t^l- O .8 I B- .a •9 _«^ i 11 1- ^^ ^ » a ^ a s K 8 ^ ;;«•« -^1 S^ CD O •<8 Si -a. 1 ^ •« to •a a — ^» 8 S -^■s ^ 2 ."5 Ci «^ — -it ",s i' e -3 1 <^ -s r^s to O TRICHINA SPIRALIS 351 tube opens. There are two digitiform appendages, one on each side of the cloaca, which serve as copulatory organs. The female is larger, 3 to 4 mm. in length and '06 mm. in breadth. There is a single ovarian tube, which is continued into a widely dilated portion, the uterus, from which a narrow tube leads to the genital opening, which is situated about the junction of the anterior fourth with the rest of the body. The embryos are passed alive, but do not appear in the faeces as they pass through the intestinal wall, and finally reach the muscles and there become encysted. They may become encysted in the intestinal walls. Man can therefore be both the intermediate and definitive host, but many other animals harbour the parasites, particularly pigs and rats. It is from badly cooked pork ^that man usually becomes infected. Pigs are probably more frequently infected from rats. To find the adults the intestinal contents of the upper part of the small intestine should be examined with a low power. They may be found by examination with a simple lens, but are easily overlooked. The mucosa must also be scraped off and examined as the worms soon penetrate into it. The encysted embryos are seen as white specks in the muscles, and are most numerous towards the insertion of the muscle. They may be found in fat and other tissues, but are less easily seen in such situations. Examination of the intestinal contents for small worms is facilitated by dilution of the stool with normal saline solution. The mixture can be well shaken, and the worms if viewed against a black background are more readily seen. With small animals the unopened intes- tines may be cut into lengths of an inch or two, and by scraping on the flattened intestine in one direction the contents and parts of the mucous membrane are pressed out of the intestine, and in this expressed substance 352 PROTOZOA then be slit open and placed between two slides and examined for encysted worms. If too opaque it may be placed in weak sodium hydrate solution, which will in a minute or two render it transparent, and the worms will be more readily seen. Protozoa. Protozoa belonging to various orders are found in the stools. Of these the Amoeba coli, a large amoeba which is passed with the mucus and in the faeces in many chronic and recurrent dysenteries, is of the most import- ance. Some observers state that it is found in normal stools, and it is certainly found in stools of patients who do not complain of either diarrhoea or dysentery. In a large proportion of these cases it will be found that mucus is passed with each stool, and in some ulceration of the colon has been present and found on post-mortem examination. In other cases amoebae are found in persons apparently healthy. In a fresh stool the diagnosis is easy, particularly in a warm tropical country, or where a hot stage is used. The large cells with active amoeboid movement often con- taining in their interior red corpuscles vacuoles, permit of no mistake. If the stool has been some time passed, allowed to cool or treated with antiseptics, diagnosis is less easy, as the amoebae, when they die, become globular, and are not easily distinguished from other large cells in the mucus. If they contain blood corpuscles or other substances taken as food, they can be more readily recognised. They stain well with any basic stain, but there is no satisfactory differential stain. For the study of stained specimens smears of the faeces or mucus should be fixed whilst still wet by placing them in a saturated solution of perchloride of mercury for ten minutes. They are then well washed in running water and dried, and can be stained by the iron alum haema- toxylin or with borax carmin. The life-history of the amoeba is not fully confirmed AMCEBA 353 Multiplication may be asexual by simple fission. This occurs in the intestine and in hepatic abscesses. Under certain conditions an amoeba will become encysted, and the contents then divide into two or four young amoebae. Asexual multiplication by simple division of nucleus and cytoplasm. Fig. 145. — Scheme of Development of Amoeba. Multiplication in encysted (oimp (autogamous.) ? sexual multiplication. The early stages of division of the nucleus (a— rf) and conjugation of the divided nuclei in pairs («), followed by further division of these products of conjugation, first into two and then into four each (/— z). The thick wall of the cyst in the later stages indicates the hardening of the cyst wall during the stages when the cysts are outside the body. These encysted forms are resistant, and retain their vitality outside the body. They are probably the important arron+c in fVip nrnnacfafion of the Darasite. The oatho- 354 ENTAMCEBA HYSTOLYTICA severe cases of dysentery the Amceba colt is not found, and it is usually absent in epidemic dysentery." It is most frequently found in relapsing or i-ecurrent dysentery. Amoebae are found in the pus of hepatic abscesses. They are very difficult to find in the pus discharged at first. If the pus be examined three or four days after the abscess is opened they are usually readily found. Schaudinn differentiates two distinct species of Amoeba in the human intestine. The one, the Amoeba coli, may be present in the intestines of healthy persons, and does not invade the tissues or pass into other organs in the body. The other, which he named Entamoeba histolytica, does invade the tissues of the alimentary canal, and may pass to various parts of the body, liver, &c. The morphological differences in the amoeba as seen in the stool are slight. In the Amoeba coli the ectosarc is not clearly differentiated from the endosarc when the amoeba is at rest. The pseudopodia are formed from both ectosarc and endosarc. The nucleus is large and rich in chromatin. Development is as described for the type in main essentials. Entamoeba histolytica has a small nucleus poor in chromatin and placed eccentrically. In the resting amoeba the ectosarc is clearly defined and pseudopodia are formed from the ectosarc only. The development also differs. Multiplication may take place (i) by simple fission, as in Amceba coli ; (2) by irregular gemination, the number of young amoebae formed being indefinite ; or (3) autogamy, in which the nucleus breaks up into a number of chromidia which are diffused through the protoplasm. Secondary nuclei then form at the periphery from which spores are produced. These are surrounded by a yellowish cyst wall and are the resistant forms. In this amoeba, therefore, the resistant encysted forms are young spores, whilst in Amoeba coli it is the mature form which is encysted and the spores are formed inside the cyst. Coccidia are said to have been found in human faeces. CERCOMONAS 355 Various flagellated organisms have been described in Ihe stools. The most important is Cercomonas hominis (fig. 146). It is a small round body with one or two long flagella. It is rarely found in healthy stools but may be common in some cases of diarrhoea. Fig. 146. Fig. 147. Flagellated organisms have also been found in thi mouth and in abscesses in connection with mouth cavity 356 INFUSORIA Infusoria are found in some cases of diarrhoea ; the best known resemble a large Paramcecium — Balantidium coli. It measures 65 to 85 fi in length. It may be found in very large numbers in the stools, and in such cases it may also be found in the intestinal walls and even in the blood-vessels ; it has been found in the pus of an abscess of the liver. It is probably pathogenic. It is a parasite found very commonly in the intestines of pigs (fig- 147)- Vegetable micro-organisms abound. Many of these belong to the coli group and include organisms which are harmless and others which are pathogenic. Many of the organisms, as, for instance, the Bacillus coli com- munis, though harmless to persons in good health as long as they are contained in the alimentary canal, can, under certain circumstances, invade the tissues and then become actively pathogenic and in some cases pyogenic. The intestinal mucosa possess considerable power of resistance even to many decidedly pathogenic organisms, and conse- quently attempts at infection by the imbibition of cultures, &c., often fails. Impaired resistance due to bad health, malnutrition, combined with enhanced virulence of an organism, is necessary in many cases for even pathogenic organisms to cause disease. The isolation and identification of pathogenic and non- pathogenic organisms in the alimentary canal is a matter of considerable difficulty and complexity on account of the large number of organisms and species of organisms usually present. 357 CHAPTER XXI. Urine. It is not proposed to consider the ordinary tests for the abnormal constituents of urine, such as albumen, sugar, and the like, but only a few special points in con- nection with the examination of urine in the Tropics. Blood is found under special circumstances as a result of parasitic invasions by Filarla nocturna and Schisto- somum hcematobium (bilharzia) respectively, and there is at least one form of tropical haemoglobinuria — black- water fever. Hcematuria can be easily distinguished from haemo- globinuria by the presence of red corpuscles in the deposit. In many cases it is easily distinguished with the naked eye, as the superjacent fluid may not be cbloured with blood in haematuria. If coloured with blood it is cloudy and not a clear and transparent red as is the solution of haemoglobin in hasmoglobinuria. In haematuria from bilharzia infection the bulk of the urine is often free from blood, but if the patient, after apparently emptying the bladder strains, the- last few- drops may be found to contain blood or mucus, and in this blood or mucus the characteristic ovum with its terminal spike and the contained ciliated embryo will be found. In all suspected cases it is therefore necessary for the patient to strain, and the few drops so passed are the most important for examination. Schistosoma have been found not only throughout Africa, in Arabia and Cyprus, but also in some of the 358 HEMATURIA that bilharziosis may become a more widely diffused disease than is at present the case. In some of the places, as in the West Indies, it is reported that the Schistosoma eggs are found only in the faeces, and that haematuria does not occur. It is believed by some that the eggs passed per rectum belong to a difFerent undescribed Schistosomum. In most cases of bilharziosis there will be a history of occasional attacks of haematuria. In these cases, by finding the ova in the last few drops of urine expressed from the bladder, the causation of the disease can be determined. Haematuria from filariasis is rarely an admixture of blood only. More often chyle is also present and frequently chyle occurs without any admixture with blood (chyluria). Coagulation of the chyle and blood fre- quently takes place, so that clots of blood-stained sub- stance, or of pure blood, are formed, or the whole mass may set as a pinkish jelly. The embryos of the Filaria nocturna may be found in the urine, but are more abundant in the blood. If scanty any small masses of blood or filaments of thread should be examined as the filariae often adhere to them. Some authors advise filtering the urine, and in the last few drops left in the filter the embryos will be found. There is no periodicity in the appearance of the filaria in the urine. If filariae are not found in the urine they may be found in the peripheral blood of the patient if the examination be made at night. Haematuria may also result from other causes, such as calculus, malignant disease, but those are not limited to the Tropics. Hcemoglobinuria, or the passage of urine coloured with dissolved haemoglobin, is the characteristic of "black- water fever." Cases of paroxysmal haemoglobinuria would, no doubt, if they occurred in an endemic area, be mistaken for blackwater fever. Haemoglobinuria is met with in Africa as a common disease, in some places HEMOGLOBINURIA 359 10 per cent, of the most susceptible portion of the popu- lation (European) are attacked annually, or one-quarter of that proportion of the less susceptible Asiatics. In some parts of India a fair number of cases are met with, but only in small proportions as compared with Africa. Cases are reported from other malarial countries, South America, West Indies, Malaya, South of Europe, &c., but the disease is rare in those countries. The urine when first passed is clear and, when diluted sufficiently, is transparent, but as it cools, and particu- larly when it becomes alkahne, a thick deposit is thrown down. The greater the dilution required to render the urine transparent the more concentrated is the haemo- globin solution in the urine, and the larger the amount of the haemoglobin the more severe and protracted will be the attack. Rate of Secretion. — In this and also in yellow fever the rate of secretion of the urine is a matter of great import- ance, as if the urine is much diminished the prognosis is grave and active measures are urgently required. The times of micturition and the amount passed each time must be observed, and the amount of urine passed at each micturition, divided by the number of hours that have elapsed since the previous micturition, will give the rate of excretion per hour during that period. Any fall in this rate is an important warning. If suppression is once established recovery will not take place in either disease. Bile in the urine may occur in some cases of malaria as a transient phenomenon. The persistent presence of bile in an acute attack of malaria is a rare but a serious and frequently fatal complication. Bile is met with in the urine, frequently in relapsing fever, and is not very rare in lobar pneumonia. There are cases of jaundice occurring in the Tropics, associated with high fever, which are neither yellow fever nor malaria. These require investigation. Nothing is i,„^„Tr. /-.f +V10 triifi riQ+iirp nf tViesp diseases. Pnssihlv 360 INDICAN Haemoglobinuric urine can be distinguished from bilious urine by dilution, when the red colour of the haemoglobin is seen. By shaking the urine and noting the pink tinge of the froth as compared to the yellow tinge of the froth of bilious urine, the distinction is readily made. The most satisfactory method for diagnostic purposes is the use of a spectroscope, when the haemoglobin bands will be clearly seen {vide Table of Spectra). In some of the cases all through, and in others at onset and end of an attack, methaemoglobin is passed alone. Such urine is a brownish colour and can only be distinguished by. the spectroscope (spectra 4 and 5). There is reason to believe that many mild cases of blackwaier fever are overlooked as the urine contains only this methaemo- globin. In this disease casts are often present in large numbers ; the casts are granular, do not often include epithelial cells, but generally contain granules of bright yellow pigment derived from the haemo- globin. Such casts are found for weeks after an attack of blackwater fever, though the urine is free from albumen. It is important to be able, in watching a case, to form an estimate of the variations in the amount of haemo- globin present. This is readily done if the first urine be diluted in the test tube to a convenient known extent. This is the standard, and the other urines found are similarly diluted if necessary till they match the standard. Indican is very commonly present in the urine of patients in the Tropics, usually in cases of intestinal disorder. It is best detected by conversion into indigo blue. The simplest method is to place a crystal of potassium chlorate on the bottom of a tube and cover this crystal with the urine. Strong hydrochloric acid is allowed to run down to the crystal without mixing with the urine. A blue ring forms at the point of junction of the two fluids if indican be present. Nitric Acid Test. — Strong nitric acid added so as to BACTERIA 361 form a layer below the urine will lead to the formation of a red colour at the junction of the fluids. If little indican is present this colour will appear in five to ten minutes; if a considerable amount it will appear at once, and if greatly in excess will be almost black. Bacteria are frequently met with in the urine. Of the pathogenic organisms, the warnings which will be given as to the danger of confusing the smegma bacillus with tubercle must be borne in mind. The typhoid bacillus may be found in the urine for prolonged periods after recovery from the disease; and so may that of Malta fever. Flakes of pus or muco-pus require careful bacteriological exammation. Often they are the remnants of a gonor- rhoeal infection and the gonococcus may be found abundantly. Sometimes they are due to tuberculosis. In all such examinations the urine first passed in the morning should be examined, as such discharges accumu- late during the night. The urine should be divided into three parts : that first passed contains the washings of the urethra. The great mass of the urine which will contain with little admixture substances derived from the kidneys and ureters, and lastly that passed after forcible expression of the last few drops which will contain discharges from the wall of the bladder and from prostatic Crypts. The bacteriology of the urine in the Tropics has received little attention. It is well to remember that urine can be used as a medium for the growth of organisms. That passed about two hours after a meal is the best, as it is not too acid. B. typhosus and many others grow fairly well. It requires boiling, filtering, sterilisation, and can be used either as a fluid medium, or by the addition of gelatine or agar made into a solid medium. Some of the diseases in the Tropics are attended with deviations from normal in the metabolic and catabolic processes indicated to some extent by changes in the 362 HEWLETT'S BODIES Other changes have been noticed in the urine of beri- beri patients and deserve close investigation. Hewlett has noted that casts, usually hyaline, but sometimes granular, can always be found in centrifugalised urine, and in addition that peculiar retractile bodies are present. These he describes as of three classes : (i) Small forms one-third to one-half the diameter of a red corpuscle, very refractile and apparently having a thick capsule with hyaline contents ; (2) larger forms, 12 to 20 /*, spherical in shape, and containing refractile granules, and appearing to contain also what may or may not be a nuclear body; (3) occasional very large bodies, 30 yttin diameter, contain- ing fine granules and a distinct nucleus with nucleolus. These cannot be dried, fixed and stained, but in normal saline in hanging drop preparations they stain neutral red, methylene blue and methyl green, but much less deeply than epithelial cells or leucocytes. None of these bodies stain with osmic acid or with Sudan III., and therefore they cannot be fat. They may by myelin bodies. They are not found in normal urine or in that of persons with nephritis. Hewlett suggests that they may be the result of peculiar degenerative changes in cells, or possibly are protozoa. Diazo-reacUon is a reaction which is constant in typhoid fever. The reaction may occur in other diseases, notably miliary tuberculosis, measles, scarlet fever and erysipelas, usually in severe cases of these diseases. The reaction is usually to be obtained about the fourth to seventh day in enteric fever, and though not conclusive as a test it is an important aid in the diagnosis of obscure forms of continued fever. Two solutions are required for the test : — Solution I. Sulphanilic acid 2 grammes. Hydrochloric acid So cc. Distilled water ... ... ... 1,000 cc. DIAZO-REACTION 363 Solution 2. Sodium nitrite 05 gramme. Distilled water ... ... ... 100 cc. One part of solution No. 2 is added to 'fifty parts of solution No. i,and mixed with an equal quantity of urine in a test tube. This mixture is then rendered strongly alkaline by ammonia. If the reaction is positive the mixture becomes carmine-red in colour, and if the test tube is shaken this colour is seen in the foam. If the colour does not appear in the foam the reaction is negative. Pancreatic Reaction. — The fresh urine must be acid and free from albumen, or, if alkaline, made acid with hydro- chloric acid. To 20 c.cm. of the clear well-filtered urine I c.cm. of strong hydrochloric acid is added. The -mixture is boiled gently in a sand-bath for twenty minutes. The flask is cooled in a stream of water rapidly and the contents are made up to 20 c.cm. with cold distilled water. The excess of acid is neutralised by adding slowly 4 grammes of carbonate of lead. The contents of the flask when cooled are filtered till a clear filtrate is obtained. If sugar is present it must now be removed by fermentation. The filtrate is well shaken with 4 grammes of tribasic acetate of lead, and again the mixture is repeatedly filtered till the filtrate is quite clear. This filtrate is shaken up with 2 grammes of sulphate of soda to precipitate the lead and the mixture is again boiled, cooled and filtered ; 7 cc. of the clear filtrate is made up to 18 cc. with distilled water, and to this is added o*8 grammes phenylhydrazin hydrochlorate, 2 grammes of powdered sodium acetate, and i cc. of 50 per cent, acetic acid. This mixture is boiled for ten minutes, filtered whilst hot and diluted, if necessary, up to 15 cc. with hot distilled water. If the " pancreatic reaction " is present a light yellow flocculent precipitate will form in a few hours. This .;ii 1- _ 364 PANCREATIC REACTION to consist of long, light yellow, flexible, hair-like crystals, arranged in sheaves. Sulphuric acid 33 per cent, causes these crystals to melt and disappear in ten to fifteen seconds. According to Cammidge, from whom this method is abstracted, a positive reaction is indicative of pancreatic disease. 