# Tropical Diseases/Chapter 2

CHAPTER II
MALARIA: THE MICROSCOPICAL EXAMINATION OF THE BLOOD
BEFORE commencing the study of malarial blood it is advisable for the beginner to familiarize himself with the microscopical appearances of normal blood, both in fresh and stained preparations. He should learn to recognize the several varieties of leucocyte; to appreciate differences in colour, size, and shape of the red cells; to recognize dirt, vacuoles, forms of crenation, and artificially produced appearances. By mastering such details at the outset important sources of fallacy will be avoided, and in the process of self-education a useful knowledge of technique will be acquired.
For a thorough appreciation of the principles on which blood examinations for the demonstration and study of the malaria parasite should be conducted, it must be borne in mind that the parasite is intracorpuscular. To see it, therefore, it is necessary, particularly for the beginner, so to dispose the corpuscles in the preparations that a proportion of them shall lie flat on the slide, in a single layer, and presenting their surfaces, and not their edges, to the observer (Fig. 16). It is mainly from ignoring this fundamental principle that so many fail to find the parasite.
Preparation of fluid blood films.—To secure this disposition of corpuscles in fresh blood the following procedure, the smallest details of which must be scrupulously carried out, is recommended:—
very fine film. It is sometimes more convenient to obtain the blood from the lobe of the ear or, in children, the great toe.
Several preparations should be made, the requisite blood being obtained by renewed gentle compression of the finger-tip.
After waiting a few minutes to allow the blood to spread out completely between the glasses, it is well to ring the preparations with vaseline. This will effectually stop all movement, all evaporation of ​the blood, and, consequently, over-compression of the corpuscles, and will thereby greatly facilitate examination.
In making these preparations care should be taken to sterilize the needle employed, otherwise grave accidents might occur. It is, of course, unnecessary to sterilize the slides and cover-glasses.
Characteristics of a successful preparation.—On holding a successful preparation up to the light, one or more areas, each made up of three zones, the different zones shading into each other, can be made out by the naked eye. Each such area includes a peripheral zone of a reddish tinge, a middle zone having a somewhat iridescent look, and a central zone absolutely devoid of colour. Successful preparations may be recognized by the presence of these zones. Preparations not exhibiting this appearance should be rejected; it is waste of time to examine them.
On examining successful preparations with the microscope it will be found that the central zone or area contains few or no blood-corpuscles. This zone may be designated the "empty zone." Proceeding outwards from this we come on an area occupied by scattered, isolated, compressed, and much-expanded corpuscles—the "zone of scattered corpuscles." Farther out the corpuscles become more numerous and less expressed (Fig. 16). Gradually, as we trace the film still farther outwards, the corpuscles are found approximated to each other, until, finally, the peripheries of the corpuscles are mostly in touch—the "single-layer zone." Farther out the corpuscles, though still lying flat, are found to overlap each other or are piled one on the top of the other—the "zone of heaped-up corpuscles." Beyond this zone the corpuscles are arranged in rouleaux the "zone of rouleaux." At the extreme margin of the preparation the corpuscles tend to break up and run together so as to form a narrow border of free hæmoglobin, the individual corpuscles perhaps being indistinguishable the "zone of free hæmoglobin." Each of these zones should be studied, for each may afford special information about the malaria parasite.
​Microscopical examination.—The beginner will save time if he gets someone who is familiar with the necessary technique, and with the appearance of the parasite in the blood, to give him one or two lessons. Accuracy and quickness can be acquired only by practice. It is a good plan to practise preparing films from one's own blood.
The examination is best conducted with a 1/12 in. oil immersion lens, a rather low eyepiece, a substage condenser, and a good but not too dazzling illumination. It is practically useless to work with any objective lower than 1/12 in., or without a substage condenser.
It is not always possible to choose, but, if practicable, a case of quartan infection should be selected for examination in the first instance. Failing a quartan, a well-defined benign tertian infection might be chosen. Failing either of these, a long-standing case of recurring malaria with marked cachexia will afford the next best opportunity. It is best to examine the patient's blood just before or at the time of rigor. In quartans and benign tertians, at the time named, there should be little difficulty in discovering large parasites; in the case of these infections, attention is called to the relatively large parasites by the abundance of coarse pigment they contain. In the blood of malarial cachectics with recurring febrile attacks it is generally an easy matter to find crescents and crescent-derived spheres, as this form of the parasite is of considerable size, carries abundance of pigment, arid possesses a very definite and striking shape.