365 CHAPTER XXII. Bacteriology. The pathogenic micro-organisms with vegetable char- acteristics are less generally studied in the Tropics than elsewhere. Much of the easier work could be done without complicated apparatus or any great difficulty. The methods now employed in British laboratories require too much apparatus and are too complicated to be used by a private worker in the Tropics, and only the simpler methods which are at his disposal are considered here. This account of the methods which can be used is therefore intended only for those obliged to use primi- tive methods and makeshifts. For the isolation and cultivation of vegetable micro- organisms artificial media are necessary, and the basis of the standard media is nutrient broth. There is much difficulty attending the making of nutrient broth from meat in the Tropics, but meat extracts, particularly Bovril or Liebig's, make an efficient substitute. In broth prepared from either of these, the organisms that will grow in nutrient broth made from meat will grow fairly well. An iron enamelled jug, measures, scales and weights, a glass rod, a funnel, and ordinary filter paper or white blotting paper is all the apparatus required. Bovril peptone, and common salt and water are the substances needed, and litmus paper, or better, phenolphthalein, which is required for the neutralisation of the broth 366 NUTRIENT BROTH Nutrient Broth. — To make the broth : Take i,ooo cc. or I Htre of water ; then take 5 grammes each of Bovril (or Liebig) and salt, and 10 grammes of peptone (Wittes' if usually used). Mix the peptone with about 25 cc. of the water and stir it well so as to form a kind of emulsion ; then to this add the remainder of the water and the salt and Bovril. The Bovril can be conveniently weighed in a watch-glass, or if Liebeg is preferred, this can be spread with a spatula on a piece of filter paper, and the watch-glass with the Bovril in it, or the filter paper with the Liebig's Extract on it, can be placed in the water with the other ingredients. The whole should now be boiled for a quarter of an hour and well stirred to ensure thorough solution. It is now ready for neutralisation. When made with Liebig the broth will be much too acid to get good growths, and with Bovril, though much less acid, may be too acid to be quite satisfactory. Moreover, the degree of acidity of different specimens varies. Neutralisation. — Litmus paper can be used in an emergency to determine the reaction of the broth, but is unsatisfactory, as many of the organic acids do not affect litmus paper, and the dibasic sodium phosphates act on litmus paper as an alkali. Many specimens of broth also have a double reaction, turning red litmjjs paper blue and blue litmus paper red, so as to leave the point of neutralisation uncertain. Where possible phenplphthalein should be used. A -5 per cent, solution of phenolphthalein in spirit is the indicator. This solution is colourless when acid or neutral, but turns a deep magenta colour with any free alkali. Carbonic acid should be expelled from a measured quantity of the broth, say 25 cc. by boiling ; to this broth a few drops of the phenolphthalein should be added, and then drop by drop the alkaline solution, till the broth turns a flesh or faint pink colour, indicating that the alkali is completely neutralised. The amount of alkaline NEUTRALISATION 367 solution has been measured, and as there are 975 cc. of broth left the amount required for the neutralisation of the 25 cc. multiplied by -y/- =, 39 will give the amount of the alkaline solution required for the neutralisation of the remainder of the broth. It is to be noted that to exactly neutralise the broth it is of no importance what the strength of the alkaline solution may be. A neutral broth so prepared ■wj;ill serve for the growth of most organisms, but the best growths are obtained with a broth slightly acid to phenolphthalein. If it be desired to use a less or more alkaline broth it is necessary to have an alkaline solution of known strength. The solutions used are the so-called " normal solutions." A normal solution is a solution of the " equivalent " weight in grammes of the substance dissolved in a litre of distilled water. If the metal of the salt be monovalent, i.e., if it is replaceable in a compound by one atom of hydrogen, the "equivalent" weight is the molecular weight in grammes. If the metal be bivalent, i.e., if it requires two atoms of hydrogen to replace it in a compound, the equivalent weight is half the molecular weight in grammes. A decinormal solution is one-tenth of that strength or one-tenth of the equivalent weight in grammes dissolved in a litre of water. A centinormal solution is one-hundredth of the same weight dissolved in a litre ; whilst a dekanormal solution is ten times as strong as the normal, or ten times the weight dis- solved in a litre. For instance, the equivalent weight of sodium hydrate, NaOH, is 23 -1- 16 + i = 40, of sulphuric acid, HgSO^, as it neutralises two molecules of sodium hydrate, is i (2 -t- 32 -|- 64) or ^ = 49. A normal solution is represented by j, a decinormal by -^ a centinormal by j^. A normal solution of sodium hydrate therefore is 40 grammes dissolved in water and diluted to 1,000 cc, whilst a normal solution of sulphuric 368 NEUTRALISATION hot with phenolphthalein ; such a broth is usually alkaline when tested by that uncertain standard litmus paper. The degree of alkalinity of a broth is measured by the number of cc. of normal alkaline solution added per 1,000 cc. of broth over and above that required for neutralisation. The minus sign — is used to indicate the alkalinity, so that — 4 would indicate that 4 cc. of a solution J of alkali had been added to 1,000 litres of the broth in excess of the amount required for neutralisation. If the broth used is still acid as tested by phenolph- thalein, that is indicated by the plus sign +. A broth described as + 10 would still require the addition of 10 cc. of ? solution of alkali per litre for neutralisation. Many specimens of Bovril broth, without neutralisations, are not more acid than this, and + 10 is a favourite reaction for the growths of many organisms. This question of neutralisation and of uniformity of reaction is a simple but important matter. The degree of alkalinity or otherwise of the media affects the properties of growths so materially that it is necessary to be particular on the point, but for mere growth of most organisms a broth neutral to phenolphthalein will suffice. After neutralisation or procuring the required degree of alkalinity or acidity to phenolphthalein, the broth should be boiled and kept at the temperature of boiling water for half an hour. It should then be allowed to cool, as it is not till it is cold that the mass of the phosphates will be precipitated. It is then, whilst cold, to be filtered through ordinary white filter paper. The broth is now prepared, but in the course of the preparation many organisms will have gained access to it from air, vessels, &c., and if left as it is these would multiply. Sterilisa- tion is therefore necessary. This can either be done in bulk or the broth can be decanted into a series of test tubes in quantities suitable for use. The procedure differs little in the two cases. If it be desired to keep the broth in bulk it should be poured STERILISATION 369 into a clean narrow-necked vessel (Erlenmeyer flask, fig. 148), which will stand heat, and the mouth of this vessel plugged tightly with cotton-wool. If it is to be divided, some 10 cc. should be poured into each of a series of clean test tubes and the mouth of each should be plugged with cotton-wool. It is better to sterilise, by dry heat, the flask or the tubes and wool before pouring in the broth. This is not absolutely essential, as the tubes, wool, and broth contained in the tubes can all be sterilised together, but is advisable. Fig. 148. For sterilisation a single boiling does not suffice, as spores are only slowly killed at the temperature of boiling water. Sterilisation. — ^To sterilise, the broth and the vessels containing it should be maintained at the temperature of boiling water for at least half an hour on three consecutive days and allowed to cool in between. This intermittent method allows the spores which have escaped the first sterilisation to develop into the less resistant organisms before the second heating, which then destroys them. The third sterilisation, which is not always absolutely necessary, is a precaution in case any spores or organisms have escaped from the two previous sterilisations. 370 VARIOUS MEDIA use and can be kept till required. The tubes, wool, and broth are all sterile and remain so for a considerable period. Organisms can only gain access to the broth by growing through the wool. This does not take place through dry wool, but in moist, warm climates, such as are met with in the Tropics, the wool gets damp and growth through it takes place. In such climates it is advisable as a routine every week to heat the end of the test tube containing the cotton-wool so as to ensure the wool being dry and to kill any organisms that have grown in it. Unless this precaution is taken the tubes soon become contaminated. The number of organisms falling on to the cotton- wool can be greatly reduced by covering the top of the tube with an inverted paper cone, such as a folded filter paper. This measure would delay the contamination of wool even in the Tropics. Glycerine Broth, &€. — For many purposes additions are made to the nutrient broth. These additions must be made before neutralisation and sterilisation ; if made after, 'the sterilisations will require to be repeated. Glycerine broth is made by the addition of 6 per cent, of glycerine. Glucose, lactose, maltose or saccharose, added in the proportion of 2 per cent, to the broth, make glucose broth, lactose broth, &c., respectively.- Solid Media. — The broth is mixed with gelatine or agar-agar in sufficient proportion for the solution to set when cooled to the temperatures at which it is desired to study the growths. Nutrient Gelatine. — The gelatine medium is made by the addition of 9 to 12 per cent, of the best French gelatine to the crude broth. Broth that has been neutralised and filtered can be used, but it is waste Of time, as neutralisation will have to be repeated. Gelatine is always acid. After the gelatine has been added in the required proportion keep in the steamer for half an hour ; neu- tralise, render alkaline or leave acid to the required SOLID MEDIA 371 extent. Allow to cool to 60° C. or less as long as the gelatine remains fluid. Whip up the white of an egg for each 500 cc. of the gelatine broth and mix well with the rest of the medium. Steam for half an hour. The white of the egg diffused through the medium will coagulate, and in its coagula- tion will carry down many of the impurities of the gelatine. Filter whilst hot through a coarse filter paper — Chardin's — which should be moistened with hot water. Store in flasks or decant into test tubes as required. Sterilise as with nutrient broth for half an hour on three consecutive days. This medium is known as nutrient gelatine, or simply "gelatine." Glucose, &c., &c., can be added to it if required, the addition being made preferably before neutralisation and always before sterilisation. Too prolonged heating causes hydrolytic changes in the gelatine so that it will not set. Extra sterilisations must be avoided where possible on this account. Nutrient Agar. — Agar or nutrient agar is made in a similar manner ; 1-5 to 2 per cent, of the powdered agar is added instead of the gelatine. It is much more difficult to filter, and where the necessary time cannot be given, a passable substitute is to allow it to cool slowly so as to permit the coagulated egg albumen and other precipitates to settle to the bottom. When cold the mass can be removed from the vessel and the lower part containing the great mass of the impurities cut off. The residue, though much inferior in appearance to the filtered product, is sufficiently clear to be translucent and can be satisfactorily used without filtration for cultures. The clearer filtered product is better. To filter it is necessary that the filter paper should be kept hot. This is best effected by placing the funnel, filter paper, and receptacle in the steam steriliser and allowing the filtra- tion to take place in the steam steriliser. 372 SEPARATION OF ORGANISMS filter paper so as to have a large number of angles and very little of the paper in contact with the glass. These papers can be bought ready folded or can be folded before use. Addition of glucose, &c., can be made as in the case of other media before neutralisation and filtration. The solid media are essential for the separation of the various organisms usually present in the animal tissues, discharges, or other substances to be examined. Separation of Organisms. — The method of procedure is based on the principle that by successive dilutions of a minute quantity of the substance to be examined the individual organisms will be so scantily distributed through the medium that they will be separated from each other by an interval appreciable to the eye. If this medium be then allowed to become solid the or- ganisms will remain well separated from each other, and if kept under conditions favourable for their growth will multiply and form in. the course of a few days " colonies " which will be visible to the naked eye. From these colonies sub-cultures can be made, and the colonies may also be examined directly. A piece of platinum wire, 3 to 4 inches in length, is inserted into the fused end of a glass rod, and a small loop is made at the other, the free end, of the platinum wire. This wire is sterilised by heating in the flame, and a loopful of the substance to be examined is taken up by this loop. A tube containing the gelatine medium melted by placing in hot water is then inoculated with this loopful and the tube is rolled between the hands to secure uni- form admixture. The amount of the substance is thus diluted by the amount of the fluid gelatine. After sterilising the needle a loopful from this tube is inoculated into a second tube and will again be diluted to the same extent. A third tube is treated in the same manner and the dilution will now be extreme. PLATING 373 In other words, provided the mixing is thorough the organisms will be so much diluted by these successive dilutions that they will be separated from each other by appreciable intervals. A fourth or a fifth dilution may be made, but is not usually required, as the third dilu- tion is in most instances sufficient. The end of each of these tubes, with the plug with- drawn, is heated in turn to destroy any organisms which may be present at the edge of the tube and the gela- tine is poured into a flat sterilised glass dish (fig. 149) — a Petri dish — which is quickly covered with another similar but larger sterilised dish. The melted gelatine Fig. 149. soUdifies as a thin sheet of nutrient gelatine, and is allowed to remain at a 'temperature of about 20° to 22° C. Some organisms will grow quickly and others slowly, and by colour, size, shape of colonies and effect on the gelatine it is usually possible to distinguish that several organisms are present. In the plate from the first tube the colonies are so numerous that they are separated from each other by too small a distance to isolate. In the plate from the second the organisms may be suffi- ciently far apart, and in the third and subsequent dilutions the colonies resulting from the growth of the widely separated organisms are usually far enough apart to be easily distinguished from each other. From these cultures can be made. As the first dilution is too little diluted for practical work and the second is usually so, it is unnecessary to 374 PLATING plates of them. The two first dilutions may be done in sterile broth or even in a weak sterile salt solution, 5 grammes to a litre, and only the third in the solid medium. This economises the solid medium, which is the most troublesome to prepare. Plating may be done with agar, but a thermometer must be used to make sure that the agar is cool enough, as if too hot the organisms may be killed. The agar will have to be heated to nearly the boiling point of water to become thoroughly fluid, and allowed to cool to about 44° C. before inoculation. It is not so easy a proceeding as plating with gelatine, but agar is the only solid medium that can be used in many parts of the Tropics, as above 22° C. the gelatine will not set. Stronger solutions, as 20 per cent, gelatine, will remain solid at 37'5° C., but these stronger gelatines are not easy to work with and frequently undergo changes during sterilisation that cause liquefaction or acid production. Unless ice and a cold incubator are available we are restricted to the use of agar for plating. A convenient method of plating in agar is to make the agar plates and inoculate when the agar has set either from the second or third broth solution, by making a series of parallel strokes with a platinum loop on the solidified surface, or, and better, by using a sterilised brush — camel's hair — and brushing lightly over the sur- face of the medium after dipping this brush in the second or third broth dilution. Excess of fluid is to be avoided by draining off from the brush against the inner side of the tube. The brush should be sterilised in a dry tube, plugged with wool, by three successive sterilisations. The platinum wire is sterilised as usual by heating in the flame. Some important organisms will not grow on any known artificial medium, and others only on special media or under special conditions. Separation of such organisms is either impossible or difficult. Standard books on bacteriology should be consulted for methods, DESCRIPTION OF ORGANISMS 375 but these will not usually be practicable for the solitary practitioner under the . conditions of tropical life and work. Description of Organisms. — Having obtained a pure culture of an organism the more important points to determine are as follows : — (i) Size, shape and arrangement. Morphological appearance. (2) Motility. (3) Spore formation. (4) Structure, Flagella, capsule, &c. (5) Staining reactions : {a) Simple stains ; {b) Gram's method ; (c) Ziehl-Neelson, (6) Growths on artificial media: {a) In broth; (6) on gelatine ; (c) on agar. (7) Conditions : {a) Essential to growth ; (6) favour- able to growth ; (c) inimical to growth. (8) Chemical products : Gas formation and curdling of milk ; acid or alkali formation ; indol formation, (9) Reaction with blood sera, particularly with the blood sera of patients suffering from definite diseases. (10) Pathogenic properties. In organisms such as B. lepra, which cannot be cultivated, only a few of these points can be determined ; and the pathogenicity has not been proved experiment- ally, with a doubtful exception, as lower animals are insusceptible. The causal relation is inferred from the constant association of the organisms, identified by their staining reactions and appearances in the lesions resulting from the disease, and from the observation that they are not found except in this disease. We propose to take briefly the methods of observing these various points. The descriptive terms used with reference to them, the relative value and the limitations to the value of each point for diagnostic purposes will also be considered. (i) The size and shape of an organism is be^t observed / / / 376 PREPARATION OF FILMS with a mordant will suffice. Films should be made on a slide or cover-glass. For this purpose with a culture in broth all that is required is to spread a drop with the platinum wire. If the culture is on agar or gelatine it should be rubbed up with a little water (sterile), and of this emulsion a portion should be spread out with a needle. Preparation of Films. — If it be desired to make a film or smear from any natural fluid or excretion it may require the addition of a little water to make a satis- factory smear. From blood, thick films may be used and decolourised by removiiig the hasmoglobin with sterile water. Tissues should be cut with a sterile knife and the cut surface rubbed on the slide. The most difficult films to make are those such as sputum containing much mucus. The' most satisfactory method is to use two slides and warm both. A portion of the mucus is transferred to one slide and this is warmed over a Bunsen flame or spirit lamp, and a second slide is warmed at the same time. The second warmed slide is used to rub the mucus on the first and is placed with the long axis at right angles to the other, and the surfaces parallel and in contact. The slides are then separated and both are again warmed in the flame with the smeared side of each uppermost. They are again, whilst still hot, rubbed together with the smeared surface of the two in contact. This process is repeated till the films are nearly dry, when they are finally rubbed ^together .harder. Good thin, dry films are easily and quickly obtained by this method. However the films are made they require fixation. This is best done by heat and is usually accomplished by passing through a smokeless flame three times, the smeared side always uppermost. Do not char the film. As many films are so thin that they are difficult to see when dry, it is well to mark the smeared side of the slide with. a grease pencil. The staining fluid is simply placed over the film for the requisite time and then SCHIZOMYCETES 377 washed off. Stains used are Loffler's blue, five to ten minutes ; carbol thionin, five minutes ; carbol fuchsin or gentian violet for organisms that do not take other stains deeply, one to ten minutes. SCHIZOMYCETES. Bacteria or Schizomycetes are unicellular vegetable organisms. Reproduction is by fission. Resistant forms called " spores " may be produced. They are differentiated by these characters from Hyphomycetes or moulds, in which spores form in specially differentiated cells, and from Blastomycetes or yeasts, which are oval or rounded bodies and in which reproduction is accomplished by budding. (i) Morphology. — The various shapes of bacteria usually described are Cocci or rounded or oval bodies, with the greatest diameter not more than twice the least. Micro- cocci is used for the smaller forms. If division takes place only in one direction the organisms may remain attached in pairs, Diplococci, or in chains. Streptococci, if a series remain attached. In other cases division takes place in two directions, and we then find the organisms arranged in squares of four or multiples of four. Such growths are called Tetrads. Others divide in three directions at right angles to each other and form cubical masses, these are known as Sarcince. A common arrangement is irregular growth in all directions, leading to an irregular mass or cluster of cocci, Staphylococci. Bacilli are cells that are rod-shaped. They are longer, at least twice as long as they are broad, and the shorter forms are distinguished from oval cocci by having the two sides parallel. By fission they may grow into long jointed rods — streptobacilli. Curved organisms are, when short, known as Vibrios, when longer and more twisted as Spirilla ox Spirobacteria. 378 MOTILITY Leptothrix. — Rod-shaped, filamentous forms showing differentiation between base and apex, but not branching. Streptothrix. — Filamentous forms showing true branch- ing. These form the connecting link between the Schizo- mycetes and the Hyphomycetes. Measurements are made as for other minute bodies. Some organisms readily change their form with varia- tions in the condition under which growth has taken place. If the variations are great the organism is de- scribed as " pleomorphic." Slight variations occur in all organisms, so that morphological characters alone are not to be rehed on. (2) Motility. — This can only be observed in living cultures, though it can be inferred for organisms which are shown to have flagella. The motion of motile organ- isms must be clearly distinguished from the oscillatory movement — Brownian movement — common to aJl minute particles suspended in fluid. True motility is best observed in a " hanging drop " preparation. This is made by making a thick ring with vaseline on a slide and taking a clean cover-glass, rather longer than this ring, and placing near the centre a small drop of the culture of living organisms to be examined. The slide is then taken up and turned so that the vaseline ring is directed downwards, and is gently brought into contact with the cover-glass so that the drop of culture on the cover is in the centre of the ring of vaseline. The cover will adhere to the vaseline ring and form a sealed chamber, and when the slide is turned over again the drop will hang from the lid of this chamber, the cover-glass, and can then be examined. If the tempera- ture is so high that the vaseline runs, lard can be substituted for it. The organisms are colourless and transparent and difficult to focus, so that the light must be reduced by nearly closing the iris diaphragm. Either \ ov ^ oil immersion objective may be used. It is well to focus first on to the edge of the vaseline ring and then move SPORE FORMATION 379 the slide towards the drop, keeping the droplets of water of condensation which usually form on the under surface of the cover-glass in focus till the edge of the drop is reached. With a little practice and a dim light the organisms can then be brought into focus and the pres- ence or absence of automatic motility determined. This property, though an important point of difference between some organisms that closely resemble each other morphologically, is subject to considerable variation, and the degree of motility in motile organisms varies from slight causes, such as slight difference in temperature, reaction of the medium, &c. (3) Spore Formation. — All the micro-organisms repro- duce by fission, but some of them also enter into a resting stage — spores. The resting form is much more resistent to agencies, chemical, heat, &c., which destroy organisms, so that spores will withstand for some time the temperature of boiling water, though the active phase of the organism is at once destroyed. Some organisms form spores very readily, others only under certain circumstances not thoroughly understood, and many pathogenic and other organisms do not form spores under any known circumstances. The spores can often be recognised in the living culture, as they are usually more highly refractile as well as rounder. For the demonstration in dried films advantage is taken of the fact that spores stain with greater difficulty, but when stained, retain their stain better than the organ- isms from which they were derived. A simple method is to stain with warm carbol fuchsin for five minutes. This much overstains both spores and bacilli. Treat rapidly with 2 per cent, sulphuric acid ; this, if done rapidly, will leave the spores stained, but remove the stain entirely from the bacilli. Wash well to remove the last traces of acid and counter-stain with Loffler's blue for ten minutes, or carbol thionin. The spores will be stained by the fuchsin and the bacilli blue by the methylene blue or thionin. 380 STRUCTURE OF ORGANISMS This method is successful for most spore-forming organisms. (4) Structure of Organisms. — Certain points in the struc- ture of organisms are sufficiently definite to be of use in diagnosis. Capsules. — Some organisms have a thick capsule which does not stain deeply with basic stains, or may not stain at all. The simplest method of demonstration is to stain the film with carbol fuchsin and to examine in water, not in Canada balsam. The organism is surrounded by a clear space, and the capsule with a defined edge can usually be seen. Welch treats the film with 2 per cent, acetic acid, which causes the capsule to swell and enables it to take the stain, and then after removal of the acid he stains with aniline gentian violet for five to thirty seconds. Cap- sulated organisms often lose their capsules in culture, but the presence or absence "of a capsule, as seen, for instance, in sputum, is of value. A cell wall is probably present in all the organisms, but it is difficult to demonstrate. In some, however, it is fairly well marked. It is best shown after the cell contents have been caused to shrink by salt solutions or iodine solution (plasmolysis). , Flagella. — Motile organisms have been shown to have flagella. They are variable in number, and whilst the vibrios have usually only one or two the motile bacilli may have large numbers. The number of flagella is of §pme value in the differentiation of species, and the presence, absence, or plan of arrangement is of differential value in grouping organisms. The methods of demonstration cannot be considered as satisfactory or easy, and there is considerable uncer- tainty in the results ; they are all troublesome. The two common methods successfully employed are Muir's modified Pitfield and MacCrorie's. By Muir's method the mordant employed is composed of :— FLAGELLA STAINING 381 Tannic acid 10 per cent, aqueous solu tion Corrosive sublimate saturated aqueous solution Alum saturated aqueous solution Carbol fuchsin ID cc. 5cc. 5 cc. 5 cc' This is well mixed, allowed to settle, and the clear fluid decanted off and centrifugalised. This mordant keeps for about a fortnight, but must be centrifugalised each time before use. The stain employed is composed of a saturated solution of alum, 25 cc, with 5 cc. of alcoholic gentian violet saturated solution. This must be prepared immediately before use. Fig. 150. The smears should be made from agar cultures, twelve to eighteen hours old, emulsified with a little distilled water. Spread very gently on a cover-glass, freshly flamed to free from grease. Hold in Cornet's forceps (fijg. 150), being careful that the film side corresponds to the fenestrated side of the forceps, otherwise mistakes as to which is the film side may occur. Allow the film to dry in air and fix by passing through the flame. Cover with the mordant and heat till it steams for two minutes. Wash well in water and dry carefully. Pour on the gentian violet stain and heat till the staining fluid steams for two minutes. Wash in water, dry and mount in Canada balsam. This method requires the use of a centrifuge, but gives a large proportion of successful results. MacCrorie's method is simpler. A single stain com- 382 SIMPLE STAINS bined with a mordant is used for staining the flagella and the baciUi are counter-stained with carbol fuchsin. The stain is composed of : — Night blue saturated alcoholic solution 10 cc. Potash alum saturated aqueous solution 10 cc. Tannin 10 per cent, aqueous solution ... 10 cc. Gallic acid i or 2 grammes improves the solution. Films from young agar cultures are prepared as above and the stain is placed on the film for five minutes and slightly warmed. It must be flushed off with running water or a thick, dirty deposit will be left. Counter-stain with carbol fuchsin, dry and mount. (5) Differentiation by Methods of Staining. — There are three main methods of diagnostic value : — (a) Simple Stains. — There is great variation in the ease with which different organisms take up stains, and this difference is sometimes of value. Some organisms do not stain uniformly, and such differences as pre- ferential affinity of stains for the ends of a bacillus, bipolar staining, is one of the characteristics of the plague bacillus. In cultures organisms frequently lose their characteristic staining reactions. Of greater value are two special methods. (6) Gram's Method is based on the fact that some organisms will retain their stain when treated with alcohol if, after staining, they are treated with a solution of iodine. A freshly prepared solution of gentian violet in aniline water is made by shaking up a few drops of aniline oil with water and filtering. To this is added drop by drop an alcoholic solution of gentian violet till a metallic film begins to form on the surface. With this stain the fixed film is stained for five minutes. Carbolic acid i in 20 can be used instead of the aniline water. If now treated with alcohol the stain would be completely removed from all the organisms. In some organisms the addition to the film of Gram's iodine solution, composed of iodine i part, potassium iodide 2 parts, and water 300 parts, for two minutes. gram's method 383 will fix the stain in these organisms so that when the film has been treated with alcohol they still retain the purple colour, whilst it is removed from evei-ything else. The organisms which retain their stains are those which are described as " staining by Gram." The alcohol is kept on till it ceases to remove any more colour and not longer, as in time it will remove the stain even from the organisms which stain by Gram. It is convenient instead of using a plain alcohol to use an alcoholic solution of eosin ^ per cent., as then organisms which do not stain by Gram will be stained faintly by the eosin. This is of most use when working with a mixture of organisms, such as is met with in many secretions, &c. For sections it has the additional advantage that it does not require such prolonged treatment with alcohol as is required if alcohol is first used to decolourise and then again for counter-staining. As the action of alcohol is so rapid when working with organisms that retain the stain less firmly, another agent that decolourises more slowly is better. The agent used is aniline oil. This decolourises the non- Gram staining organisms as effectually as alcohol. It is used in the same way but can be left on longer. If counter-staining is desired the counter-stain must be used before staining by Gram. The use of aniline oil instead of alcohol is better for sections. Counter-staining with eosin or bismuth brown should be done first. After staining with Gram and adding the iodine, blot and treat with aniline oil. This will dehydrate as well as remove the stain from non- Gram staining Organisms. Then wash off the aniline oil with xylol and mount in xylol balsam. Most of the pyogenic cocci, the organisms usually found in suppuration, stain by Gram. Many of the organisms associated with intestinal and other diseases do not stain by Gram. (c) Ziehl-Neelson's Method. — A comparatively small 384 ziehl-neelson's method number of groups of organisms are described as acid fast, because when once stained they retain the stain even after treatment with fairly strong, 25 per cent., solutions of the mineral acids. Hydrochloric, nitric or sulphuric acids are those used. The method employed is to use a strong basic stain such as fuchsin in a i in 20 aqueous solution of carbolic acid, and either to stain in the cold for some hours, or more conveniently to warm until the carbol fuchsin steams, and then keep warm for five minutes. This is conveniently done on the slide. The film is fixed as usual and covered with the carbol fuchsin. The slide is placed on a copper which has been warmed in the flame and left there, fresh stain being added if evapor- ation is too rapid or the stain shows signs of boiling. The stain is flushed off and replaced by a 25 per cent, solution of sulphuric acid. The pink colour disappears and is replaced by a yellow. The film is again washed and if still pink again treated with sulphuric acid. This is repeated till on washing at the most a faint pink colour returns. The specimen is well washed in water to completely remove the acid and counter-stained with Loffler's blue for five minutes. Wash, dry, and either examine directly by placing a drop of oil on the film or mount in Canada balsam. The acid-fast organisms retain the red colour of the fuchsin whilst other organisms are stained blue by the methylene blue which is used as the counter-stain. In tropical work it is important only to use fresh carbol fuchsin. The solution keeps well in England, but in the Tropics it deteriorates , so that sometimes in a week or so, and at others in some months, it ceases to stain well. Colour-blind people will find it well to use gentian violet instead of fuchsin. An alcoholic solution of gentian violet is added to the i in 20 carbolic acid to make the stain, and Bismarck brown is used as the counter-stain. The more important members of the acid-fast group TUBERCLE 385 cannot be cultivated in the simpler media. It will therefore be convenient to consider these organisms here. There are four main groups of the acid-fast organisms, which will be considered under the heading of the best known member of the group : — Tubercle ; lepra ; smegma ; Timothy grass. Some forms of the Streptothrix group are also "acid- fast." The tubercle group includes the organisms found in tuberculosis, in mammals, birds and reptiles. The organisms can be cultivated on blood serum and nutrient glycerine agar, or in glycerine veal broth. Growth is slow and much affected by the temperature. The preferential temperature is that of , the animal from which the organisms were obtained. The mammalian, avian and reptilian tubercle bacilli therefore grow at different temperatures and are pathogenic to mammals, birds and reptiles re- spectively. Some authorities hold that they are modifications of one and the same organisms and that they can, by suitable methods, have their characters altered so that the differences disappear. By most authorities the three ar£ considered to be specifically distinct, and some go further and do not admit the specific unity of the tubercle organ- isms in different mammals. Koch holds that bovine and human tuberculosis are distinct, on the ground that their pathogenicity varies. Tubercle bacilli in man are found in the secre- tions or excretions from an infected organ and there- fore may be found in sputum, urine, &c. They usually set up a granulomatous new growth which has a marked tendency to caseate and break down. The organisms may be present in large numbers, but in some situations, such as the skin, bones, pleural effusions, &c., they are usually found only in small numbers. They are found but rarely in the blood. 25 386 LEPROSY The Lepra Bacillus is the only representative known of this group. It cannot be cultivated on artificial media, and all experiments at inoculation of lower animals have failed. The organisms are found in extraordinary numbers in leprous tubercles in the skin, and when these ulcerate, in the discharges from these ulcers. Before ulceration the bacilli can be readily demonstrated, by compression with a clamp of a tubercle or portion of infiltrated skin. On pricking this, serum loaded with the bacilli will exude. The bacilli are not found in ulcers or sores in purely nerve leprosy, as in that form they are present in the nerve sheaths and the ulcer or necrosis is not due to the breaking down of a leprous granuloma, or of tissues infiltrated with the organisms. One of the most constant natural discharges to contain the bacilli is the mucus discharged from the nose. It is also one of the earliest manifes- tations in many cases, including some of nerve or anaesthetic leprosy. By some it is believed that the earliest and most constant lesion of leprosy of all forms is a deposit in or below the nasal mucous membrane. Various differences in size, staining reaction, &c., between lepra and tubercle bacilli have been de- scribed, but they are not sufficiently marked or constant to be of diagnostic value. The most im- portant diagnostic point in films of mucus is the aggregation of the bacilli into small, dense clumps in leprosy, in many cases still retaining the outline of the cell in which they grew. In sections of skin the extraordinary profusion of the organisms, as well as the aggregation into compact masses, is characteristic of lepra bacillus as opposed to human tubercle. In some of the lesions of avian tubercle a similar grouping may be found, whilst in internal organs in man there may be diffuse growth of the lepra bacilli. SMEGMA 387 Smegma Bacilli. — This group probably includes several species. In most specimens of sniegma the organisms, though truly acid fast, are decolourised by alcohol, that is, they are not alcohol fast. Some varieties, however, do not lose their stain in alcohol and are like the tubercle, both acid and alcohol fast. This organism has been the cause of frequent mistakes in diagnosis, as- urine easily becomes con- taminated with this bacillus, and it may readily be mistaken for that of tubercle and a diagnosis of urinary or renal tuberculosis given. In the majority of cases the use of alcohol, as well as of acid, will prevent this mistake, but as some specimens of the smfegma, including Lustgarten's so-called syphilis bacillus, are also alcohol fast, the possibility of the confusion should be avoided by using the catheter. The fourth group, of which the Timothy grass bacillus, or B. phlh, is taken as the type, are the only organisms of this group which grow readily on' almost any medium. They are found on several species of grass used as fodder and may be found in enortnouis numbers in the faeces of cattle. As a consequence they are often found in milk and prodiicts, such as butter and cheese, derived from milk. Several varieties or species have been de- scribed: This group is of economic importance, as cattle have been condemned as tuberculous, on the grounds that acid-fast ba^cilii were found in the stools, and milk ; butter and cheese have also been condemned on this insufficient reason. These or- ganisms are not pathogenic. (6) Growths on Artificial Media. — With organisms that can be cultivated, the growths on artificial media (nutrient broth, gelatine or agar), differ in some cases sufficiently to be of diagnostic value, and in any case the character of the growth is one of the properties of an organism that requires description. Cultures may be made on plates as in the separation 388 CHARACTERS OF CULTURES of different organisms, or, and more conveniently, in tubes. The growths in fluid media are made by taking on a sterilised platinum loop a portion of the culture and inoculating the tube. The nature and character of the naked-eye appearance in the broth at varying periods should be described. The temperature at which the cultivation is made must also be noted where incu- bators are available. Blood heat, 37° C, and 21° C, are the most convenient, and terms like "room temperature" should be avoided. In many tropical countries the temperature of the air will range from 25° to 30° C, and satisfactory growths of the more important organisms can be obtaine'd. In a description of the growths at these temperatures a result intermediate between those at incubator temperatures will be obtained. All cultivations must be carried on in the dark. The points to be observed in a broth culture are the surface, whether covered with Or free from a film or pellicle. In the body of the fluid note if the fluid is turbid and the degree of turbidity, if not turbid whether quite clear or with floating particles ; the presence or absence of a precipitate and, if one be present, whether it is composed of a uniform fine deposit or if in separate masses. Any change in colour, and bubbles from formation of gas, must be further noted. On solid media the growths may be observed on plates or in tubes. In tubes the growths can be seen on sloped cultures by drawing the inoculated platinum loop over the surface of the medium obtained by placing the tube, whilst the medium was still liquid, in ,a sloped position and allowing it to set, or in stab cultures. In these the medium is allowed to set with the tubes vertical. An inoculated wire, not a loop, is plunged steadily into the depths of the medium and withdrawn without splitting the medium. The appearance of the separate colonies is niost im- portant. There are great diversities in the appearance of growths on solid media, and an acQurate. series of CULTURES 389 defined terms for descriptive purposes is much needed. Such a series, of descriptive terms has been drawn up by Chester, but many of the terms will probably not be generally accepted and they are used at present by few bacteriologists. The observer should note and describe the size of the of the individual colonies, their shape, the character of the edge, their elevation, whether raised or depressed, and give, a detailed account of the character of the surface. The macroscopic appearances of the colony : if transparent, whether highly refractile or not, and if not clear whether opalescent, finely or coarsely granular, or irregularly blotchy. Any colouration, either of the colony itself or the surrounding medium, must be noted. In some organisms the different colonies remain distinct even when in contact, whilst with other organisms adjoin- ing colonies readily grow together or become confluent. In growths on gelatine the presence or absence of signs of liquefaction in the surrounding medium is a point of the first importance. In stab cultures any surface growth must be noted, as well as the growth in the line of puncture. It may be uniform, finely or coarsely granular, composed of numerous fine or coarse colonies which remain discrete; and are not confluent, or of large masses. Extension into the gelatine in the neighbourhood of the puncture may take place, and the character of these extensions, whether as knots or as fine filaments, or in an irregular, arborescent manner, is worthy of attention. If liquefaction, in a gelatine medium, has taken place it will be well shown. It is most abundant in the upper part of the line of puncture when the organism requires oxygen, but with organisms that grow best in the absence of air will be more conspicuous in the depths. Air bubbles along the line of puncture, indicating formation of gas and any colouration of the growth or of the medium, must be noted. The amount of growth that takes place in a given time 390 CONDITIONS AFFECTING GROWTH as compared with other organisms, or similarly the relative amount of liquefaction, gas formation, &c., in the time, is an aid in distinguishing allied or similar organisms, though liable to be modified with different strains of the same organism. There is no cultural characteristic that cannot be modified by frequent subculture, culture under different conditions of temperature, reaction of medium, and other influences. The cultures of some organisms vary more than those of others. The information gained as to the character of growths, though of considerable value, has to be considered with other properties of the organisms. Cultures on milk, potatoes, &c., are often of more diagnostic value for special organisms. (7) Conditions Affecting the Growth of Organisms. — One of these, the effect of oxygen, is of special practical. value. Some organisms will only grow in presence of oxygen ; such organisms are strictly aerobic. Others will not grow at all in the presence of oxygen, these are said to be strictly anaerobic. The largest number of bacteria are intermediate between the two and are termed facultative anaerobes. Aerobic organisms grow readily under the ordinary conditions, as even in stab cultures there is usually sufficient oxygen pi^esent for the commencement of growth. , Anaerobic organisms are most easily grown in stab cultures of glucose, agar or gelatine, or in glucose formate agar made by adding "02 per cent. sod. formate to glucose agar (Kitisato). The tubes must have been freshly boiled to expel air from the medium, and the stab should be made with a fine needle so as to carry down as little air as possible. After the needle is withdrawn the upper part of the medium should be heated so as to melt it and seal the opening made by the needle. It must be remembered that though the growth of anaerobic organisms does not take place in presence of air, the organisms, and particularly the spores, may CHEMICAL PRODUCTS 391 retain their vitality and grow if transplanted into more favourable conditions. (8) Chemical Products of Organisms, — These vary both with the nature of the organism and the character of the medium. Gas formation is one of the easiest to deter- mine, but it is also necessary to have in the medium some substance from which the gas can be formed. The sugars are valuable for this purpose, and it will be found that whilst one organism will form gas from either glucose or lactose another will form gas only from glucose. Another manifestation of chemical ^ictivity is the formation of acid or alkali. Formation of acids and gases are of particular importance, as so many of the Fig. 151. organisms found in the intestine either form gas and acid from glucose or form acid only. Formation of Gas. — ^The formation of gas can be shown in most stab preparations as bubbles of gas form along the needle track. It is better shown by melting the gelatine, or, better, glucose gelatine, and rotating the tube alternately in opposite directions after inoculation. Such a preparation is known as a " shake culture," and after it has set and grown, bubbles of gas will be formed all through the medium. . A better method that can be used with fluid media is to place in the medium a small inverted tube, Durham's 392 GAS AND ACID tube. During sterilisation of the medium the gas will be expelled from this small tube, which will be completely filled with the fluid medium. If the tube be inoculated with an organism that forms gas from the medium the gas formed in the small inverted tube will accumulate in it and cause it to float (fig. 151.) Gas formation from glucose is one of the characteristics of some of the commoner intestinal bacteria. Formation of Acid. — Acid formation can be shown by using a neutral or slightly alkaline medium coloured with litmus ; the formation of acid is shown by the change in colour of the litmus. Most of the intestinal bacteria form acid readily. An ingenious application of these properties of the intestinal organisms is that of MacConkey for the detection of faecal contamination of water, milk and other substances. He uses Durham's tubes and employs a medium con- taining bile salts. Bile salts inhibit the growth of many organisms, but are favourable to the growth of intestinal bacteria. The medium he employs is corhposed of : Peptone, 20 ; salt, '5 ; sodium taurocholate, "5 ; water, 100 ; to which is added glucose or lactose in the proportion of "5 per cent. The medium is neijtral and is coloured with neutral litmus. A Durham's tube is placed in the test tube containing the medium and during the three sterilisations required will be filled with the medium. A measured amount of the water, &c., to be tested is added and the tube incubated, preferably at 42° C, for twenty-four hours. Organisms which in this meditim produce acid and gas may be suspected to be possible inhabitants of the intestinal tract. Other pathogenic and non-pathogenic intestinal or- ganisms form acid only. Of organisms other than these many/ will not grow in the medium at all, or if they do grow form neither acid nor gas. If, therefore, neither acid nor gas is formed, the