When the beginner has learnt to recognize the larger forms of the parasite, he will have begun to appreciate what sort of body he has to look for; thereafter he should be able to educate himself, and to pick out the smaller and intermediate forms.
In proceeding to make his first examination of a liquid blood slide, the beginner, in the first instance, should confine his attention to the "single-layer zone." Field after field of this he must pass in review, carefully scrutinizing the interior of every ​blood corpuscle, every leucocyte, and every pigmented body, even though it be not included in a corpuscle. He must not expect to find parasites in every corpuscle, or even in every field; and he certainly must not expect, as the beginner usually does, to find in every slide the beautifully regular segmenting form or "rosette body" or the weird-looking flagellated body made familiar to us by so many illustrations. Such bodies, though really present somewhere and in some form at one time or another in every case, are among the least common of the many phases of the malaria parasite; they are met with only under very definite and not very constantly encountered conditions, and are not very often seen at an ordinary clinical examination.
In most cases the parasite is discovered in the first field or two examined; but in not a few instances dozens of fields may have to be scrutinized before a single parasite is found. Therefore no examination can be said to be complete, in a negative sense, until at least half an hour has been spent over several suitably prepared slides.
The intracorpuscular forms most frequently met with have the appearance either of small specks of pale protoplasm, or of larger masses of pale protoplasm containing grains of black pigment. Close watching discovers that the former are endowed with amœboid movement, and that they continually change shape and position in the affected corpuscles. As these movements are an important test of the parasitic nature of the body sought, they should be carefully looked for. The smallest protoplasmic specks look like washed-out smudges of dirty-white paint, half hidden by the hæmoglobin; they are sometimes hard to see. Their parasitic nature can readily be determined by their movements; by their soft, ill-defined margins; and by the fact that they tend now and again, on first removal from the body, and permanently later, to assume the appearance of tiny white rings which show up very distinctly in the hæmoglobin of the corpuscle. These features readily distinguish them from the sharply defined, clear, ​motionless, non-parasitic vacuole (Fig. 17). The other common forms—the larger or smaller intracorpuscular pigmented parasites—occupy anywhere from a sixth to nearly the entire area of the affected corpuscles. They are recognized by their pale protoplasm; by the black hæmozoin particles scattered about or, if towards the period of rigor, concentrated in their interior; and by their more or less active amœboid movements. In quartans and tertians, but especially in the former, segmenting rosette forms are seen occasionally.
Examination of blood for flagellated bodies.—When the student has become familiar with these appearances, and has thoroughly seized the fact that the segmenting forms are to be found only or usually during, just before, or soon after the rigor stage of fever, he should endeavour to follow up the initial steps of the exogenous, sexual or mosquito phase of the parasite. So far as ordinary preparations permit, this phase is best studied in the "zone of heaped-up corpuscles" and in the "zone of rouleaux"; because in these zones the parasite, not being subjected to pressure, has more freedom to undergo its evolutionary change into the flagellated body.
In ordinary quartans and tertians flagellated bodies are but seldom encountered. The best time to find them in such cases is said to be during the hot stage of the fever. In cases of crescent infection they are much more frequently met with, as, in this form of malaria, flagellated bodies are usually more numerous, appear at any time of the clinical cycle, and persist in the circulation perhaps for several weeks after fever has disappeared.
In most cases of crescent infection the gradual evolution of the flagellated body from crescent through oval and sphere can, with patience, be easily followed.
Diagnostic value of the "zone of free hœmoglobin."—The zone of free hæmoglobin is of value as enabling the practised observer to pronounce very rapidly on the presence or absence of pigmented parasites in the blood. The relatively large quantity of blood in each field of this zone, and therefore the ​proportionally large number of parasites in any given field, lends itself to this, as does the fact that the hæmozoin shows up very distinctly in the homogeneous sheet of free hæmoglobin.
Phagocytosis and pigmented leucocytes.—Striking examples of phagocytosis are often witnessed in malarial blood. So soon as a malarial parasite, whether spontaneously or as a result of pressure, escapes from the blood corpuscle in which it had developed, it becomes exceedingly liable to attack by the phagocytes. More especially is this the case with the flagellated organism; this body seems to have a powerful attraction for the phagocytes, which are often seen to travel long distances to attack it.