evi- dence is strong that there is no living faecal contamina- INDOL FORMATION 393 tion, and not that of the commonest of the intestinal organisms, B. coli communis. If acid alone is formed it is doubtful whether there is such contamination, as B. coli communis must be absent. If acid and gas are both formed there is strong probability that the water, &c., is contaminated with organisms that are inhabitants of the intestinal tract. Indol formation is another important chemical pro- duct of some bacteria. A simple medium is required, and plain peptone water made by boiling lo grammes of peptone and 5 grammes of salt in a litre of water is usually employed. This should be filtered and sterilised as usual. The tube of this medivim should be inoculated with the organism to be examined and incubated for at least twenty-four hours. Other tubes inoculated at the same time are incubated for longer pei^iods. To this culture a little pure sulphuric acid is added ; a red colour develops in a few minutes if indol and a nitrite have been formed. If the colour remains unal- tered;^ three or four drops of a "05 solution of sodium nitrite should be added to the mixture, and if a red colour now develops indol alone has been formed. Yellow-fuming nitric acid which contains traces of nitrites may be satisfactorily used instead of nitrite. Amongst other chemical products are ammonia, alcohol, phenol, sulphuretted hydrogen, and the substances which cause curdling of milk. Effects on certain Aniline Colouring Matters. Neutral-red Agar. — This medium consists of ordinary agar, to each 100 cc. of which -3 gramme of glucose and I cc. of a saturated aqueous solution of neutral- red have been added before the medium is poured into tubes. In this medium B. typhosus and B. dysenteries grow without changing it, while B. coli communis and the paratyphoid baqilli, in twenty-four to forty-eight hours 394 SERUM REACTIONS decolourise the medium and produce a greenish fluores- cence, forming gas at the same time. Stab cultures or shake cultures may be employed. Drigalski and Conradi's Medium. — To prepare this medium, 2,000 cc. of 3 per cent, nutrient agar are treated with 20 grammes nutrose, then with a solution of 30 grammes of lactose in 260 cc. litmus solution. The procedure is as follows : The litmus solution is boiled for ten minutes in the steam steriliser, the lactose added, and the mixture again boiled for ten minutes. The litmus lactose solution is cooled to 40° to 50° C, and the nutrose agar cooled to 70° C. is added to it. The mixture is rendered alkaline with hot 10 per cent, soda solution to the extent that on shaking, the froth formed is distinctly blue after a few minutes' standing. Finally, 20 cc. of O'l per cent, freshly prepared solution of crystal violet are added, and the medium sterilised in the usual way — it should be bluish-violet when solidified. On this medium B. typhosus produces small transparent colonies, while B. coli communis produces larger colonies, brilliant red and non-transparent. (9) Reaction of Organisms with various Blood Sera. — Certain pathogenic organisms effect a change, in the blood serum of persons infected with these organisms, so that the serum contains substances which, when mixed with a living culture of the organism, cause loss of motility of the bacteria, and also cause them to aggregate in little clumps or masses. This aggregation is called agglutination, and the serum which causes this agglu- tination is said to contain agglutinins. The application of the test is simple. The. blood serum, free from red corpuscles, is obtained, as has been already described, and diluted with sterile broth to a known extent, ten, twenty, thirty or more times. Some- times the blood is sent in capillary tubes or the serum^ has not well separated. In such cases, to obtain clear serum it is necessary to use the centrifuge (fig. 152). This diluted serum is mixed with an equal volume of a SERUM REACTIONS 395 living active culture of the organism to be tested and the mixture examined as a hanging-drop preparation. Loss of motion of the organisms and agglutination should take place within a certain time limit and a control made by using the same dilution of normal blood serum with more of the same culture. This action of the serum on Fig. 152. — Centrifuge. the organisms is specific and affords a means of proving the co-relation of the organism and disease. It is true that strong undiluted normal serum will cause in some cases a similar agglutination, but not with the great dilutions which will act in serum from a person with disease. 396 SERUM REACTIONS The converse of this test is to use a culture of an organism to test the serum reaction of a patient suspected to be suffering from the disease which the organism can cause. Typhoid and Malta fever are the diseases in which the action is most decisive. This appHcation of the principle is known as the agglutination test, or the Griinbaum-Widal or Widal reaction. In the application of the hanging-drop method the serum may be diluted in Wright's tubes or by a number of loopfuls of broth being added to a loopful of serum and well mixed. To obtain a dilution of i in 20, one loopful of serum is mixed with nine loopfuls of broth, and of this mixture one or two loopfuls are mixed with one or two loopfuls of an active culture. If higher dilutions, say i in 100, are required, it would be inconvenient and tedious to mix one loopful with ninety-nine of broth. It is easier to make a dilution of I in 10 and dilute one loopful of this dilution with nine of broth, which gives the same dilution more quickly. The gradual loss of motility and aggregation of the organisms can be watched under the microscope in the hanging-drop preparation. Many observers prefer the macroscopic demonstrations of the same effect. This is done by aspirating into a tube a mixture of serum diluted to the required extent with broth mixed with an equal quantity of active broth culture. The mixture of culture and diluted serum is drawn up into a narrow tube and placed vertically in the incubator. The organisms will lose their motility and aggregate into a mass and fall to the bottom of the fluid, leaving the superjacent fluid clear and free from turbidity, in marked . contrast to the control, which will still remain turbid. This method is known as the " sedi- mentation test." There are many fallacies which may occur in con- nection with these tests. The culture must be an active one and recently made. A control with serum of normal blood must be made and the dilution must be sufficient. PATHOGENIC PROPERTIES 397 Some strains of the organisms agglutinate more readily than others, and even with diluted serum of normal blood, agglutination may take place if the organisms are grown on unsuitable media, or if the cultures are too old. The change in the serum may be a persistent one, so that a positive reaction in the case of a pei'son who has had a previous attack of typhoid or Malta fever gives no information as to his present condition. (10) Pathogenic Properties. — The pathogenic properties of an organism are shown by the effect of inoculating a susceptible animal with the organism in pure culture if possible. Where that is not possible, with fluid contain- ing as few other organisms as possible. In some instances, as in tuberculosis, the similarity of the lesions produced by a similar organism in animals indicated the variety likely to be suitable for inoculation to animals. In others a series of animals had to be used to find a susceptible host. Rats, guinea-pigs and rabbits are the animals most commonly used, but in other cases monkeys, dogs, cattle and horses have had to be employed. No such experiments can be made under the Vivisection Acts without a licence, and in any case there are so many difficulties and fallacies that without a thorough study oi these' and of the methods employed the results obtained would be valueless. Material used for injection may be :— (i) Pure cultures of an organism. (2) Products of bacteria in solution such as toxins. (3) Fluid excretions, secretions and portions of diseased tissues. (4) Blood. The injections are usually made with strict antiseptic precautions into the subcutaneous cellular tissues. Witl fluid cultures there is no special difficulty. Cultures or solid media require to be emulsified with sterile salin( solution. Solid tissues, portions of spleen, &c., shoulc be rubbed up in a sterile mortar with a little sterili broth and then injected. Occasionally a sm^ll mass o 398 INJECTIONS solid tissue is inserted into an aseptic pocket made under the skin for the purpose, and the wound closed by a sealed dressing such as gauze and collodion. In other cases the injection is intramuscular or intra- peritoneal. In intraperitoneal injections of small animals a big fold should be taken up between the finger and thumb, care being taken to include the whole thickness of the abdominal wall and to see that no intestine is included. Whilst still held this fold should be transfixed with the needle of the hypodermic syringe containing the substance to be injected so that the point just pro- trudes. ' The finger and thumb are now removed, the abdominal wall will flatten out and the point of the needle will be in the abdominal cavity. In this way the injection can be made without any risk of injuring the intestines. The results of inoculations are not always conclusive. The resulting disease, even in a susceptible animal, may show very' little resemblance to the disease caused by the same organism in man or other animals. An organism that is pathogenic may by successive cultures lose its virulence, and become with some animals; non-pathogenic, whilst on the other hand, if passed- through a series of animals, the virulence may be increased. Koch's postulates are : — (i) The organisms must be found in the tissues, fluids or organs of the animal affected with the disease. (2) The organism must be isolated and cultivated outside the body on suitable media for successive generations. (3) The isolated and cultivated organism on inoculation into a s-uitabld animal should reproduce the disease. (4) The same organism must be recoverable from the inoculated aiiimai. These \n- the main are still considered sound, though not practicable for all organisms, as some cannot be cultivated 5 for others no susceptible animal is yet MYCETOMA 399 known, and in some of the lower animals, though a disease is produced by the injections it bears little or no resemblance to the human disease under investigation. Streptothrix madurce. — This organism is the cause of a disease of special importance in the Tropics — madura foot. It occurs in India, Straits Settlements, East Africa, British Guiana, and Cuba, and is probably to be met with all through the Tropics. Clinically it is a chronic disease which causes much , swelling of a firm fibrous nature and destruction of the tissues, with the formation of sinuses discharging watery or oily-looking fluid. In some cases in this discharge, white, black or pink granules, visible to the naked eye, are found, and these are masses of branching filaments with mycelial arrangement. In other cases these granules are very rare, and only the branching filaments of the mycelium are found ; in still other cases these may also be absent, though in sections of the tissue the mycelial clumps are to be seen. • The organism is characterised by the formation of dense clumps of mycelium formed of the branching filaments of the streptothrix. The ends of the filaments at the edges of these masses degenerate and become swollen, forming the so-called clubs. The organism will grow on any of the ordinary media, forming limpet-shaped masses. In general character the mycetoma resembles the streptothrix of actinomycosis, but it_ does not stain by Gram, in sections, does not liquefy gelatine, and the "clubs" are rounder. The clumps take any of the ordinary basic stains, including hsemotoxylin, and either this stain or carbolthionin is to be recommended to show the organism in sections. The clumps of the mycelium set up changes in the tissue so that the growths are surrounded by a mass of newly formed tissue of the granulomatous type. At the periphery of the masses of this growth is much badly 400 HYPHOMYCETES formed fibrous tissue, and the centre is pften broken down. It is in the breaking down of this granulomatous tissue that the mycelium clumps are liberated and are discharged with the fluids from the sinuses. HYPHOMYCETES (MOULDS). Of the various fungi which attack the skin and hair some are widely distributed, though more common and luxuriant in their growth in the Tropics. Pityriasis versicolor comes under this head. Others, like Favus^ may be common in some places, but as in temperate climates, are of limited distribution. The commoner fungi attacking the hair of the head are unknown in many tropical countries. The only fungus of this class at present recognised as peculiar to the Tropics is a cutaneous ringworm, Tinea imbricata, characterised clinically by the large size of the epidermal scales which are partially detached and the tendency to form geo- metrical patterns. For the demonstration of the fungi causing these various affections the older method consisted in soaking the hair or scales in a 7 per cent, solution of caustic potash, which rendered the keratin clear and transparent, whilst the fungus was less affected and could bp clearly seen. This method causes swelling of the fungus and spores, and therefore is not suited for the differentiation of the varieties or species of fungi. A modification of Gram's method of staining gives more useful results but is slow. The hair or scale is stained in gentian violet aniline water for five minutes and dried with blotting paper. It is then treated with Gram's iodine solution for two minutes and again dried with blotting paper. It is then covered with aniline oil to which a little iodine has been added and left till the fungus can be seen. It should be examined from time to time under the microscope, as though the process is slow, ultimately even the fungus will be decolourised. FUNGI 401 Do not wait till all the tissue is clear, but when nearly so, treat with aniline oil and clear in xylol. Mount in xylol balsam. The points to observe are the arrangement of the growth, whether- inside or outside the hair, scale, &c., the presence and the size of the spores. The nomencla- ture of these fungi is based on these points. According to the seat of growth of the fungus it is an ecto- or endothrix, and microsporon or megalosporon according to the size of the spores. These ringworms are true fungi. The fungi are multi- cellular organisms composed of filaments, either simple or branched, or jointed or unjointed. These filaments are called hyphce, and if they project into the air are aerial hyphce, or down into the substance of the medium they are known as sub-aerial hyphce. They frequently form a compact mass — a mycelium — and if this form is a hard, dense mass it is known as a sclerotium. Sexual reproduction as well as reproduc- tion by fission has been proved to occur in most members of the group. These fungi include the ordinary moulds, and some, such as ergot, form compounds which, when eaten, are poisonous. In addition to the cutaneous fungi which cause the true ringworms (Tinea), fungi may be found in mouth, ear or nose, as well as in pulmonary cavities. These are secondary growths. Occasionally, particularly in bird- rearers, who take uncooked grain in their mouths, a true pneumono-mycosis occurs. It is possible that some cases of madura foot, the black variety, are due to a fungus and not to a strep- tothrix. The tropical fungi, aerial and otherwise, have so far received little attention, and should offer a fruitful field for research. Most of the fungi grow readily on nutrient media, but best if a sugar be added. Maltose is the most suitable for the fungi of the ringworms. 26 Q o § H tn m ^ Ui < S o H !" S5 CQ l-H hJ JS , 1 B a"" S U ^ -^ o o o s — "1 S 1 ■73 ir; >» c " ■2 a a M 1 s B , a „ 6/1° ™ U o -S 3 c o. a en Z D 1X4 O J2 „ 'S .§■?- >, 1 .J3 4-. = g 1 1 1 |-^ a >, w C 1 1 1 ;^ .. tiJO u c u,S a| 1 1 1 1 « o Vi ' T3 TJ W O C B g~ 1 'rt S 3 *- O )H S 'o 3 ^ fU ^ 1 Mil <6 M S in S ed • - 2 >." a. II S it 1 ¥oU .«-> I I : *5 1 8 : S : a o 1 1. M' Oq 05 05 05 S S S o-o o (A '-; U "^ E' B S t s S „ „- a =^.s S I S "■agog. bio « T3 >, g cor3ii™aes a am M s.s .c a, o o „ 3. a a .M w n e o 2 5 S 5 o .c o ^ o >.• O " Q c (O ••« gj '•3.S5 m e H « o 5 (A .0 U "i O 3 " g S " c o 2°.2 ■^ j-s S.S as to S ... I " (fl .-" O .^ TO c.S-S o s .a 3 .0 ^ c « u .0 ^ Q Ul T3 4_, B u "ts «.y s s s-s .. "■ >,.2 as B « B rt B « 610 >f B O » ° — MB «l 0.2. o a + + a o H •" 2.C + " o ""3 S.S .. 3 — ■"13 u « 1 - S: j: .- C ' 2 S « S 3 g i* o - o-n ■a Ok o *j o o " o Si o + + <*) «5 .2-S>3 .S-g : §-gS.2„ O M o o O J-.C ?,"" G ■" o a 2 Si,"" D. 9 3 SfC 0.5"° O « K 5 « Ph n S r3 o . „ . . . _s : : : : : c Co J£ , ^ O-S ■3^5 .3 = -ge « a ?i o o = Ji -■■ «--!- I- C >,T3 — jO O — O o K h 'a s a s !i1 CI bfl-a c § > ^ •2 5 S^'s M o rt flJ S "(3 'o J3 11 •£ S 13 S <; J" o B a,x in a 'S ei << C ri .£3 ill S ^ M.ac C «. w ' to J3 3i2 = 2 3 S S-S "So. S .S 3 bA C O S.^ o bjo o a, -a £ 2:- S' u ri u < < - ."S I I I + + + + + + + I S: S; r3 tj isoa ^ ^'^'^ con- versely the smaller the value of g the greater will be the limits of error. For example, if in 100 cases of beri-beri 10 have been fatal, and it is required to determine the limits of error in assuming this proportion to hold for the next 10,000 cases : — Applying the formula. Here g = 100, m = 10, n = go. IQ I ., V 2 X 10 X 90 icx> — 100 X 100 X 100 = ro+ 2 X -0428=4+ -0856 ■0856 represents the variations per unit. So that in the next 10,000 cases — There may be 1,000 + 856 = 1,856 deaths ; Or, 1,000 - 856= 144 deaths. This formula only deals with the mathematical relation- ship of the figures. Further allowance has to be made for errors of observations and the numerous uncertain factors met with in any statistics. A mere consideration of the effects of the extent of the possible mathematical error in dealing with small figures, suffices to indicate the care that is necessary and the multitude of observations that are requisite before formulating a definite conclusion. In spite of the magnitude of this error, of the numerous possibilities of errors of observation, and even of the fact that mere increase in the number of observations may only multiply the same error or doubtful point, as the same source of error may be included in each observation, the acquisi- tion and use of statistics is of high value, and often indicates a correct conclusion. The liability to eiTor diminishes but does not destroy this value. It is customary to indicate all results in percentages, EVIDENCE 431 and no doubt this method renders comparison easy ; but a consideration of the formula of probable error shows that I in 4 is by no means the same thing as 25 in 100. The number of observations made must be included in any account, and whenever possible these observations should greatly exceed the 100. The consideration of the magnitude of the probable mathematical error under the most favourable circumstances should lead to as great an exactitude as possible and avoidance of other and avoidable sources of error. Value of Evidence. — Considerable judgment as well as caution is requisite in obtaining information other than that derived from personal observation. As regards occurrence of diseases, parasites, &c., much of the information received must be taken with great caution, as it is often from laymen and untrained ob- servers. Even more in the Tropics than in England such persons hold theories either of their own or derived from others, and are anxious to bring forward only facts which are in support of these theories. It is well in making enquiries to be careful to limit the enquiry to points that are within the power of any ordinary observer. It is not well to discard altogether such evidence, as on many important points information can be derived, and in some of these the liability to error is no greater than with a professional observer. Various points in connection with malaria might be well taken as illustrations, both of the value of such infor- mation and the errors that are likely to occur as a result of too much confidence in such information, as well as of the general methods which have been adopted deter- mining etiological and other factors. These points comprise : (i) As regards Individuals. — Their susceptibility to the disease and the effects of the disease, including liability to relapses, length of period of intermission between relapses, and any evidence of the acquirement of immunity. (2) As regards the Population in General. — Suscepti- 432 STATISTICS OF MALARIA bility, and any factors, age, race, or habits, influencing it. Mortality per i,ooo of the population at various age periods ; and case mortality in treated and untreated cases; liability to any special, immediate or remote, complications ; effect on general health ; any evidence of acquisition of immunity. (3) As regards the Place. — This should include enquiries as to any special house, village or district, as well as the country in general, where the disease is more or less prevalent than the average. Seasonal variations and their effects, particularly rainfall, temperature, and any cause affecting level of sub-soil water. Any facts known as to the prevalence of the known main factor — in the case of malaria, prevalence of Anophelince — in the spread of the disease. Some numerical estimate, endemic- index, of the liability to infection. Most of these points can be determined to some extent by careful enquiries, though the results must be con- firmed by observation, or where possible by the adoption, as a check, of other methods. The results obtained in this way, though not to be implicitly relied upon, will be a valuable guide to the direction of researches required in a district or country. Liability to Infection. — Enquiries as to individuals necessitates a selection of cases, and information of a reliable nature can only be obtained on every point from few persons. In the case of new-comers the date of arrival in a country and the subsequent movements, with approximate dates, are usually to be trusted. The date of the first attack of malaria can generally be obtained. Sufficient information about the attack, such as the character and duration of the " fever " ; the effect of quinine, and absence of any other cause of pyrexia, such as septic infection or pneumonia, must be ascertained to render it probable that the attack was malarial. Any form of indisposition in the older residents is so frequently called malaria that less reliance is to be placed on these than on the history of the first attack RELAPSES 433 In malaria it must always be remembered that relapses are so common that a second attack, even at an interval of several months, does not prove a second infection. The Liability to Relapses is more difficult to determine, but with a fair number of individuals it can be ascer- tained, and great individual variations will be found. In newcomers three weeks to a month is a common interval, whilst in others the period may be as long as four or six months. In this connection careful enquiries as to the habits as regards quinine are of great import- ance, as if quinine is taken constantly, even in small doses, the relapse is often postponed till the quinine is discontinued. Increase in the Interval between Relapses. — Any obser- vations as to increase in the interval between the relapses, with increased length of residence or diminution in the severity of the attacks, may indicate that a degree of immunity has been acquired, and the length of residence required for this, though shorter in all in the more malarial districts, is, to a great extent, an individual peculiarity. As regards the population in general, it is essential that the actual numbers of the different races represented be known before any use can be made of totals, such as number of deaths, admission to hospital, &c. This warning may appear superfluous, but it is not. In pub- lished reports one of the commonest errors is to speak of a disease as being more or less prevalent in a district on the ground of the number of cases seen, not as it should be, on the proportion of the susceptible population attacked. It is in connection with blackwater fever and yellow fever that such errors are most common. Age Incidence. — Personal observations should be made on unselected cases and the number of cases examined mentioned in the table, with the percentages. Ages cannot be ascertained with certainty, especially m coun- tries where the differences, in season are not very marked. With children age has to be estimated from the size, 28 434 MORTALITY teeth* and development. In adults knowledge of local history and notable events, the dates of which can be fixed, are of considerable value. Age periods of five years are usually taken, but it is of the utmost import- ance in malarial investigation to subdivide the first quinquennial period and further subdivide the first year into quarters. The first quarter should be subdivided into months. Malaria is rare till the end of the first month. As an age period the first ten years should never be taken as a whole, as such different results are obtained in a village, or in a series of obsAvations, if a large proportion are, say, under four, or only a small proportion, flonclu- sions drawn from the incidence of malaria in the first ten years of life, taken as a whole, are often misleading. Mortality is best estimated at the rate of so many deaths per 1,000 per year, as then the results can be compared. If dealing with short periods, as, for instance, one week, the death-rate would be the proportion of deaths per 1,000 of the population in that period, multiplied by 52. If a long period, say ten years, is * Ages at which teeth are cut in Europeans. The differences in native races have not yet been worked out. Table kindly supplied to me by Mr. K. W. Goadby. Temporary Dentition. Central incisors 5th to 8th month. Lateral incisors 7th to loth 5) First molars 1 2th to 14th » Canines 14th to 20th » Second molars 20th to 30th )» Permanent DENTI TION. Ui per jaw Lower jaw Central incisors 77 years 7 years. Lateral incisors 8 ?) ... 8 » Canines .. II tt ... 10 )5 Premolar I .. 10 n ... 10 JJ Premolar II II )i ... II JJ Molar I .. 6i » ... 7 51 Molar II .. 12 J) ... 12 J) Molar III .. 24 V ... 24 )) MORTALITY 435 taken, the death-rate would then be represented by the number of deaths per 1,000 divided by 10. The factors necessary are the number of persons of the required class alive at the commencement of the period, the number of deaths of this class who died from the disease which it is desired to investigate in the period, and the length of the period. Case Mortality is the percentage representing the pro- portion of cases terminating fatally. The number of cases of the disease and the number of deaths from the disease are the only two factors requisite. If it is desired to compare the " case mortality " in differelit years or other periods of time, cases occurring in those periods only must be included. In malaria untreated and treated cases must be considered separately, and the treatment mentioned as the case mortality is so much reduced by effective treatment. Remote or Indirect Mortality is the mortality due to remote complications, visceral changes and increased liability to other diseases, or to the tendency which malaria appears to have to aggravate some diseases. Our knowledge of this branch of the subject is most inaccurate and requires complete revision. The effect on general' health varies greatly in different conditions, and under circumstances little understood. Splenic enlargement, anaemia and diminished rate of growth are the most definite. Susceptibility to tuber- culosis appears to be induced by chronic malaria in countries where tuberculosis is prevalent. The effect on the general health, apart from the actual attacks, whether mild or pernicious, varies according to race. Period of natural incubation and its variations can be determined from the histories of patients, and then must be limited either to first attacks or to other attacks in which a long interval has elapsed. The most common history given is of some immediate antecedent. Ex- posure to chill, constipation, change of residence, par- ticularly from a warmer to a cooler place, and even 436 IMMUNITY cessation of travelling, are given as the causes of the attack. These causes are not to be taken as those of the infection, though they may determine or accelerate the manifestation of the disease. The time of actual onset of symptoms can usually be told with certainty, but the time of infection is diffi- cult to determine. The frequency with which travelling in one form or other enters into the causation is usually to be ascribed to passing through a highly malarial district, or even to spending some hours in a house where infected mosquitoes are to be found. With a sufficient number of cases it is sometimes easy, as in the case of a steamer, or in persons travelling over known i-outes, to fix on the date of infection as the date on which a halt was made at a notoriously malarial place. Such cases show the wide limits of the period of natural incubation, often longer than those which have been determined experimentally by feeding infected mosquitoes on susceptible persons. The evidence of immunity is to be considered under two heads : (i) Age incidence of the disease in natives and cessation of attacks with advancing years. (2) In newcomers the residential period during which attacks occur, and any evidence, by the diminishing frequency or severity of attacks, that some immunity is acquired. With malaria it is important to consider whether there are periods in which from climatic conditions infections do not take place. In the case of individuals, if there are periods during which they are not resident in places where malarial infection is possible. Immunity is destroyed or diminished by such periods, so that if they are long immunity is not acquired at so early a period, or at all. There is evidence that immunity is not of long duration in malaria, but more exact observa- tions are required on this point. In any consideration of immunity the liability to infection— endemic index— must be taken into account, as with a low endemic index individuals only, not a class, will acquire immunity. ENDEMIC INDEX 437 (3) As regards Place. — In considering any place it is important to bear in mind that malaria is a local disease, and that even in houses close together one will be more malarial than another. Still more so are different quarters of the same town or district, and the localities where the disease is most prevalent vary from year to year. These differences, and the causation of the variation in the differences, require local investigation in all cases. Seasonal variation may act in "two ways, first by ren- dering the conditions more favourable for the multipli- cation of Anophelince, and secondly by presenting condi- tions more favourable for the development of the malaria parasites in the mosquitoes. Rainfall, both the amount and distribution, i.e., whether in frequent light showers with short intervals, or heavy downpours with long intervals, is of great importance, but the effect may vary with the same monthly rainfall, as occasional heavy showers flush out and destroy mosquito larvae, whilst the same amount of rain falling slowly will merely increase the size and maintain the same breeding places. The level of the subsoil water may be more affected by distant rain than by the local rainfall, and thus distant rainfall may be a cause of the unhealthiness of a place. Rain on mountains or hills behind a station is an example of this. Where there are snow-covered mountains, as in Equa- torial Africa, the water supply is dependent on the melt- ing of this snow, and therefore a high temperature increases the water supply of a large district. A high temperature within certain limits causes more rapid breeding of mosquitoes, causes them to require food and therefore to attack men more frequently, and is favourable to the rapid development of the malaria parasites, and so in all these ways will favour the spread of malaria. The species of Anophelince present and of those most numerous in the district should be determined, and these species of mosquitoes should be tested as to the readiness with which they may become infected by the malaria parasite. Different species vary greatly in this 438 ENDEMIC INDEX respect, and even with the same species infection seems to occur with varying difficulty under different circum- stances. As a rule mosquitoes reared from larvae are not as easily infected as those of the same species caught in the adult stage. The possible or known circumstances affecting the development of the parasites are the tem- perature, the age of the mosquito, whether impregnated or not, and the nature of the food previously taken by the mosquito, and probably other conditions, which all require local investigation. Endemic Index. — A numerical estimate of the liability to malarial infection is an important ' factor to deter- mine. The number of malarial attacks, or of hospital admissions for malaria, from a known number of persons, is of little value, even if the diagnosis is confirmed in every case by blood examinations, as these admissions will include recurrences and relapses, which will vitiate the figures, e.g., a man infected once with malaria may have a dozen attacks of malaria as a result of this single infection in a year, or he may have only one. In the first instance he would appear in returns as 12, in the second as i, though in both instances for our purpose they should be represented as i. If first attacks only are included this difficulty does not occur, and first attacks have the further advantage of being usually severe, and in persons who have not yet acquired the habit of treating themselves and who therefore come under medical observation. For an estimate of the liability to infection, or endemic index, by this method, the factors to ascertain are the dates of first attacks of malaria occuiTing during the course of the observations, verified by blood examina- tions or in other ways, effect of quinine, &c., and the length of residence previous to the attack, and the number of newcomers during the period who have escaped infection. As a separate estimate a statement by as large a proportion as possible of the resident population as to the length of time they had each ENDEMIC INDEX 439 resided in the country before their first attack of malaria. These figures usually lead to much the same result. Reliance has to be placed on histories only, and errors may occur, though each factor is one which most of the residents are capable of observing. By this method the length of residence in weeks or months that is ordinarily required for an attack of malaria is determined. The period of incubation we know varies, but is commonly from ten days to three weeks, and this period should be subtracted from the length of residence required for an attack of malaria to obtain the period of residence required for infection. Where bodies of men are working together and are under medical observation, as in regiments, gangs of workmen, &c., this method is, I believe, the best and simplest, and includes no sources of error that are not common to other methods. In such an estimate all persons who were born and have lived in malarial countries for prolonged periods should be excluded ; also those who have contracted malaria in other malarial countries. For these exclu- sions there are two reasons — (i) to avoid including relapses, and (2) to avoid including persons who may be immune. A somewhat similar method is, to determine the pro- portion of untreated natives who harbour the ' parasites of malaria. In this method the ages must be known, and unselected children, including those appai-ently in good health, must be examined. Children should form a large proportion of the cases. This method has been extensively used, and an arbitrary standard, ten years, has been selected ; the proportion of children under 10 years of age with malarial parasites is then taken as the index. A more satisfactory method is to determine the propor- tion at different ages. Thus in one district, whilst 86 per cent, of the children under 2 years of age were in- fected with parasites of malaria, only 28 per cent, of those 440 SPLEEN TEST from 5 to lo harboured them. If, therefore, in such a place most of the examinations were made in young children, a much higher index would be obtained than if most of the children were over 5. In many of the determinations no further information than "children under 10 years of age" has been given, and in some of them the number of children examined is very small. It is not very easy in some places to get a sufficient number of children for examination, but with patience it can generally be done. As these cases are untreated, many of them, if not most, will have had the parasites for considerable periods, and therefore the figures only indicate antecedent, perhaps remote, infection. If young children were examined monthly till parasites were found, the liability to infection under native conditions would be determined more accurately. In making any series of blood examinations the time selected should be during a period of settled weather. If examinations are made during a change, particularly from hot to cold, the para- sites will be more easily found, as the effect of chill is to favour the development of the parasites. Examinations made at such times will therefore show a higher index than those made in settled weather. The Spleen Test, or^ the proportion of persons with enlarged spleens, is useful if age and race are taken into account. It is of more value amongst negroes than amongst other races, as the negro spleen does not con- tinue to enlarge after immunity has been acquired in the same way that the spleens of many individuals of other races do. The test can be used easily, as there is nothing in the examination to excite alarm or frighten the children, and can be made more quickly than any other examination. It indicates only antecedent, probably remote, infec- tion, and is less certain proof of antecedent infection than the presence of parasites. A large proportion of enlarged spleens between 2 and SPLEEN TEST 44I 5 years ot age is an indication of a high endemic index. If the presence of malaria in a district is proved, the absence of enlarged spleens in negro adults, or a low proportion between 10 and 15, is equally a proof of a high endemic index, whilst if the proportion of enlarged spleens in adult negroes is appreciable or large in those between 10 and 15 the endemic index is low.* The determinations obtained by the spleen test are less liable to be influenced by meteorological conditions than the test by blood examinations ; they are easier, and can be made in a larger number of cases, but otherwise are less accurate, as the conditions that lead to splenic enlargement after malarial infection vary and are not thoroughly understood, and splenic enlargement may be due to other causes. Another proposed method of estimation of the index is by determining the proportion of the Anophelince that are found to be infected with the parasites of malaria. For this method to be of value the mosquitoes must be selected from different houses and places in equal pro- portion, as it will be found that there are great variations in this proportion in adjoining houses and at different times. One good "crescent case" will infect almost every AnopheHne of certain species that bites the patient, whilst only a small proportion of those that bite the more numerous poor crescent cases will be infected. Anophelince in European houses are rarely found to be infected, whilst in an overcrowded native house, where there is no protection of the inmates from mosquitoes, a large proportion of infected Anophelines may be found. The proportion of infected mosquitoes is not the real test so much as the number of -infected mosquitoes, so that in these estimates the number of Anophelince that bite a man per hour is also required. A high endemic index, as determined by the other * With no other race but the Negro can such conclusions be drawn with certainty. ( Vide page 23.) 442 ENDEMIC INDEX methods, will be found in places where Anophelince are very numerous, even when the proportion infected is very small. It must always be remembered that a place with a large number of Anophelince of species known to be efficient carriers of the parasite, even if free from malaria at any one time, has the potentialities of a high "endemic index" if the place be occupied by newcomers or other persons susceptible to malarial infection. This is the reason that railway and engineering works are so often attended with outbreaks of malaria, even when con- ducted in places that previously were not considered to be very malarious. Anophelince are present and perhaps numerous ; in any case many new breeding-places are formed during excavations, and the mosquitoes become numerous. Gangs of workmen are crowded together in temporary huts, and they are not protected from mosquito bites. The workmen will include susceptible newcomers, and frequently some persons harbouring parasites. A single good "crescent case," often a man with no symptoms of malaria, will infect a number of mosquitoes, and in the course of some ten days or so these mosquitoes will infect any susceptible persons who sleep in the hut. The earliest numerical estimate was arrived at by determining the proportion of persons at different ages whose organs contained malaria pigment. This method can only be adopted under circumstances where post mortem examinations can be obtained in both children and adults. The results indicate antecedent malaria infection. The method, though fairly good, is only of limited value, as the large number of post-mortem examina- tions required can only be obtained in few places. These, then, are the main methods for the determina- tion of the endemic index : — (i) By determining the length of residence required to render malarial infection probable in susceptible new- comers. CHARTS 443 (2) The ages at which the largest proportion of natives harbour the parasites of malaria. (3) Ages at which splenic enlargement is common. (4) Percentage of persons dying from all causes with malarial pigmentation of the organs. (5) Number of infected Anophelince. Other evidences, though too complicated by other factors to be used numerically, are a high infantile death- rate amongst the natives, particularly a high death- rate from convulsions in infants over six months ; a high European death-rate ; and, we are inclined to add, the occurrence of blackwater fever. Graphic representations in the form of "charts" are useful as indicating the main results of any enquiry, as they are easier to follow with the eye than columns of figures or rows of statistics. The essential of a good chart is that it should be capable of translation back into figures, i.e., a chart should be such that it can be read. The principle of charting on a plane surface in two dimensions is that the horizontal line represents one factor, usually time or periods of time, whilst the vertical represents the other factor. Each of the factors should be represented according to scale in order that it can be read. This point is often overlooked, even in the familiar temperature charts, in that whilst the height of the temperature is recorded correctly in the vertical columns, the distances measured horizontally between the points representing the different observations are equally spaced, so as to look neat, whilst the real intervals of time are irregular, e.g., temperatures taken at 2, 10, 12, 3 should not be recorded on a four- hour chart, as if the time intervals were equal, but should be so recorded that the distances measured on the hori- zontal line are unequal in the proper proportion. In that case the chart can be correctly translated back into figures, otherwise, if represented as equi-distant the translation would read 4, 8, 12, 4. 444 CHARTS The limit to the translation of the chart is the scale of the chart; where the intervals allowed for time are small, translation can only be approximate. In blood charts it is usual to represent the heights as percentages of normal, as real figures would require such enormous charts if it were desired to represent graphi- cally on the same scale both red and white corpuscles. In such cases it is better to keep to the figures or to use different vertical scales for the more numerous and more scanty elements. It must be clearly indicated what each line on the chart represents. CHART I. Negroes (Native Africans). — Hausa and Yomba Children, 320 ; Hausa Adults, 100. Compiled from Official Report, Lagos, of W. H. G. H. Best. Ate in Vears. 2 5 10 15 20 25 30 35 40 | k ^ 90 - ~ -^1 , 4"! V. ^ go - V \ J'an- ] Is-SO- \ f 1 S ? 20- \ Q cj in- \ 1^^ '° V Too many lines on one chart are difficult to follow, and the only cases in which it is advisable to have two or more lines is when it is desired to compare two or more results. The different methods of determining the endemic index of malaria are conveniently rendered graphically and serve as illustrations of the method. Chart I. is compiled from an official report of W. H. G. H. Best, of the Lagos Medical Service, and formerly of the London School of Tropical Medicine, which is almost the only report published that gives sufficient details for the determination of the age incidence. CHARTS 445 No cases are given under 3 months of age, and those under 6 months are very few. The chart shows clearly that vmder the conditions of a native life a large propor- tion of children are infected in less than six months, and practically all ■ in less than a year, whilst the number of infected children after five years is so small that the majority must have acquired immunity. CHART II. Negroes (Native Africans), Central Africa. — 714 Native Children under 15, and numerous Adults. Age in Years. 12 6 ID 15 20 26 30 36 40 60 Chart II. shows the age incidence of enlarged spleens in Central Africa, and on chart III. are shown the same cases subdivided into two widely different groups ; in the one district Europeans often pass their first year without getting ipalaria, whilst in the other few escape for more than a few weeks. Th^e earliest age incidence of enlarged spleen and the earlier period at which it ceases to be common in the more malarial districts are well shown. The weakness of the spleen test is that a considerable proportion even of untreated cases of malaria do not show marked splenic enlargement, and it is probable that 68 per cent, of enlarged spleens indicates universal infection as much as 90 per cent, harbouring parasites would do. 446 CHARTS CHART III. Negroes (Native Africans), in a most Malarial District IN Central Africa. Residence Required for Probable Infection with Malaria, under Six Weeks. Native African, in less Malarial District. Residence for One Year does not render Infection Certain. vSrs" '2 5 10 15 20 25 30 c ID ■^12 70- ss I ■0^60- y °&a.n- \ M«,o- J f V' »^ S 2°^ i \ \ 1" V :==. Chart IV. indicates the proportion of pei'sons at different ages with malarial pigment in the spleen in a moderately malarial country. Two years' exposure was required for probable infection. It shows malarial infec- tion later, and less complete immunity. CHART IV. Negroes (Native Africans), Compiled from Post-mortem Examinations in British Guiana. Age in 1 Years. ' 2 5 10 15 20 25 30 35 40 50 60 Over 60 ^C -c « P V' A 01 S V s. l&°° \ o£ V 5fE30- N S M \ S 20 !> in - 1 ^..^ The line commences at one month, no pigmentation being found earlier. The next point is "under six months." CHARTS 447 The liability to malarial infection as determined by the first method, the length of residence required for probable infection, would be simply charted for different districts by representing for each place in the vertical lines the number of months requisite for the infection of three- fourths of susceptible newcomers, or the percentage of persons who would be infected in a period of six months or a year, as is considered to be most convenient. CHART V. Compiled from Reports by Travers A.im'V^ atso^.