Pigmented leucocytes that is to say, leucocytes containing grains or blocks of hæmozoin are very often encountered; they can best be seen in the single-layer zone during, or shortly after, fever. Leucocytes may sometimes be observed to include the hæmozoin set free by the falling to pieces of the segmented parasites. Often they derive their hæmozoin from the remains of some sphere or flagellated body which they may have engulfed subsequently to the preparation of the slide. In peripheral blood the phagocytes are rarely, if ever, seen to attack the parasite so long as it is inside a blood corpuscle.
Both the large mono- and, very rarely, the poly-nucleated leucocytes may contain malarial pigment. Care, however, must be exercised in drawing conclusions from the discovery of black material in these bodies; in imperfectly cleaned slides, fragments of dirt, which the leucocytes rapidly take up, are apt to mislead.
Pigmented lymphocytes.—According to Metchnikoff, the lymphocyte has no phagocytic action in malaria. This observation I believe to be correct. Several writers, however, have described and figured what they regard as malarial pigment in the lymphocyte. This, I am convinced, is founded on an error in interpretation, and has arisen from ignorance of the fact that in all bloods, healthy and malarial alike, from 20 to 50 per cent, of the small mononucleated lymphocytes contain, lying in the narrow zone of ​cytoplasm, one or two minute round dots of intensely black material optically indistinguishable from hæmozoin. I am not aware that this appearance has been described hitherto, but multiplied observation has convinced me of the accuracy of my statement. The discovery, therefore, of what looks like a speck of intensely black pigment in the lymphocytes must not be regarded as evidence of malarial infection.
Diagnosis of vacuoles.—The beginner may have a difficulty in determining whether certain appearances in the corpuscles are vacuoles, or whether they are parasites. The following hints may help him to
Fig. 17.—Vacuolated and crenated blood-corpuscles. (After Laveran and Blanchard.)
a correct decision: Vacuoles (Fig. 17, a, b, c, d, e, f, h, i, j, k, l) are well defined, clear, and have sharp edges; they may change form slightly, but they have no true amœboid movement, carry no pigment, and, of course, do not stain. Intracorpuscular malaria parasites, on the contrary, are dim and, as a rule, ill defined; they have soft, shaded-off edges; possess amœboid movements; when large they carry hæmozoin grains; and, of course, they take the appropriate stains. It is hardly necessary to indicate the points of diagnosis from leucocytes, or from cupped, folded, or crenated (Fig. 17, c, f, g, i, j, k, l) corpuscles.
Moribund and fragmented parasites.—Moribund—it may be fragmented—free parasites (Fig. 18) are ​often a source of confusion to the beginner. Their nature is frequently misunderstood; they are sometimes erroneously termed "sterile bodies," an expression at one time frequently applied to the crescent-derived spheres (gametocytes). They are, in fact, mechanically-freed parasites expressed from blood corpuscles by the compression to which the blood is subjected between slip and cover-glass. The longer blood is on the slide—particularly if evaporation be not prevented by vaseline-ringing of the cover-glass—the more closely will the cover-glass approximate to the slip, the greater will be the pressure on, and consequent thinning and spreading out of, blood corpuscles and parasites, and the greater the liability to damage of these very delicate
Fig. 18.—Degenerating vacuolated plasmodia.
bodies. Frequently the artificially-freed parasites are broken into small fragments. The entire, as well as the fragmented parasites, on becoming free in the liquor sanguinis, tend to assume a spherical or disc-like form; at the same time the protoplasm of which they are composed seems to become diffluent, and the hæmozoin is resolved into a number of minute dust-like particles possessing active, brownian movement. Some of the spherical or disc-shaped bodies with dancing hæmozoin particles are really crescent-derived spheres or other forms of gametes. These are parasites which have escaped from corpuscles in a normal way, but which have become arrested in their evolution in consequence of the abnormal conditions in which they are placed in vitro; others are the remains of flagellated bodies, the microgamete filaments having broken away.