— yournal of Tropical Medicine, July 3, 1906. Indicates number of cases admitted with malaria from Klang. Ditto from surrounding districts, where no measures had been taken. YEAR 1901 1902 1903 1904 1905 | CASES 350 300 250 ZOO ISO 100 50 25 < V \ / / \ V- ■^ • 4 '" \ 'n •% \ ^o > \ ^^ — -^ 1 In 1901, 176 Government ofEcials had a total of sick leave amounting to 1,026 days, whilst in 1904, 281 had only 71 days' sick leave. In some charts the distances on the horizontal line have no meaning, and it is simply for convenience that the horizontal spacings are made ; a series of vertical columns packed together or widely and irregularly separated would have the same meaning but cause confusion. The convenience of such charts is that various points can be indicated on the same chart and compared. 448 CHARTS The success that has attended prophylactic measures in many places has been marked. Some of the observa- tions are imperfect, and in many of them several methods have been employed and have been associated with improved treatment of the disease, and therefore the actual results do not depend entirely on a reduction of the endemic index. Two illustrations of the results obtained are selected for charting and criticism. One is that recorded by Travers and Watson for the town of Klang in the Malay Peninsula. The figures CHART VI. Deaths from malaria in the drained area. Deaths certified as from other diseases. YEAR 1300 1901 1902 1903 1904 1905 | DEATHS 120 1 10 too SO 80 70 60 50 / y / \ ( A \ \ y \ \ ■ ■■ ■ ■ ^ m iM » M »< ^ aa ** ^' "^ During the same period in the surrounding undrained districts the death rate certified as from fever increased from 173 in 1900, to 351 in 1905, and from other diseases from 133, to 271 in 1905. reported are the admissions to hospital for malaria verified by blood examination, and the deaths attributed to malaria, in both, for a series of years. The populatioi^ is known to have increased, but as the amount of increase is not known it is taken as stationary, and to that extent the results appear rather less striking than they really are. KLANG EXPERIMENTS 449 The measures adopted were to intercept by drains, running across the base of a hill behind the town, the water that would otherwise have joined the town subsoil water, and by the provision of numerous deep drains to lower the level of the subsoil water in the town. Later hollows were filled in. The effect has been to diminish the number of certain species of Anophelince ; species such as Myzomyia Rossi are still very abundant, but these are not efficient carriers of malaria. These works were commenced in 1901 and continued since, the greater part being supplied early in 1902, and were carried out by the Government in accordance with recommendations made by a Committee composed of three medical men and three engineers. The main results are indicated in the charts (V. and VI.). As a control the surrounding district where no anti-malarial measures were adopted is used. It will be seen that whilst in the towns both the number of cases of malaria and of the deaths was greatly reduced though the population had increased, in the surrounding districts there was an increase in both these items. The increase in fact was due to an increase in the population. One striking feature is that the deaths from other causes diminished as well as those from malaria, indicating the indirect influence of malaria on the prevalence and severity of other diseases. This is shown in Chart VI. The numbers are considerable as the population is over 4,000, and the cases of malaria were 334 in 1901, and average twenty-nine from 1903 to 1904 with a popu- lation known to be greater. Applying Poisson's formula, we find that dividing the population 4,000 into two groups, one group, 334, consisting of those who con- tracted malaria, and another group, 3,666, consisting of those who did not contract malaria, the limits of error are represented by + 2 >y ^x 334x3,666 yj^j^_ — 4,000 X 4,000 X 400 ^ = + '01 23715 per unit ; Or, + 49-486 for a population of 4,000.. 29 450 CENTRAL AFRICAN EXPERIMENTS So that the number of cases of malaria per annum in this population, if the conditions had remained con- stant, might have varied between 334 + 49 = 383, and 334 - 49 = 285. As will be seen, the reduction to 29 in 1903 is manifestly outside the limits of mathematical error, and indicates a marked improvement in the condi- tions by which malaria was spread in this district. Any error therefore, if it existed, is one due to errors of observations, and the " control " is a sufficient check on this. Chart VII. indicates the incidence of disease in a series of Mission stations in central Africa for a period of years. From 1900 onward various anti-malarial measures have been taken. These measures varied in different stations and were carefully planned by the medical officer, Dr. Howard, in accordance with the local conditions. As charted the effects are most striking, as the proportion invalided or who died during the five years when these measures were practised was less than quarter of that in the preceding five years. The actual numbers are very small, as in the second period there were only an average of 24*4 workers and a total of 122 in the five years. Of these 7 died and 8 were invalided = a loss of 15 ; in 1901 to 1905 there were 182, of these 2 died and 4 were invalided = a loss of 6. If we apply Poisson's formula to these figures we find that the ratio in the first period involved a probable error of "059552 per unit ; in a population of 182 in a similar period the number of deaths might have been 21, or a minus quantity. The reduction to two there- fore falls within the limits of mathematical error. This result, therefore, though striking, must be taken rather as an illustration than as a proof of the value of such methods. Many statistics require correction before charting. Of these corrections some are obvious and easily made. The number of cases is useless unless the total number of the population that are susceptible to the disease is BLACKWATER FEVER STATISTICS 451 also known. In dealing with inhabitants of different races any difference in the susceptibility has to be noted and allowed for. Blackwater fever is a good illustration in point, and so many erroneous statements are made in connection with it that it well serves as an illustration. It occurs CHART VII. Compiled from Report by Howard, Published London School of Tropical Medicine, February, 1907. Deaths per 1,000. Number invalided per 1,000. CALCULATED PER 1000 120 110 100 90 80 70 60 50' 4-0 30 20 10 1887 88 89 90 91 92 93 94 95 96 97 98 39 1900 1 2 3 4 5 1 ' 1 1 1 1 1 1 1 1 u. » ^ -« __ _« ._. 1 1 ■-■ -■J 1 1 1 1 1 In this chart the figures are so small that fluctuations from year to year are considerable, and the result is better shown by averaging for periods of five years. The value of the evidence is considerable, but the small figures available reduce this value to a gr^at extent. in Tropical Africa, in India, and in the West Indies, amongst other places. All races are probably susceptible, though this is doubted by some as regards some negro races : in any case the susceptibility varies, and the negro susceptibility is so slight that not one in many thousands will get the disease under conditions where some 8 per cent, of the Europeans are attacked. The Indian is 452 BLACKWATER FEVER STATISTICS certainly susceptible, but . only about one-fourth as susceptible as the European. These variations in racial susceptibility require much further study. We must know, therefore, both the number of Euro- peans and the number of cases that occur amongst them in each district before we can compare the prevalence of the disease in each district. Similarly the number of cases amongst Indians and the number of Indians must be known, and the proportion in the two races must be kept distinct. If this is done it will be found as a general rule that in the most malarial district in Africa the prevalence of blackwater fever is the greatest, though the actual number of cases seen may be no more than in a more thickly populated but less malarial district. It will also be found that it is only in Africa that a large proportion of susceptible persons are attacked. In other countries, where perhaps as many cases may be seen, the number of susceptible persons is much larger. Another correction, an important one, has to be made. Unfortunately the amount of the correction is dependent on a variable factor — the period of incubation of the disease. More cases of blackwater fever probably occur in England than in any one small district in Africa, but these cases are all in people who have returned from Africa and acquired the infection there. In these cases there is no doubt that the infection should be attributed to the part of Africa from which they came. Here the matter is easy, but in Africa itself it is so often found that persons develop the disease who have been travelling, that it is a matter of great difficulty to attribute the disease to the correct place of origin. In many cases the place where the disease develops is certainly not the place where it was acquired. The correction to be applied here is essential, but can only be an approximate and arbitrary one in the present state of our knowledge, as the period of " incubation " is unknown, and probably variable. It is better to take the place of residence a SECRETION RATES 453 fortnight before the attack as the more probable place to be implicated in a large proportion of the cases. Charting is often useful to represent the secretion or excretion rates either of definite substances, such as urea, or the volume of a mixed fluid, such as urine. Here times are represented by the distance measured horizontally, and amounts, weights, or volumes by the height measured vertically. The only difficulty is that however it may be secreted urine as well as other fluids are only passed at intervals, and it is the rate at which urine is being formed, not that at which it is being passed, that is of importance. The only available method is to divide the number ol ounces of urine passed, or, if necessary, drawn off by catheter, by the intervals measured in hours between the successive micturitions ; the result will give the average rate per hour, assuming that the bladder is equally empty after each micturition. Such charts are of a special value in diseases like blackwater fever, in which there is a tendency to sup- pression of urine, and may indicate the periods of greatest danger. The geographical and topographical distribution of disease, of parasites and of certain insects is of consider- able importance. Maps should be drawn to the required scale and the places where an examination shows that the condition to be charted is present marked with a plus, -f-, and where absent negative, — . Places where no observations have been made should be clearly indicated, for if, as is sometimes done, they are repre- sented as negative, most misleading conclusions as to the distribution of the disease will be drawn. It is usual to represent the incidence of a condition by shading, and the depth of the shading indicates also the prevalence of the condition. In determining the incidence of a disease in a town or village a plan must be drawn up and the houses or groups of houses infected indicated as above. 454 GRAPHIC REPRESENTATION Extraneous conditions, such as wells, streams or other sources of water, must be shown, and when dealing with a question such as malaria, known to be carried in a certain way, other conditions favouring the prevalence of such carriers as Anophelince, must also be indicated. These maps and charts enable the conditions to be quickly understood, and are therefore of considerable value if accurate and carefully drawn up. With a little ingenuity almost anything can be repre- sented in a graphic manner, or charted. The value of a chart is the ease with which relations are shown and from which conclusions can be deduced. They show no more and prove no more than the figures or facts they represent, but by many are more easily followed. If, therefore, a chart does not represent matters more clearly than the figures the chart is useless. 455 APPENDIX. Table I. — Average Weights of Organs in Ounces. Europeans (Quain) Negroes (British Guiana) Indians Chinese ... Brain 49'S 42-04 41-9 47-3 Heart . II . IO-8 ■ 8-7 ■ 9 Lungs • 4S ■ 237 . 27-2 . 26-1 Spleen , 6 .. 6-9 .. 18-4 .. 14-8 .. Liver S3 477 48-4 43 '4 Kidney .. II ■• 9'9 .. 8-9 .. 87 Table II. — Variation in the Weights of Lungs with the Time after Death. Examination made after death Within 3 hours .. 4 ., 6 „ 12 „ 18 Over 18 Average weights 18 23 27 29" I 34-8 44-1 No. of cases in which the lungs weighed 20 0/.S. or less 71 9 31 4 3 21 ozs. to 30 ozs, 21 32 37 47 3 31 ozs. to 40 ozs. 4 26 37 52 9 41 ozs. and over I 3 6 35 33 Table III. — Average Weights of Brain in Negroes and Ages : 16 to 20 Negroes ... 44-3 Indians ... 40 Indians at Different Ages. zr to 30 31 to 40 41 to 50 46 ... 45-4 ... - 43 41 '2 41-3 40-8 51 and over 41-5 40-5 Table IV. — Proportion of the Spleens in Indians and NBgroes Weighing 15 ozs. and Over. Ages Indians Negroes 20 to 25 ... 30 per cent. ... 32 per cent. 26 to 45 ... 52 „ •• 16 „ Over 45 ... 39 „ ,.. is „ Table V. — Variations in the total number of leucocytes and in the relative proportion of the different kinds of leucocytes occur in healthy persons to a moderate extent. The number of leucocytes in healthy adults is rarely under 6,000 per cmm. or over 12,000. In new-born children the number is much greater — up to 20,000, and in pregnancy is increased up to 15,000. During active digestion there is an increase in the number of leucocytes. Variations in the relative proportion of the different forms of leucocytes readily occur in healthy children ; but in adults, with the exception of the lymphocytes, such variations are comparatively small. 456 TABLES— CLASSIFICATION OF DIPTERA The following table gives examples of the relative proportions of the dif- ferent leucocytes in certain diseases, as well as the number of leucocytes per c.mm. usually met with in such diseases. No. per cmm. Poly- morpho- nuclear Per cent. Lympho- cytes Per cent. Large mono- nuclear Per cent. Eosino- philes Per cent. Pneumonia ... Great increase up to 60,000 85 to 95 'S s I Sepsis Increase up to 30,000 or 40,000 75 to 90 IS to 25 5 to 10 I to 8 Liver abscess... Increase varies, often slight, 12,000 to 20,000 75 to 85 15 to 25 5 to 10 2 to 4 Typhoid Slight increase at most 50 to 65 25 to 40 Stois I to 3 Malta fever . . . ij It ») SO to 65 25 to 40 StoiS I to 3 Relapsing fever Great increase up to 50,000 75 to 90 10 10 20 5 to 10 I to 2 Malaria No increase ; decrease during apyrexia 45 to 65 IS to 25 IS to 30 I Trypanosomia- sis Kala azar No increase 50 to 65 20 to 30 15 to 20 2 to 4 Marked decrease — 50 to 60 25 to 35 IS to 20 2 to 4 1,000 to 3,000 Anchylostomia- Usually increased, es- 66 to 70 10 to 20 5 to 10 10 to 50 sis pecially in early cases It must be remembered that multiple infections are common. In relapsing fever lung complications ate so common that possibly the leucocytosis is due to this. The tendency to leucocytosis and an increase in the polymorphonuclear leucocytes due to any disease will mask the mononuclear increase due to malaria. Table VI. — This schedule of the classification of the Diptera differs in some details from the more usual, which is that adopted in the text. It has the advantage of being clearer. Sub-order I. Orthorrhapha — The pupa escapes from the larval skin either through an anterior T-shaped opening, or (rarely) through a posterior transverse slit : adults without a frontal lunule : — I. Nematocera. Flies with four- or five-jointed palpi and many- jointed antennae, the segments of which, except the basal two, are similar and are often fringed with long hairs : — i. Nematocera vera. Antennje long and frequently with whorls of hairs : legs long and slender : abdomen usually long and slender. Examples, Craneflies, Midges, Gnats, Mosquitoes. Chironomidas. CLASSIFICATION OF DIPTERA 457 ii. Nemaiocera anomala. Atitennse of many small segments, but short and without whorls of hairs : abdomen usually stoutish : legs shorter and stouter than in Nemaiocera vera. Examples, March-flies, Buffalo-gnats. Simulidae. 2. Brachycera. Flies with one- or two-jointed palpi, and usually short three-jointed (sometimes four- or five-jointed) antenna? : — iii. Brachycera anomala. Third segment of antennae ringed as if composed of several small segments fused together : body without strong bristles. Examples, Horse-flies, Soldier-flies. Tabanidffi. iv. Brachycera vera. Third segment of antennae not ringed, but usually bearing a bristle or style (antennae sometimes four- or five-jointed) : usually with strong bristles. Examples, Snipe-flies, Robber-flies, Dance-flies. Sub-order II. Cyclorrhapha. The pupa escapes from the larval skin through an anterior circular opening : adults with a frontal lunule : antennae short, usually three-jointed, the third segment with a bristle or style : — 1. ASCHIZA. Flies without a frontal suture. Examples, Syrphus flies, Big-eyed flies. 2. SCHIZOPHORA. Flies with a frontal suture. Examples, Bot flies, Muscids, Glossina, Stomoxys. Sub-order III. Pupipara. Larva nourished within the parent and not born till it is ready to change into a pupa. Examples, Tick-flies, Hippoboscids, Bat-ticks, Bee-louse. VII. — Instruments and Reagents. Microscope, with two eye-pieces, 2 and 4 ; three objectives, f in., ^ in., and 5^ in. ; oil immersion lens ; substage condenser, and iris diaphragm and mechanical stage, micrometer eye-piece with scales or with squares, micromillimetre scale, camera lucida. Watchmaker's glass. Portable microscope. Direct vision spectroscope. Slides, No. 2 quality. Cover-glasses, No. i quality, to be packed in oil. Needles in handles. Cork felt. Entomological pins. No. 20. Forceps. Cornet's forceps. Mounted platinum wires. Test tubes, thick and best quality. Durham's tubes. Watch glasses. Petri dishes. Photographic trays, half- and full-plate. Erlenmeyer's flasks. Funnels. Glass tubing. Glass rod. Beakers. Burette, 50 cc. Evaporating dishes and copper dish for boiling slides. Spirit Bunsen. Primus kerosene lamp. 458 INSTRUMENTS AND REAGENTS Glass measures, 500 cc, 100 cc. and 10 cc. Scales. Gramme weights. Paraffin oven. Paraffin moulding dish and blocks. Microtome. Steam steriliser. Hot-air steriliser and incubator. Iron enamelled jugs. Mounting and Imbedding Reagents. — Alcohol, cotton wool, methy- lated spirits, oil of cloves, xylol, Canada balsam, glycerine, Farrant's ■ solution, glycerine jelly, ether, chloroform, celloidin, paraffin wax, HoUis' glue, or shellac. Stains. — Hasmatoxylin crystals, hsematein, methylene-blue (Hochst's pure medicinal), thionin, gentian violet, fuchsin, carmine, picrocar- mine, toluidine blue, night blue, bismarck brown, methyl violet, eosine, both soluble in alcohol and soluble in water. Gram's stain made up. Leishman's stain. Burroughs' and Wellcome's tabloids. Griibler's stains are the best. Other Reagents. — Acids : Hydrochloric, nitric, sulphuric, picric, osmic, tannic, carbolic (pure), gallic, acetone. Agar-agar, alum, ammonia, alcohol, methyl alcohol (pure for analysis), borax, creasole, iodine, filter paper, filter paper (Chardin), formalin, gelatine, glucose, lactose, lithium carbonate, lysol, mercuric chloride, naphthaline, peptone, platinum chloride, potassium ferrocyanide, potassium iodide, potassium bichromate, phenolphthalein, sodium carbonate, sodium citrate, sodium hydrate, sodium sulphate, sodium taurocholate. Plate 1 ? < 1 . ^' 10. 14. . 17 ai. 11. ^i -^ 9 IH 15. ■%> 18 . 19 . ■I*,- '<■'■■• ■- V lit) 25 . 24. A Isrzi, del , BaJe Sc DtmifilssoiLl^ Li,h PLATE I. Stained with H^ematoxylin or Eosine and Hjematoxylin. Figs. I, 2. Normal variations in red blood corpuscles. 3, 4. Nucleated red blood corpuscles. 5. Blood plates. 6, 7. Abnormal variation in size and colour. 8. Abnormal shapes, poikilocytes. 9. BasopMlic granules. 10. Polychromatic red corpuscle. 1 1 . Lymphocy te . 12. Large mononuclear leucocyte. 13. Polymorphonuclear leucocyte. 14. Eosinophile leucocyte. 15, 16, 17. Myelocytes. 18. Quartan rosette. 19. Young tertian parasites. 20. Half-grown tertian parasite. 21. Rosette benign tertian. 22. Malignant tertian (sub-tertian), ring form. Double infection of corpuscles. 23. Crescent. Gamete maUgnant tertian (sub- tertian). 24. Young halteridium. 25. Sporulating halteridium. 26. Tr)rpanosome of fish. 27. Trypanosoma Brucei (tsetse fly disease, nagana). 23 PLATE II. Malignant Tertian Parasites (Sub-Tertian) Stained WITH Carbol Thionin. Figs. 1. Young form, rings. 2. Half -grown parasite. 3. FuU-grown parasite. 4. Sporulating parasite. 3a, 4a. Are fuU and sporulating parasites, as seen in sec- tions of organs shrunk by the spirit and other processes. S, 6, 7, 8. Development of the gametes of maUgnant tertian. 9, 10. Benign tertian parasites, half-grown and sporu- lating, II, 12. Quartan parasites, half-grown and sporulating. 13 to 16. Sporozoa of cattle and horses. Embryos of Filaria Stained with Hcsmatoxylin. 17. F. nocturna. 18. F. Persians. 19. F. Demarquaii. c Plate H. V'r .4 > 3a %i n) 4a. ?-•=' . 5. 6 . 7. 9. t ;4^, 10 r^. la . 14 15 . 16 . ^^K«,| 18 |iia>«MMS;-, A TerzL. df^l. Bale 8c DamelssoiL L''"'- 1 Plate ni . 4 4. 6 . 12. ;^ 10. If® ? V J3. ■'m. 14. 17. -■!-^- ;;?: i^-" 15. 19. A.Terzi. del ' 16 . 20 ^ «» 18. ■:.:,'~;jf' :■-:■ ii. Bale A-DanielssouL'-.^ lith. PLATE III. Stained with Leishman's Stain. 1. Normal red corpuscle, 2. Blood plates. 3. Lymphocyte. 4. Large mononuclear leucocyte. 5. Polymorphonuclear leucocytej 6. Eosinophile leucocyte, 7. Mast cell, 8. Transitional form. 9. Abnormal mononuclear cell found in certain diseases, including trypanosomiasis. Myelocytes showing various types of granules, Halteridium. Small drepanidium in red corpuscle. Same drepanidium in plasma. J 8 19, 20. Large drepanidium in various stages. Degeneration of red corpuscle caused by drepa- nidium (Schuffner's dots). 10 to 13- 14. IS- 16. 17- ', 19. 20. 21. PLATE IV. Stained with Leishman's Stain. Stages of benign tertian parasite. Gamete benign tertian. Characteristic degeneration of red corpuscles con- taining benign tertian parasites (Schuffner's dots). Stages of quartan parasite. Stages of malignant tertian (sub-tertian) which are seen in peripheral blood. Male gamete, maUgnant tertian (sub-tertian). Female gamete, malignant tertian (sub- tertian). Double infection with malignant tertian (sub- tertian) parasites of a red corpuscle ; baso- philic granules ia red corpuscle. Trypanosoma Lewisi (rat). Trypanosoma hominis (Congo). Spirillum of relapsing fever (stained with carbol fuchsin). 24, 25. Amoeba colt. Figs. I to 5. 6. 7, 8 . 9- 10 to IS- 16, 17- 18. 19- 20. 21. 22. 23- J- XCI.L.D J. V .*••■■• •. ■■'•, • -ife' ^■ ■^- %;_ 3. . i .»"' 4 . e :^v. 6 . * \ -•'-•V 10. 11. 12 . 13. >>. ll. #- » ,-, -> 15 . 16 . 17. 18. 1 19 20. 2:i. "»■)? ..J "iff. o « 6 2a. -U";i' "■' INDEX. Abnormalities, racial, 22-23 Abscesses : — Blood changes caused by, 57 Hepatic : — . Leucocytosis in, 422, 456 Organisms in pus of, 345, 3S4> 3S6 Pus discharged in fseces in, 323 Acanthocephala, classification of, 350 Acarina (mites and ticks), characters and disease-carrying genera, 286-287 ; examination of, 290-292 ; dissection of, 292 ; internal anatomy, 292- 293 ; as disease carriers, 293 ; life-history, 293 ; classification of, 294- 296. Acartomyia, 221 Acetone, use of, in paraffin imbedding, 32 Acid formation by bacteria, 392 ^des, 198, 202, 224 jEdeomyia, 223, 225 ^deomyina, classification of, 211-212; eggs of, 252 ; larvae of, 258, 261, 262 /Edeomorphus, 224 j^dincB, differential characters of, 210-211 ; genera of, 224-225; man attacked by, 225 ; habitat of, 225-226 ; eggs of, 226 ; larvae of, 226 ; breeding places of, 226 Africa : — Blackwater fever in, 452 Cellia pkarceensis in, 2l8 Central: — Anti-malarial measures in, success of, 450-451 Filariasis in, 224 Malarial infection in, charts illustrating, 444-446 Myzomyia funesta in, 217 Pyretophorus costalis in, 217 Ticks in, 296 East :— Madura foot in, 399 Ticks in, 296 Filaria occurring in, 128 Haemoglobinuria in, 358-359 Jigger-flea in, 281 Schistosoma found throughout, 357 Sleeping sickness in, 108-109 460 INDEX Africa — continued. South :— Epidemic jaundice of dogs in, 100 Mai de Caderas in, loS Texas fever in cattle in, 100 Tick fever of. III Trypanosoma dimorphum in horses in, lo8 West, AnophelincB of, 217 Agar, nutrient, 371 Agglutinins, 140, 144, 394 Agglutination test, 396-397 AgrionidcB (dragon-flies), larvae, 150, 255 Air, estimation of bacteria in, 424 Alcohol : — Fixation by, 65, 70 ; by absolute alcohol, Ji, 65 ; by alcohol and ether, 70 Formol, as reagent in tropical work, 27-28 Hardening reagent in tropical work, as, 26-27 Aldrichia, 214 Alimentary canal, organisms in, 356 Algeria : — Anopheles maculipennis found in, 216 Auchmeromyia found in, 189 Amblyomnta, 296 Amblyomma hebrcerum, 294 America : — Screw- worm fly in, 186 South :— Jigger-flea in, 281 Mai de Caderas in, 108 Mosquitoes of, 218 Texas fever in cattle in, 100 Tropical: — Jigger-flea in, 281 Malaria carrier in, 218 Amabte, 352-354 Ammonia, albuminoid, detection of, in water, 409, 413-414 Ammonia, free, in water, Wanklyn's process for detection of, 408-409 Amphistomum, 342-344 Amyloid degeneration, demonstration of, 309 Anaemia : — Ankylostomiasis, of, see that title Counts of red blood corpuscles in, 421 Pernicious, blood changes in, 59-60, and note., 421 Pigment deposit in cases of, 303, 304 Post-malarial, 435 Anguillula intestinalis, 349 Animals, parasites in blood of, 100-105 Ankylostomes, 24, 348. INDEX 461 Ankylostomiasis :— Aneemia from blood changes in, 57-58, 421 ; fatty degeneration of, 308 Blood in faeces of, 320 Leucocytic variation in, 456 Yellow pigment in, 305 Ankylostomuvi duodenaU, occurrence of, 316, 346 ; characters, 346, 348 ; male and female, 347 ; eggs of, in fseces, 328-329, 331 Aneurisms, verminous, in horses, &c., 117 Anopheles, differentiation of, 202, 214, 215 ; wing scales of, 204 ; anomalous character of genus of, 213; malaria parasites carried by, 215 ; sali- vary glands of, 241 ; eggs of, 254 ; pupoe of, 269 " Anopheles pool," 267 Anopheles bifurcatus, 2 1 6 Anopheles maculipennis, 163, 215-216 ; Nuttall's articles on, died, 269 Anophelina : — Breeding places of, 265, 267 Characters of, 207-210 Classification of, 211 Disease carriers, as, 75, 225-226, 244-245, 437-438, 441-442 Eggs of, 253 Feeding time of, 153 Genera of, 212-215 Head and scutellum of, 198 Identification of species, 227 Larvse of, 255, 259, 261, 263, 264 ; carriage of, 269 Measures to diminish numbers of, 449 SpermathecEe, number of, in, 242 Annett and Dutton, died, 239, 249 Anthomyia, larvae of, in man's intestines, 154 Anthomyida, characters and genera of, 189-191 Anthrax, propagation of, 154, 167, 171 Ants destructive to mosquitoes, 272 Apes, anthropoid, micro-organism pathogenic to, 403 Aphaniptera (fleas), order of, 162 ; characters, 193 ; intermediate hosts of parasites, 273 ; diagnosed from other Diptera, 155, 273 ; capture and examination of, 273-274 ; cleaning and mounting of specimens, 274 ; anatomy of, 275-277 ; metamorphosis of, 276-277 ; Pulidda, 153. 277-280; Sarcopsyllidte, 278, 280-281 ; classification, 277-281 ; parasitic on rats, 279-280 Aphides, destruction of, by larvte of hover-flies, 175 Aponomma, 296 Appendicitis, leucocytosis in, 422 Aptera, 150 Arabia, Schistosoma found in, 357 Arachnoidea, 148, 149, 286 et. seq. Araneid(z, 286 Argas, 294 ' Argas miniaius, 293 462 INDEX ArgasincB, characters of, 288-290 ; number of ova, 293 ; differential characters and genera, 294 Arribahagia, 214 Arsenic in water, detection of, 408 Artkropoda, 148, 149 Ascaris lumbricoides (round-worms) 344-345 ; eggs of, in fseces, 328, 331 Asckha, 457 Asia : — Cellia kochii found in, 218 Stegomyia scutellaris found in, 223 Asilida (robber-flies), characters and structure of, 156, 161, 172-173 Assam : — Cimex rotundatus in, 285 Hamogregarines in dogs in, 102 Atyloius, 169 Auchmeromyia, 187-189 Autumno-KStival fever, crescent bodies in, 243 Bacilli : — Definition of, 377 Living, in fluid, estimation of numbers of, 424 Bacillus anihracis, 402 B. coli communis, 393, 394, 403 B. difhtheriiE, 403 B. dysenterica, 403 B. gartner, 403 B. lepra, 402 B. paratyphoid, 403 B. festis, 273, 403 B. pklei, 402 B. pyocyaneus, 403 B. septicemia and B. hcemorrhagica, 403 B. smegma, 402 B. tetanus, 402 B. tuberculosis, 402 B. typhosus, 393, 394, 403 Bacteria (see also Vegetable micro-organisms) : — Aerobic, anaerobic and facultative, 390 Cultivation of, at " room " temperature, 20 Differential characters of, 377. Tissues, in, 311 Urine, in, 361 Balfour, cited, 103, 104, 266 Bancroft, cited, 249 Bats, parasites of, 100, 193 Bees, parasites of, 193 Benign tertian parasites : — Asexual cycle, 87 ; length of, 79 Corpuscle containing, effect on, 87 INDEX 463 Benign tertian parasites — continued. Diagnosed from benign quartan and malignant tertian, 94 Gametocyte form, 88 ; changes in, 91 Sporulation of, number of spores at each, 79 ; site of, 80 Zoological position of, 105 Benign quartan parasites : — Asexual cycle, 86 ; length of, 7g Corpuscle containing, effect on, 86 Diagnosed from benign tertian and malignant tertian, 94 Gametocyte form, 88 ; changes in, 91 Figment deposits in, 86 Sporulation, site of, 80 Zoological position of, 105 Bentley, cited, 102 Beri-beri, urine of, 361, 362 Berkeley, W. N., cited, 254 Best, W. H. G. H., official report of, cited, 444 Bile in urine, 359-360 Bile acids in faeces, 321 Bile pigments in faeces, 321 Bilharziosis : — Blood changes in, 58 Geographical distribution of, 357-358 Binctia, 225 Birds :— Bed-bugs, attacked by, 285 FilaritE in, 250 Hippoboscidce parasitic on, 191 Lice of, 150 Parasites in blood of, 98-100 Trypanosomes found in, 106-107 Bironella, 214 Blackwater fever : — Distribution of, and racial susceptibility to, 451-452 Excretion charts in, 453 Haemoglobinuria characteristic of, 358 Methsemoglobin in, 136 Pigments in, 305 Prevalence of, 433 Secretion of urine in, 359 Tonicity of blood in relation to, 139 Urine of, 360 ; blood in, 357 Yellow pigment evidencing, 304 Blanchard, cited, 210 and note Blastomycetes (yeasts), 404 ; differentiated from Schizomyceies, 377 Blepharoceridce., 163 Blood :— Animal parasites found in, 69, 117 Colorimetric estimations of haemoglobin in, 425-428 464 INDEX Blood — continued. Chemical reaction of, 135-136 Circulating, sporulation of parasites in, 80 Coagulation time, estimation of, 135 Composition of, 40, 134 Corpuscles, see Red blood corpuscles and Leucocytes. Counts, method of, 416-420 Crescents in, diagnosis of, 96 Destruction of, pigments evidencing, 303-304, 305 Examination of, for pathogenic bacteria, 132-133 Faeces, in, 319-320 Films :— Ciianges in shed blood, method of showing, 91-92 Decolourised, 73-74 Dried, preparation of, 48-51 ; fixation, 51 ; staining, 52 et seq. ; value of, in recognition of parasites, 69-70 Fluid, preparation of, 41-44 ; appearances in, 44-47 ; staining of, 47- 48 ; value of, in recognition of parasites, 69-70 ; appearances mistaken for non-pigmented parasites, 93-95 Fresh, examination of, 41 ; elements normally present in, 44 ; crescents in, 243 Isotonic strength of, 138-139 Laked, 140 Myelocytes in, 423 Parasites in, occurrence of, 40 ; recognition of, 93-97 Protozoa, examination for, methods, 69-74 Quantity of, method of obtaining, 134-135 Specific gravity of, 135 Spectroscopic examination of, 136-137 Tonicity, estimation of, 138-139 ; range of variation, 139-140 Blood plasma, trypanosoraes in, 69 ; parasites in, io5 et. seq. ; prevention of coagulation, 134 Blood platelets : — Examination for, 47 ; Hsematoxylin stain for, 53 Recognition of, 96 Romanowsky's stain, effect of, 66 Blood serum : — Culture medium, as, 146 Hypertonicity of, 140 Opsonins in, function of, 146; estimation of opsonic index, 146-147 Precipitins formed by, of related animals, 144-145 Substances due to infection of micro-organisms in, 144 Wright's tubes for obtaining and diluting, 141- 144 Blood-vessels, trematodes in, 337 Borax methylene blue for detection of protozoa, 70-7 1 Bothriocephalus, 337 Bouche, cited, 189, 190 Brachycera, differential characters and genera, 457 INDEX 465 Brachycerous flies, 156 Braddon : — Method of preparing fresh fluid blood films, 42-44 Solution for staining fresh blood films, 47 and note Brain : — Demonstration of parasites in, 82-84 Pigmentation in, natural and malarial, 81-82 Sporulation of malignant tertian parasite in, 80, 81 Weight of, in Negroes and Indians, 22, 455 Brauer, cited, 160 Brazil : — Filaria occurring in, 129 Malaria carrier in, 218 Mosquitoes of, 220 British Guiana : — Filarim occurring in, 128 Madura foot in, 399 Broth, glycerine, 370 Nutrient, preparation of, 366 ; neutralisation of, 366-369 ; sterilisation of, 369 Buffalo, parasite of tsetse-fly disease harboured by, 108 Bunsen burner for spirit, 3, 4 Burma, Cimex rotundatus in, 285 Calliphora (blue-bottle flies), genus and characters of, 154, 178, 179 Colorimetric estimations, 425-428 ' Cambridge rocking microtome, 37, 38 Camera lucida, 13 Cammidge, Hied, 364 Canada, Anopheles viaculipennis found in, 216 Canada balsam, mounting of mosquitoes in, 227-228 Canaries, proteosoma in, 98 Carbol fuchsin : — Decolourised films, for staining, 74 Tropical use, for, 389 Trypanosomes demonstrated by, 109 Carbol thionin, micro-organisms in tissues demonstrated by, 71, 8385 Cat, Opistkorchis felineui, the host of, 343 Catageiomyia, 220 Cathcart's microtome, 33 Cattle :— Mai de Caderas in, 108 Micro-organisms pathogenic to, 402-403 Parasites of, 193 Rhodesian fever in, causation and transmission of, 100 Timothy grass bacillus of, 387 Texas fever, causation and transmission of, 100 Tsetse-fly disease in, 107 Cecidomyidm (gall-midges), 162 30 466 INDEX Cell protoplasm : — Cloudy swelling of, 307 Degenerations of, fatty, see thai title ; amyloid, 309; fibrous, 309-310 Cellia, 198, 215, 218 Cellia argyrotarsis , 247 Celloidin imbedding, 33 ; cutting of sections, 38 Centrifuge, 395 Ceratophyllus, 278, 279 Ceratopogon, 164, 165 Ceratopsylla, 279 Cercomonas hominis, 355 Cestodes (tape-worms) : — Canine, 337, 338 Classification of, 350 Definitive hosts of, 332, 333 Diagnosis of species, 336-337 Eggs of, in faeces, 330 Human, 332 Structure of, 333-336 Chad, Lake, Auchmeromyia around, 189 Chagasia, 214 Charts : — Distribution and incidence of disease, parasites, &c., indicated by, 453-454 Malarial incidence, &c., represented by, 444-453 Principle of making, 443-444 Chester, cited, 389 China: — Myzorhynchus sinensis found in, 218 Trematodes found in, 343 Chinese : — Meckel's diverticulum in, 22 Organs of, average weight of, 455 Splenic enlargement in, 23 Chironomidce (midges), 164-165 Chloroform vapour, mosquitoes killed by, 232 Chlorosis : — Blood changes in, 59-60 and note Reduced haemoglobin in, 421 Chlorides in water, 414 ; detection of, 412 Christophers, cited, loi, 102, 103, 292 Chromatin, stains for demonstrating, 62-64, 65 Chrysomyia, characters and occurrence of, 178-179, 1 86- 1 88 Chrysops, 169, 171, 172 Chrystya, 214 . Cimex lectularius and C. rotundatus, 284-285 Cimicidce, characters and genera of, 283-285 Cobb, cited, 124 Cocci, 61 Coccidia, metamorphosis of, 311-312; found in man, 312; demonstration of in tissues, 312-313 ; in faeces, 354 INDEX 467 Coccidiidea, 105 Coccidium cuniculi, 312 C. bigeminum, 312 C. hominus, 312 Coleoptera, 151, 152 Congo, the : — Auchtneromyia of, 1 89 Ticks of, 296 "Congo floor Maggot," 187 Connective tissue, filarice in, 123, 124, 128, 129 Copepoda, 299 Copper in water, detection of, 406-407 CorethridtE, classification of, 211 Corethrina, differential characters, 207, 208; classification of, 207, 211; genera of, 212 Cotylogonimus , 342, 343 Cover-glasses for tropical use, 16-18 Crescents, recognition of, in decolourised films, 73 Cropper, cited, 46 Crows, parasite of, 98 Crustacea, 299 Ctenocefhalus, 278, 279 Ctenacephalus serraticeps (dog-flea) 273 Ctenopsylla, 279 Cuba, madura foot in, 399 Culex :— Clypeus of, 198 Differential characters of, 221, 222 Eggs of, 210, 252, 254 Genera of, 222 Head and scutellum of, 198 Larvae of, 261 Occiput, scales of, 202 Pupae of, 269 Wing of, 163, Z04 Culex dorsalis, eggs of, 252, 254 C.fatigans, 222, 244, 247 C. pipiens, 219, 222 Culicid larvae, cannibal, 255 Culicida, see Mosquitoes Culicina : — Differentiation of, 202, 208, 209, 210, 219-222 Eggs of, 252, 2S3 Feeding time of, 153 Larvae of, 261 Culture making, 26 ; blood serum as culture medium, 146 Cyclol&ppteron, 204, 214 Cyclops, fresh water, 299 Cyclorrhapha, characters and genera of, 160-161, 457 468 INDEX Cydorrhapha aschha, characters and subdivisions, 161, 175 C. schizophora, characters and subdivision of, 161, 175, et seq. Cynomyia, 178 Cynonyia vtortuorum, 178 Cyprus, Schistosoma found in, 357 Cysts, contents of, boiling method of examination 29 Daddy-long-lbgs (Tipula), wing of, 158-159 Danielsia, 221 Davainea, 337 Delhi boils, parasites of, 116, 315 Deinocerites, 224 Demodex follicularum, 298 Demodicidm, 287, 298 Dendromyia, 225 Dendromyinm, characters of, 202, 210, 225 Dengue fever, 250 Dentition tables for Europeans, 434, note Dermacentor, 296 ' Dermacentor reticulatus, loi Dermatobia, 154, 177 D. noxialis, lj6 Desert-rat, see Jerboa Desvoidea, characters of, 219, 220 ; larvje, 261 Desvoidy, cited, 178, 189, 190 Diarrhoea : — Stools of, 355, 356 Tropical, 321-322 Dibothriocephaloidea, 337, 350 Dibothriocephalus fuscus, 338 Dicrocaelium, 342, 343 Diplococci, 377 Diflogonoporus, 337 Diptera : — Bite, virulent, of, 153 Characters and structure of, 151, 155-160 Classification of, 160 et seq., 456-457 Disease germs carried by, 1 54 Feeding times of different species, 153 Fleas, see Aphaniptera Hosts of human parasites, as, 154-155 Larvae of, 152, 160 Man, means of harming, 153 Parasitism of, internal and cutaneous myiasis, 153-154 Pupae of, 160 Young, living, production of, 160 Dipylidium, 336-337 Dipylidiuni caninum, 273, 338 INDEX 469 Dog:- Definitive host of some tape-worms, as, 332, 337 Epidemic jaundice of, causation and transmission of, loo-ioi Filaria immitis in, 117, 123-124, 249 Hamogreiarines in, in Assam, 102 Parasites of, 193, 299 Piroplasmosis of, transmission of, 293-294 Trematodes, the hosts of, 343 Dourine, trypanosome of, 108 Dragon-flies, larvse of, hostile to larvse of mosquitoes, 150 Drepanidium, Schiiffner's granules found with, 88 Dysentery, stools of, 322, 323, 352, 355 Egypt, Cotylogonimus heterophyes found in, 343 Elephantiasis, 123 Emphysematous changes in organs, 24 Empididce, 174-175 Endocarditis, septic, leucocytosis in, 422 England : — Anopheles maculipennis found in, 216 Blackwater fever in, 452 Entamceba histolytica, 354 Enteric fever, urine of, 362 Entozoa, examination for, 24 Eosin stain for myelocytes, 59 Eosin and hsematoxylin stain, blood parasites shown by, 60 Eretmapodites, 204, 220 Erysipelas, urine of, 362 Europeans : — Dentition tables for, 434 Organs of, average weights of, 455 Fasces : — Analysis of, 324-325 Bile pigments and acids in, 321 Bulk of, in native races and Europeans, 322-323 Coccidia in, 354 Contamination of water, milk, &c., by, detection of, 392-393 Eggs in, numerical estimation of, 425 Examination of, macroscopic, 318 et seq. ; microscopic, 327-331 Flagellated organisms in, 355 Larvae oi AnthomyidiE in, 190- 191 Mucus in, 319-320 Parasites and their ova in, 326-331 Protozoa in, 352 Reaction, normal, 322 Spirochcetce found in, 112, 355 Fasciola, 342, 343 47° INDEX Fasciola hepatica, life-history of, 341-342 Fasciolidce, eggs of, in fseces, 330 ; description of, 339 ; human genera of, 342 ; subfamilies of, 350 Fasciolopsis, 342, 343 , Fats in faeces, 325-326 Fatty degeneration ; — Estimation of extent of, 308 Occurrence of, 308-309 Stains demonstrating, 307-308 Favus, 400 Feltinella, 2 14 Fever : — African relapsing : — , Carriers of, 284, 293 Leucocytic variation in, 456 Spirochseta of, iii. Watery motions of, 323 Texas fever, transmission of, 293 Fibrous degeneration, demonstration of, 309-310 Ficalbia, 225 Filaria bancrofii, 126, 127; intermediate host for, 217 F. demarquayi, 130, 131 F. immiiis, mosquitoes as hosts of, 194, 249 ; intermediate hosts of, 216 F. medineHsis (guinea-worm), 299 F. nocturna : — Development of, in mosquitoes, 247-248 ; species of mosquitoes carrying, 222, 247 Urine, in, 357, 358 F. ozzardi, 126, 127 F. Persians, 130, 131 Filaria : — Adult forms, 117 ; occurrence of, 123-124 Avian, 124, 250 Carriers of, 218, 224 Development of, in mosquitoes, 194, 247-249 Differentiation of human, 128-129, 132 Diptera as intermediate hosts of, 155 Measurement of, 124-125 Occurrence of, 316 Preparation of specimens, 129-132 Filarial embryos : — Avian, growth of, 123 , Blood, fresh, in, 117-118; in blood plasma, 106; enumeration of, in blood, 423 Differentiation of species, 121-122 Discharge of, through the skin, 117 Dried films, examination of, in, 119-120; staining of, 120 Fresh, examination of, n8, 119 Mosquitoes, development in, 123 Periodicity, 121, 122 INDEX Filariasis, blood changes in, 58 Finches, parasite of, 98 Finlaya, 221 fishes : — Parasites of, 313 Trypanosomes in, 106 Fixation and hardening : — Macroscopical specimens, of, 24-26 Microscopical specimens, of, 26-29 Flagellata, 115 Flagellating bodies, stained specimens of, 92 Fleas, see Aphaniptera Fleischl, von, calorimetric estimation of hjemoglobin, by, 426-42S Flies, classification of, by antennae, 156 Flemming's solution, 29 Forest-flies, young of, 160 Formalin, use of, in museum preparations, 25 Formaldehyde, fixation by, 51 Formol alcohol, as reagent in tropical work, 27-28 Formosa, 218 Fowls, parasites of, 193 Fox, the host of Opisthorchis noverca, 343 Foxes, flying, parasites in blood of, 100 Fresenius, " Quantitative Analysis" cited, 414 Fungi, tropical, 400-401, 404 Gad-flies, see Tabanida Galgey, 123 Gamasidce, 287 Gametocyte : — Changes in, 89-91 Chromatin in, 78, 91, 92 Genesis of, 93 Shape of, in different species of parasite, 88-89 Gastrodiscus, 342-344 Gelatine, nutrient, 370-371 Giles, cited, 242 Giksia, 221 Giemsa's method of staining films, 65-66, 72 Glass, deterioration of, in the Tropics, 12-13 Glossina : — Differential characters of, 178, 182 Genus and characters of, 178 Internal anatomy, 184 Larvae of, 183 Mouth-parts of, 183 Occurrence of, 183 Synoptic table of species, 184-186 471 472 INDEX Glossina morsitans, io8, 184 Glycerine jelly, mounting of mosquitoes in, 228 Glycerine, nutrient, 370 Gmelin's reaction of bile pigment, 321 Goadby, K. W., dentition tables of, 434, note Goats : — Heartwater in, transmission of, 294 Micro-organism pathogenic to, 403 Goeldia, 225 Gonococcus, 403 Goudot, cited, 177 Gower's hEemocytometer, 418 „ „ slide, 415, 416; as substitute for micrometer slide, 13 ,, hsemoglobinometer, 426 ,, solution for demonstrating agglutinins in serum, 144 Grabhamia, 220, 221 Gram's method of differentiating micro-organisms, 85, 382-383 Granuloma, sclerosing, spirochsetse found in, 112 Gregarinoidea, 105 Griibler, 62 Griinbaum, cited, 419 Griinbaum-Widal reaction, 396-397 Guinea-pigs, micro-organisms pathogenic to, 402-403 Guinea- worm, intermediate host for, 299 Hadrus, characters of, 169; example of, 171-172 Hamaphysalis, 296 Hcemaphysalis leachi, 100, 293-294 Hcematobia (house-flies), 178, 182 HmmcUopota, 169-171 Hsematoxylin stain : — Blood films, for, 52-56 Demonstration by, of, brain parasites, 82-83 > filarise, 120 ; filarial embryos, 122 ; malarial parasites, 85 ; protozoa, 70 Haematoxylin and eosin stain for detection of blood parasites, 60 ; pro- tozoa, 70 Haematuria, causation of, 357-358 ; distinguished from hEemoglobinuria, 357. Hoemoglobin : — Colorimetric estimations of, 425-428 Dissolved, in serum, 140 Occurrence of, 138 Solution of, see Blood-Tonicity Spectrum of, 137 ; of reduced haemoglobin, 136, 137 Hsemoglobinuria : — Geographical distribution of, 358-359 Tropical, 357 Haemoglobinuric fever, ansemia after, 421 Hmmogogus, 224 INDEX 473 HtBtiiogregarina, occurrence and development of, 102-104; in blood plasma, 106 ; zoological position of, 105 Hceniogregarina balfouri, 104, 105 H. gerbilli, 104, 105 H. ranarum, loj Hemolysis, ansemia due to, 421 Hcemoiporidia, development of, 74-75 ; structure of, type of young form, 76-77 ; sexual cycle, 98 ; zoological position, 105 Hair, demonstration of fungi of, 400-401 Hair follicles, parasites of, 298 Halteridium in birds, 98-100 ; zoological position of, 105 ; in blood plasma, 106 Hearson's incubator, 19 Heart :— Dogs', filarial in, 123-124 FilaricB in, 129 Hemiptera, 151, 152, 281-285 HeptaphUbomyina, 205, 211 Hermann's solution, 29 Herpetomonas, 115 Hewlett, cited, 362 " Hielscher's tubes," 313 Hippoboscidce, 191-193 Homalomyia, 182-191 Herder's method of preparing fresh fluid blood films, 44 Horse : — Definitive host of larvse of Gastrophilus equi, 154 Parasites of, 193 Surra fatal to, 108 Trypanosoma dimorphum in, 108 Howard, 450 Howardina, 221, 224 Huhceoteomyia, 221 Huppert's test for bile pigment, 321 Hyalomma, 295 Hyalomma csgyptium, loi Hydrotea, characters of, 189 ; latvK of, in fteces, 190 H. ciliata, wing of, 191 Hylomyia, characters of, 190; larv^ of, in fzeces, 190-191. Hymenoptera, 150- 15 1 Hyphomycetes (moulds), 400-401, 404 ; differentiated from schizomycetes, 377 ; connecting link between schizomycetes and, 378 Hystrichopsylla, 279 Incubator : — Advantage of, in tropical countries, 4, 19-20 Home-made, 4, note India : — Cimex rotundatus in, 285 474 INDEX India — continued. Epidemic jaundice of dogs in, loi Madura foot in, 399 Malaria carriers in, 217 Myzomyia rossii in, 216 Surra, occurrence of, in, 108 Ticks of, 269 Trematodes found in, 343 Indian field-rat, Hamogregarina gerbilli in blood corpuscles of, 103, 104 Indian plague commission, work of, 273 Indian sparrow, proteosoma in, 98 Indians : — Brain weight at different ages in, 455 Organs of, average weights of, 455 Splenic enlargement in, 23, 455 Susceptibility of, to blackwater fever, 451-452 Indican in urine, 360-361 Indol formation by bacteria, 393 Infection, carrying of, by diptera, 154 Infusoria in faeces, 356 Insecta : — Metamorphosis of, 1 51-152 Structural characteristics of, 149 Sub-divisions of, characteristics of, 149-151 Instruments and reagents, 457-458 Intestines : — Blood stasis in, consequent on malarial infection, 85 Cestodes in, 332-337 Examination of, in the Tropics, 24 Gaseous distension of, 24 Nematodes in, 344 Trematodes in, 339 Iron pigment, blood destruction evidenced by, 305 Iron in water, detection of, 405-406 Isotonic solutions, 138 Ixodes, 295 Ixodida, characters of, 287 ; sub-families of, 287-290 Ixodince : — Characters of, 288-291, 294, 295 Genera of, 295 Ova of, number of, 293 Piroplasmoses carried by, 293 James, Hied, 103, 215 Janthinosoma, 204, 220 Japan : — Larval form of B. mansoni found in man in, 332 Trematodes found in, 343 INDEX 475 Jaundice, haematogenous, 140 Jays, parasite of, 98 Jerboa, parasites in red blood corpuscles nf, 103, 104 ]igget-f[ea. {Sarcopsj//ia penetrans), 154, 278, 280-281 Joblotina, 198, 202, 211 Journal of Hygiene, cited, 269 Journal of Tropical Medicine, cited, 447 Jung's slide microtome, 38 Kaiserling's method of preparing specimens, 25 Kala-azar : — Flagellate forms discharged from intestinal ulcers in, 116 Leishman-Donovan bodies found in, 97 Leucocytosis in, 422 Leucocytic variation in, 57, 456 Parasites of, 315 ; carriers of, 284 Yellow pigment found in, 303 Kasan, Russia, bed-bugs from, 281; Kerteszia, 214 Kidneys : — Fatty degeneration in, 307, 308 Yellow pigment in, 314 Koch, cited, loi, 385 ; postulates on the pathogenicity of organisms, 398 Laboratory, tropical, description of, i-S Lasioconops, 221 Laveran, cited, 115 Lead in. water, detection of, 406 Leicesteria, 222 Leishman-Donovan bodies : — Chromatin masses in, 114, 115 Demonstration of, 113-114, 315 Genus of, 115 Occurrence of, 97, 113, 314 Pathogenesis of, 116 Leishman : — ^ ModiScation of Romanowsky's stain, 62-63; for demonstrating brain parasites, 82; Leishman-Donovan bodies, 114; myelocytes, 68; protozoa, 72-73 ; Schiiffner's granules, 87-88 ; structure of malarial parasites, 77-78; trypanosomes, 109-110 Gametocytes of benign tertian and quartan recognised by, 91 Leitz camera lucida, 13 Lepidoptera, 151, 152, 191 Lepra bacillus, 386 Leprosy : — Amyloid degeneration in, 309 Carriers of, 284 476 INDEX Leptida, 173-174 Leptince, 174 Leptothrix, 378 Leucocytes : — Abnormal elements resembling, 58-59 Animal blood, of, parasites found in, 102-103 Differential count of, method of making, 58, 417-426, 422 Eosinophile, 56 Fresh fluid films, appearances in, 46 Hsematoxylin stain for, 54-56 Increase in total number of, 422 Large mononuclear, 55, 56 Leishman-Donovan bodies in, 97, 113 Lymphocytes, 55, 56, 67 Mononuclear, 67 Pigmented, 78 Polymorphonuclear, 55-56 Romanowsky's stain, effect of, 67 .Variations in numbers and relative proportions of, in health and disease, 455-456 ; variations after malaria, 56-58 Varieties of, in normal blood, 54-56 Leucocytosis, 422 Leucocythsemia, 58, 60 Leucocytozoon cams, 103, 104, 105 L. funambuli, 104, 105 Lichen hypertrophicus, 306 Limatus, 202, 225 Linguatulida, 286, 298-299 Liver : — Fatty degeneration in, 307, 308 Leishman-Donovan bodies in, 113, 114 Melanin in, in malarial infection, 302, 303 Puncture of, 315 Trematodes in, 337 Lophoceratomyia, 220 Lophoscelomyia, 214 Louis Jenner stain for blood films, 61-62 ; protozoa demonstrated by, 72, 73 Louse, sporogony of Haemogregarina gerbilli in, 104 Low, cited, 238, 269 Lucilia (blue-bottle flies), genus and characters of; 178, 179, 1S4, 186, 187 Lungs : — Filarial embryos in, 122 Leishman-Donovan bodies in, 113 Negro races, of, 23 Post mortem, weight of, in Tropics, as compared with European standards, 22; variations in, with time after death, 455 Trematodes in, 337 Lutz, cited, 211 INDEX 477 Lymphatic system : — Filarics in, 123, 128 Leishman-Donovan bodies in, 113 Preparation of cultures from, 26 Lymphocytes; — Hsematoxylin stain for, 55 Romanowsky's stain for, 67 MacCallum, cited, 98 MacConkey, cited, 392 MacCrorie's method of fiagella staining, 381-382 Macleaya, 220 Macrogametes, 90, 98 Macroscopical specimens, preparation of, 24-26 Madura foot, organism of, 399, 401, 402 Magalhaes, cited, 117 Mai de Caderas, 108 Malaria : — Age incidence of, 433-434 Bile in urine of, 359 Blackwater fever, occurrence with, 452 Blood changes in, 59 Cause of death in acute, 81, 84 Cerebral symptoms in, 80, 81, 84 Endemic index, 436 ; determination of, 438-443 ; spleentest for, 440-441 ; graphic representation of methods of determining, 444-447 General health, effects on, 435 Hosts, definitive and intermediate, of, 75 Immunity from, 436, 445 Incubation, natural, period of, 435-436 Individual susceptibility, line of enquiry as to, 431 Infection, liability to, 432-433 Influence of, on prevalence and severity of other diseases, 448, 449 Leucocytic variations in and after, 57, 422, 456 Mortality from, 434-435 Parasites of, see Parasites Pigment of, 301-303 ; demonstration of, 304-305 Place, influence of, 432, 437 ; chart showing residence required for probable infection with, 446-447 Pneumonia coexistent with, 423 Prevalence of, estimation of, 433 Prophylactic measures for, success of, charts illustrating, 447-449 Relapses, 433, 43S Seasonal variation in, effect of rainfall, 437 Splenic enlargement in, 445 ; pigmentation in,. 446 Stools of, 323-324 Malay Peninsula and Archipelago, malaria carrier in, 218-219 Malaya, mosquitoes of, 218, 220, 222 478 INDEX Malignant tertian (sub-tertian) parasite : — Asexual cycle, length of, 79 Corpuscle, containing, effect on, 88 Development, asexual and sexual, phases in, 89 Diagnosed from benign tertian and quartan, 94 Gametocyte form of, 88-89 Male and female crescents, 90-92 Pigment deposits in, 86 Sporulation, number of spores at each, 79 ; site for, 80 Zoological position of, 105 Malta fever : — Agglutination test in, 396-397 Bacillus of, in urine, 361 Leucocytic variation in, 57i 45^ Mammals, parasites of, 191, 298, 299 Man : — Coccidia found in, 312 Filarise developed in, 249 Infection of, with Trichina spiralis, 351 Intermediate host of echinococcus, 332 Micro-organisms pathogenic to, 402-403 Parasites of, 189, 193, 284, 298, 299, 313 Trematodes, host of, 343 Mansonia, occiput scales of, 202; wing scales of, 204, 219; differential characters, 221, 223 ; genera and habitat, 223-224 ; number of sperma- thecae, 242 ; eggs of, 254 ; pupae of, 269 Mansonia uniformis, M. albipes and M. annulipes, 224, 227 Mast cells, Roraanowsky's stain for, 67 Mashonaland, mosquito of, 211 Mastigophora, in human blood, 69 ; parasites of class of, in blood plasma, 106 ; spirochata of relapsing fever belonging to. III; Leishman- Donovan bodies belonging to, 115 Measles, urine of, 302 Measurements, microscopic, 415-416 Characters of, 207 Classification of, 211 Eggs of, 253 Genera of, 212 Head and scutellum of, 198 Larvae of, 258, 259, 261 Occiput scales of, 202 Pupal stage of, 259 Megaropus, 296 Megaropus annulatus, 293 Meigen, cited, 177 Melanoconion, 220, 221 Melanin, 301-303 Melophagus ovinus, 192, 193 INDEX 479 Membranous colitis, 319 Mercury, perchloride of, saturated solution of, fixation by, 51 Merozoites, 74 Mescostoides Hneatus, 338 Methsemoglobin, spectrum of, 136, 137 Methylene blue, combination of, with eosin, 61-63 Methylic alcohol as a fixative, 72 Mice, micro-organisms pathogenic to, 402-403 Micrococcus melitensis, 403 Microfilaria nocturna and M. diurna, 121, 122 Microgamete^, 90, 98 Micrometer eyepiece, measurements by, 415-416 Micro-organisms, vegetable, see Vegetable micro-organisms Microscope for tropical use, 5-6 ; parts of, 7-S ; illuminating apparatus, 8, 11-12; cost of, 9; lenses, 9-13; testing of, 9-13; measurements, 13-15, 415-416; dissecting, 15; warm stage, 15; slides, 16; cover- glasses, 16-18 Microscopic examination : — Fresh specimens, of, 26 Preparation of tissues for, fixation and hardening methods, 26-29 ! imbedding, 29-33 Microtomes : — Freezing, 33-36 Paraffin and celloidin sections, for, 37-38 Mimomyia, 225 Mirrors, silvered, deterioration of, in the Tropics, 12 Monkeys : — African and Asiatic, parasites in blood of, 100 Micro-organisms pathogenic to, 403 Relapsing fever reproduced in, in Uganda, of, piroplasma found in, loi Monogenia, 341 MonostomidcE, 342 Mosquitoes : — Alimentary canal of, 232-237 Bacilli in, 250 Breeding places of, 258 ; permanent, 265-267 ; temporary, 267-268 ; artificial, 268 Carriage of, 269-272 Characters of, 162-163 Classification of, 206-213 ; Theobald's, 206, 211-213 ; proposed, 261 Definitive hosts of malaria parasites, as, 75, 90, 99, 244-247 Differentiation of species, 162, 194-195, 202, 206, 226-227 Diseases acquired by larvae and distributed by adult, 250 Dissection of, 229242; alimentary canal, 232-237; stomach, 239-241 ; salivary glands, 241-242; genital organs, 242 Eggs of, 252-254, 263 ; obtaining, 254-255 Examination of external characters, method, 227-228 480 INDEX Mosquitoes — continued. Filaria developed in, 123, 247-249 Food for, in captivity, 272 Genital organs of, 242 Gregarines in, 250-25 1 Head appendages of, 199 Hosts of different parasites, as, 75, 99, 194, 244-247 Killing, means of, 232 Larvae of, destroyed by larvse of dragon-flies, 150; metamorphosis of, 151 ; breeding collection of, 255-258 ; duration of larval stage, 259 ; anatomy of, 259-265 ; respiratory syphon attached to 8th segment 259-261, 264 Microscopic examination of, 197 Mounting of specimens, 195-196, 227-228 Occiput, scales of, 202 Order of, 161 Proboscis of, 229-232 Proteosoma infection transmitted by, 98 Pupje, collection of, 258 ; duration of pupal stage, 259, 263 ; anatomy of, 262-263 ; description of, 269 Scales, types of, 197-199 Sections of, 237-239 Stomach of, 239-241 Structure of, 194, 199-202, 232-237, 239-241, 242 Sub-families of, 207-212 Wings of, 203-205 Yellow fever carried by, 250 Motuca fly of Brazil, 172 Mounting and imbedding reagents, 458 Mouth, SpirochiztiE ioxaiA in, 112; mucous membranes of, normal pigmenta- tion of, 306 Mucidus, 204, 219, 220 Muir's method of flagella staining, 380-381 MuUer's fluid, 28 Murray, cited, 283 Musca (house-fly), 178 Muscidce : — Characters and genera of, 178, et seq. Halteres of, 159 Muscidc2 acalyptratcE, 175 M. calyptrce, 175, 182 Museum preparations, methods, 24-26 Myelocytes, 58-59 ; Leishman's stain for, 68 ; Romanowsky's stain for, 67-68 Myiasis, internal and cutaneous in animals and man, 154 MyxosporidcE, 313 Myzoinyia, anomalous character of genus of, 213 ; differentia characters, 214, 216 Myzomyia culicifacies, 2 1 7 M^funesta, 217, 244, 247 INDEX 481 M. rossii, 216-217 Myzorhynchella, 214 Myzorhynchus, 215, 217-218 M. barbirostris, 218, 247 M. sinensis, 218, 247 Nagana, trypanosoma of, 107 Natal, auchmeromyia in, 189 Necator, Americanus, 346-348 Negroes : — Blackwater fever, susceptibility to, 451 Brain weight at different ages, 455 ; age of maximum weight, 22 I^ungs, deeply fissured, in, 23 Organs of, average weight of, 455 Parasites in livers of, 299 Splenic enlargement in, 23, 440, 455 Nematoda, 69, 117; in blood plasma, 106; in tissues, 316-317; human, 344; treatment of specimens, 344 ; classification of, 350 Nemaiocera, characters and genera, 156, 157, 456 Nematgcera vera and N. anomala, 456-457 Neocellia, 215 Neopsylla, 279 Neosporidia, 105, 314 Nerve degeneration, demonstration of, 309-310 Nessler's solution, preparation of, 409 Neuroptera, 150, 151 Newham, H. B. G., analysis of water by, 404-414 Nigeria, A. watsoni found in, 343 Nitrates in water, 414; detection of, 410-412 Nitrites in water, detection of, 410 Norway rat, parasite of, 104 Notter, cited, 414 Nutrient agar, 371-372 ; nutrient broth, see Broth ; nutrient gelatine, 370-371 Nuttall, cited, 145, 269 Nycteribida, 193 Nyssorhynchus, 2151 218 Ochromyia (Cayeror Senegal fly), 178 Oestrida (warble-flies), 175-177 Old Calabar, 218 Oliver's colorimetric estimation of haemoglobin, 426 Ookinet, 90 Opisthorchis, 342, 343 Opsonins, specificity, 146 ; estimation of opsonic index, 146-147 Organs, average weights of, 455 ; variation of weight between Tropics and European countries, 22 Oriental sores, parasites of, 315 Ornithodoros, 294 3^ 482 INDEX Ornithodoros moubata and 0. savignyi, characters and distribution of, 296 ; transmission of, 293 ; larvae of, 293 Orth's fluid, 28 Orthoptera, 150-15 1 Orlhorrhapha, character of, 160, 456 Orthorrhapha brachycera, charactersand sub-divisions, 161, 168 et seq 0. nemocera, characters and sub-divisions, 161-167 Osmic acid mixtures as fixatives, 29 Owl, little, trypanosome in blood of, 99 Oxyhsemoglobin, spectrum of, 136, 137 Oxyuris vermicularis, 330, 331, 345 Palestine, Anopheles maculipennis found in, 216 Pancreatic duct, obstruction of, 324 Pangonia, 169, 171 Pangonina, 169 Pangoninus westermani, 337, 343 Paramphistomida, 339, 342-344, 350 Paraffin imbedding, 30-32 Paraffin sections, cutting of, 37-38 ; fixation on slide, 38-39 ; treatment of, 39 ; of mosquitoes, 237, 239 Parasites : — ' Animal, found in human blood, 117 Animals, in blood of, 100-105 Birds, in blood of, 98-100 Faeces, in, 326-331 Human malaria : — Amceboid movement of, 76, 77, 79-80 Asexual cycle of development, 75-76, 79 Chromatin, varying arrangements of, 77-78 Classification of, 78-79 Carriers of, 211, 217, 218, 225-226 Corpuscle, containing, effect on, 86-88 Development of, in stomach of mosquito, 75, 194, 244-247 Diptera as definitive hosts of, 154-155 Enumeration of, in blood, approximate method, 423-424 Flagellating and non-flagellating bodies, 243-244 Hosts, definitive, of, 216, 217, 244-247 Melanin deposited by, 301-303 Pigment deposits in different forms of, 79, 85^86 Sporulation of, 76, 79, 80 Staining, methods of, 47-48, 61 Structure of, appearances in fresh blood, 75-76; in stairjed speci- mens, 76-77 Zoological position of, 105 Intestinal, 332 et seq. Ova of, measurements of, 331 Sporozoites, development of, 245-247 Tissues, in, 311 et seq. Zygotes, development of, 244-245 INDEX 483 Parrots, parasite of, 98 Patton, cited, 103, 284, 285 Pediculida, 281-283 Pediculus capitis and P. vestimenti, 282-283 Pedipalpi, 286 Pelvis, parasites in veins of, 117 Peniastomiidce, 286 Penlas.'omum constrictum, 299 Pettenkofifer's reaction for bile acids, 321 Phagocytosis, 146 Philippines, occurrence of surra in, 108 Phlebotomus, 165-166 Phoniomyia, 225 Pkthirius inguinalis (crab-louse), 283 . Phylum nemathelminthes and P. plalyhelminthes, 350 Pigeons, parasite of, 98 ; lice of, 281 ; micro-organisms, pathogenic to, 403 Pigment deposits in malaria, 301-303 Pigs, infection of, with Trichina spiralis, 351 ; parasites of intestines of, 356 Pine Islands, anthrax in, 171 Piroplasma bigeminum, 100, loi, 105 P. canis, 100, loi, 105 P. equi, lOI, 105 P. maris, loi P. ovis, loi, 105 P. paruum, 100, lOJ Piroplasmata, occurrence and development of, 100-102 ; zoological position, 105 ; in blood plasma, 10 ; carried by ticks, 293 Pityriasis Tjersicolor, 400 Plasmodium (Hsemamseba), 105 P. falciparum, see Malignant tertian parasite P. kochi, 100, 105 P. malaria, see Benign quartan parasite P. prcecox, see Proteosoma P. vivax, see Benign tertian parasite Pneumococcus, 402 Pneumonia : — Leucocytic variation.iri, 57, 422, 456 Malaria co-existent Vfi^i, 423 Watery motions in, 323 Poisson's formula, 429-430, 449-450 Porocephalus armillatus, 299 Portal system, parasites in veins of, 117 Post-mortem examinations in the Tropics, 21-22 ; special observations as to, 22-24 Precipitins, value of, in classification of animals, 144-145 " Primus " kerosene smokeless burner, 4 "Primus" lamps, 19 Proteosoma of birds, mosquitoes the host of, 194, 222, 244 ; occurrence of, 98 ; zoological position of, 105 484 INDEX Protozoa : — Acquired by acquatic larvae and distributed by adult, 25 1 Classification of, causing malaria, 105 Demonstration of, in tissues, 84 Examination of blood for ; methods,^ 69-74 Stools, in, 352 Tissues, in, 311 Pseudoscorpionida, 286 Psorophora, 204, 220, 241, 254 Psychodidoe (owl-midges), 165-166 Pulex cheopis, 278-280 Pulicidm (fleas), characters and genera of, 278-280 Pupipara : — Antennae of, 1 56 Characters and genera of, 161, igi-193, 457 ; young of, 162 Pyretophorus, 214, 217 Pyrelophorus costalis, 217, 247 Pyrexia, cell degeneration in, 307 Python, Royal, parasite in lungs of, 299 Quartan parasite, see Benign quartan parasite Queensland, Texas fever in cattle in, 100 Quinine administration in malaria, 433 Rabbits : — Coccidium honiinis parasitic to, 312 Micro-organisms pathogenic to, 402-403 Rats :— Fleas, species of, found on, 280 Micro-organisms pathogenic to, 403 Norway, parasite found on, 104 Parasite in leucocytes of, 103 Parasites of, 1 93 Plague disseminated by, 273 Relapsing fever reproduced in, iii. Trichina spiralis harboured by, 351 Trypanosomata of, 107 Reagents, 458 •• Rectum, chronic ulcerations of, 319 Red blood corpuscles : — Bodies simulating ring-forms in, 46 Counts of, method, 416-420; in anchylostomiasis, 421 ; in ansemia, 421 Crenation, 45-46 Hsematoxylin stain for, 53 Hsemogregarina in, of animals, 102-104 Malaria parasites developed in, 75-76 Modifications of, by different malaria parasites, 86-88 Nucleated, 53 INDEX 485 Red Blood Corpuscles — continued. Protozoa, examina,tipn for, 69 Sporozoa in, 69 Tonicity, estimation of, 138-139; range of variation, 139-140 Vacuoles, recognition of, 45 Renal casts, boiling method of examination of, 29 Reunion, Island of, 285 Rhabdonema intestinale, 349 Rhipicephalce, 295, 296 Rhipicephalus annulatus, lOO R. appendiculatus , 100 R. australis, 100, loi R. bursa, 10 ( R. evert si, loi R. sanguineus, loi, 104 Rhodesian fever of cattle, 100 Ringworms, cutaneous, tropical, 400-401 Robber-flies, see Asilidce Rogers, cited, 115, 315-316 Romanowrsky's stain : — Colours observed with, 66-67 Modifications of, 62-64 S Leishman's, see Leishman Structure of malarial parasites demonstrated by, 77-78 Rondani, cited, 178 Ross, P. H., cited, 74, 98, lOI, 216, 218, 250 Ross and Milne, cited, in Sabethes, 225 Sabethoides, 225 Sabetina, 210 andnote Sambon, cited, 299; box designed for carriage of mosquitoes by, 270-271 Sand-flies, see Simulida Sarcocystis, 314 Sarcoma, melanotic, pigment of, 306 Sarcophaga, 177 Sarcophaga carmaria, 177 Sarcophagida, 177-178 Sarcophila, 178 Sarcopsylla, 280-281 S. penetrans, see Jigger-flea SarcopsyllidcE, characters and genera, 280-281 ; compared with the Pulicidts, 278-279 Sarcoptidce, 287 Sarcosporidia, asexual development of, 313-314 ; demonstration of, 314 Scarlet fever, urine of, 362 Scharlach, R., fat demonstrated by, 307 Schaudinn, cited, 99-100, 112, 354 486 INDEX SchistosomidcB, 339, 344, 350 Schistosomum hamatoUum and S. jafonicum, in human blood-vessels, 69, Ii7i 337; in submucosa of bladder or rectum, 316; eggs of, in fseces, 328, 329, 330-331 ; blood in urine from, 3S7-3S8 Schizogony, 74 Schizomycetes, differential characters of, 377 ; morphology, 377-378 ; con- necting link between hyphomycetes and, 378 Schizonts, 74 Schizophora, 457 Schmidt's reaction for bile pigment, 321 Schiiffner's dots, 66, 102 Scorpionidce, 286 Screw- worm (Compsomyia macellaria), 154, 159 Scurvy, leucocytosis in, 58, 422 Scutomyia, 221 Sebaceous glands, parasites of, 298 Section cutting, 33-38 Sepsis, leucocytic variation in, 456 Sheep :— Heartwater in, transmission of, 294 Maggot in, 186 Piroplasma ovis in, loi Trematodes, the host of, 343 Sheep "tick," 155; young of, 160 Shipley, cited, 125, 344 Siberia, trematodes found in, 343 Simulidce, 165, 166-167 Skin, pigmentation of, 306 Skusea, 221, 224 Sleeping sickness, trypanosoma of, 108-I09 Slides for tropical use, 16 Smegma bacillus, 361, 387 Snail, fresh- water, intermediate host of Fasciola hepatka, 341 Solid media, 370-372 Somaliland, ticks of, 296 Somerville, cited, 414 Soudan III., fat demonstrated by, 307 Sparrow, Indian, proteosoma in, 98 Specific gravity of organs, determination of, 308 Spirilla, 377 Spirochtsta : — Blood plasma, in, lo6 Demonstration of, in Pathogenic symptoms of different varieties, 1 1 1 - 1 1 2 Stools, in, 3SS Ticks as intermediate hosts of, 293 Trypanosomes, relationship to, Schaudinn's theory, 112- 113 Spleen : — Diffluent, 24 INDEX 487 Spleen — continued. Enlargement of : — Age incidence of, in Central Africa (chart), 445 Different races, in, 23 Negroes and Indians, in, 455 Post-malarial, 435, 440 Leishman-Donovan bodies in, 113, 114 Malarial, 23-24 Melanin in, in malarial infection, 302, 303 Preparation of cultures from, 26 Puncture of, 314, 315 Putrefactive changes in, 23 Sinuses of, sporulation of parasites in, 80 Yellow pigment in, 304 Sporoblasts, 74 Sporogony, 75 Sporozoa, in human blood, 69 ; classification of, 105 Sporozoites, 74, 245-247 Sprue, stools of, 322, 324 Squirrel, Kathiawar, hcemogregarine in leucocytes of, 103 palm, Leucocytozoon funambuli found in, 104 Staining solutions also fixes, 72-73 Stains, list of, 458 Staphylococci, 146, 377, 402 Statistics in tropical work, difficulties as to, 429 Stegomyia : — Breeding places suited to, 268 Captivity, keeping in, 272 Differential characters of, 220, 222-223 Distribution of, 22^-223 Eggs of, 252, 253, 254; manner of laying, 210 ; carriage of, 269 Genera of, 223 Larvse of, 222, 226, 258, 261 Occiput scales of, 202 Scales of head and scutellum, 198, 219 ; of clypeus, 198 ; of wing, 204 Stegomyia calopus, 223 i'. /aj«ate, 223, 247 ; diseases carried by, 250-251 ; eggs of, 253-254 S. scutellaris, 223 Steriliser : — Hot air, 19 Steam, 18 Stethomyia, 213-214 Stomoxys (stable-flies), genus and characters of, 178, 179; mouth-parts, 179- 180; dissection, 181; internal anatomy, 181-182 Straits Settlements, Madura foot in, 399 Streptobacilli , 377 Streptococci, 377, 402 Streptothrix, 378 S. madurce, 399-400 488 INDEX Streptothrices of actinomycosis, 402 Strongyloides intestinales, 316, 331, 349 Submucosa, Leishman-Donovan bodies in, 113 Subtertian, see Malignant tertian Surra, loS Sutton, cited, 414 Swift's microtome, 34-36 Symphoromyia, 174 Syphilitic lesions, spirochatm found in, 112 SyrphidcB (hover-flies), 175 Tabanidm : — Biting species of, 169 Characters and subdivisions, 156, 168-172 Diagnosed from Pangonina, 169 Feeding time of, 153 Order of, 161 Sections of, 169 Tabanus, 168, 169, 171 TachinidcE, 19 1 Tallquist's haemoglobin scale, 428 Tceniorhynchus, 219, 221 Tape-worms, see Cestodes Telosporidia, 105, 313 Tetrads, 377 Texas fever in cattle, 100 ; transmission of, 293 Theobald :— Cited, 202, 203, 204, 205, 206, 211 Classification by, of jSdince,\2,z^ and note ; of Culicidce, 21 1 ; of Culicince, 219-220 Diagrams after, 198, 200, 201 Monograph of the Culicidce by, cited, 226, note Mounting of mosquitoes, method, 227 Subdivision of Anophelince, by, 215 Theobaldia, 220, 221 Therioplectes, 169 Thoma-Zeiss' hsemocytometer, 418; as substitute for micrometer slide, 13; pipettes of, for blood counts, 417 ; slide of, 415, 416 Thysanoftera, 151 Ticks, see Acarina Timothy grass bacillus, 387 Tin in water, detection of, 407-408 ; Brucine test, 407 Tinea imbricata, 400 Tobacco smoke, mosquitoes killed by, 232 TcBnicB, canine, 338 Tania echinococcus, 332 Tcenia saginata, 337 Tcenidce, 350 Toisson's fluid, 144, 417 Toluidin blue stain, for detection of protozoa, 71 Travers and Watson, reports of, cited, 447 Trematoda : — Blood-vessels, in, 6g Classification of, 337-339, 342-344, 350 Human, 337-339 Structure of, 339-341 Tissues, in, 316 Trichina spiralis, 331, 349-351 Trickinella spiralis, 316, 317 Trichinosis, blood changes in, 58 Trichocephalus dispar (whip-worm), 346; eggs of, in faeces, 328, 331 Trichoprosoponina, 211 Trombididce, 287 Tropical work : — Evidence, precautions as to, in, 431 Freezing in, 34 Laboratory, tropical, description of, 1-5 Post-mortem examinations in, 21-22; special observations, 22-24 Statistics in, need for precautions as to, 429-431 Trypanosomiasis, yellow pigment found in, 303 Trypanosoma dimorphum and T. gambiense, 108 T. leioisi, 273 T. noctua, life-cycle of, 99-100 T. Iheileri, 108 Trypanosomes : — Blood plasma, in, 69, 106 Demonstration uf, 109 Mammalian, pathogenic action of, 107-109 Multiplication of, no Recognition of, in decolourised films, 73-74 Spirochatce, relationship to, Schaudinn's theory as to, 112- 113 Staining of, 109- no Structure of, 109-110 Transmission of, IIO-III Trypanosomiasis, leucocytic variation in, 57, 456 Tsetse fly disease, trypanosoma of, 107-108 Tubercle bacilli, human and other, 385 ; diagnosed from Lepra, 386 ; demon- stration of pathogenic properties of, 397-398 Tuberculosis : — Miliary, diazo-reaction of urine in, 362 Post-malarial susceptibility to, 435 Tunis, propagation of malaria in, 216 Typhlopsylla, 279 Typhoid fever : — Agglutination test in, 396-397 Bacillus of, in urine, 361 Diazo-reaction of urine of, 362 Leucocytic variation in, 57, 456 32 490 INDEX Ulcers, spirochata found in, 112 United States, Anopheles maculipennis found in, 216 UranotanincB, 224, note, 225 ; latvse of, 261 Urine : — Bacteria in, 361 Bile in, 359-36° Blood in, 357 Diazo-reaction of, 362-363 Filaria nocturna in, 358 Haematuria, causation of, 357-358 Hsemoglobinuric, 358359 ; distinguished from bilious, 360 Indican in, 360-361 Methsemoglobin in, 360 Pancreatic reaction of, 363-364 Spectroscopic characters of, 136 Tropical diseases, of, 361-364 Urobilin, 136 Vegetable micro-organisms : — Acid fast, 384 et seq. , 402 Capsulated, 380 Chemical products of, 391-394 Cultures of, 387-390 ; conditions affecting growth of, 390 Description of, 375-376 Differentiation by methods of staining, 382 et seq. Films, preparation and staining of, 376-377 Flagellated, demonstration of, 380-382 Media for cultivation of, 365-372 Morphology, 377-378 Motility, 378-379 Pathogenic properties, testing of, 397 Reaction with various blood sera, 394-397 Separation and plating of, 372-375 Spore formation, 375 Verallina, 224 Vertebrates, cold blooded, hsemogregarina in, 102 Vibrios, yjT, 403 Vincent, cited, 57 Viper, nose-horned, parasite in lungs of, 299 Visceral capillaries, sporulation of parasites in, 80 Ward, H. B., table of canine tape-worms by, 337, 338 Water :— Analysis of, methods, 404-405 ; chemical analysis, 405-4J4 Hardness of, estimation of total, 412-413 Purity of, 413-414 Supply of, in laboratory, 2-4 Weil's disease, 359 INDEX 491 West Indies : — Filaria occurring in, 129 Mosquitoes of, 218 Schistosoma found in, 357-358 Screw- worm fly in, 186-187 While blood corpuscles, see Leucocytes Whitelegge, cited, 414 Wolf, parasites of the, 299 Wright, J. H. :— Blood coagulation time, method of estimating, 135 ' Cited, 146 Modification of Leishman's stain, 62 Tubes, for estimating tonicity of blood, 139 ; for obtaining and diluting serum, 141-144 Wyeomyia, 225 Yaws, spirochceta found in, 112 Yeasts, 377, 404 Yellow fever : — Fatty degeneration in, 308 Mosquitoes the host of parasites of, 194, 250 Prevalence of, 433 Secretion of urine in, 359 Stegomyia calopus, carried by, 223 Yellow pigment of malaria and some other diseases, 303-304 ; blood destruc- tion evidenced by, 305 Zambesi, filariasis on the, carrier of, 224 Zenker's fluid, 28-29 Ziehl-Neelson's method of differentiating micro-organisms, 383 et seq. Zinc in water, detection of, 407 Zygotes, 90, 244-245 Zygotoblasts, 74 Zygotomeres, 74