​The parasite as a means of diagnosis.—All of these multiform appearances the student must learn to recognize and interpret. Skill in this is merely a matter of time, practice, and reflection. Given these, the student should be able not only to diagnose by the microscope malarial infection, but also to recognize the type of any particular infection, the period of the fever cycle, and, it may be, the severity of the case. For diagnosis in malaria, therefore, skill in the microscopical examination of the blood is of the utmost value, and no pains should be spared by the practitioner in malarious countries to acquire it. In acute untreated malaria the parasite may be detected practically in every case. Thus in 616 cases Thayer and Hewetson—except in two or three instances where the patient's blood was examined only during convalescence—found it in every instance. The best authorities are equally emphatic on this point. Personally, I can assert that since I became familiar with the subject I have never failed to find the parasite in any acute untreated malarial case I have had a proper opportunity of examining. Whenever in a case of acute disease, supposed to be malarial, I have failed to do so, the case has turned out to be of quite another nature.
Bearing of quinine on microscopical diagnosis.—It is of little use to examine the blood for the intra-corporeal forms of the malaria parasite after full doses of quinine have been taken; the drug rapidly brings about the disappearance of this phase of the parasite. The crescent alone is unaffected by drugs, and in suitable cases may be found for days after the patient is cinchonized.
On staining malarial blood.—As a general rule, the beginner should work only with unstained preparations of fresh liquid blood. To the unpractised, staining is full of pitfalls. In such circumstances it must not be relied on for purposes of responsible diagnosis. To the experienced microscopist and diagnostician, and for the study of the morphology of the parasite, staining is of the highest value; moreover, some such method must be employed should ​permanent preparations be desired, or should it be inconvenient to make an immediate examination of the blood. Many methods have been devised; here only one or two can be given.
Preparing the film.—It is usual to spread the blood on cover-glasses; for many reasons I prefer to spread it on slips. There are various ways of preparing the films; the following I can recommend:—
Fig. 19.—A method for the preparation of blood films. (From the Malaria Number of the "Practitioner," March, 1901.)
However spread, after the blood has dried, whenever it is desired to stain it, either at once or at any future and more convenient time, it should be "fixed" by dropping on the film a little absolute alcohol and ether in equal parts, or absolute alcohol alone. Cover- glass films may be dropped into a small wide-mouth bottle containing the fixing agent, or they may be fixed by passing them through the flame (not a good method), or by placing them for some hours (one to three) in a warm dry chamber at a temperature of 105° to 120° C. When alcohol is used, after ten to thirty minutes or longer it must be dried off before staining.
Stains
The stains usually employed belong to two categories: (a) those which stain the protoplasm and the nucleus the same colour; (b) those which, by tinting them differently, differentiate the protoplasm from the nucleus.
(a) Of the former I would recommend the following:—
Löffler's methylene blue.—Concentrated alcoholic solution of methylene blue 30 parts, solution of caustic potash (1/1000) 100 parts. Stain for thirty seconds, wash in water, dry and mount.
Carbol-thionin.—Saturated alcoholic (60 per cent.) solution of thionin 20 parts, watery solution (2 per cent.) of carbolic acid 100 parts. The mixture should be kept at least a fortnight before use. Stain for five minutes, wash and mount.
(b) The many methods of obtaining differential staining of protoplasm and nucleus depend on the circumstance that, when solutions of certain kinds of methylene blue and eosin are mixed, a third red dye, with a special affinity for chromatin, is formed. They are all modifications of the original Romanowsky method. They are a little uncertain and troublesome to use, their success depending on the purity of the chemicals employed and careful attention to detail. The beginner is advised to become proficient, in the first instance, with the simpler methods given above. Having attained this, he should practise some form of the Romanowsky method, for, besides displaying the intimate structure of the malaria parasite, in consequence of the intense tinting of the nucleus which it effects it greatly facilitates the finding of the smaller forms of the parasite, not always an easy matter, especially in the case of the very minute young subtertian or malignant parasite. Of the three methods given below, Leishman's, everything considered, is the best. Both it and Jenner's method have the advantage of dispensing with preliminary fixing.
Jennets stain.—Special care must be taken that there is no trace of alkali or acid on the glass on which the film is spread; it must, therefore, be washed in distilled water and stood in absolute alcohol. The stain is made by dissolving Grübler's water-soluble eosin (yellow shade) and medicinal methylene blue in pure absolute methylic alcohol, which must be free from acetone and other impurities; the solutions are then mixed in the proportions of 125 parts of 1/2 per-cent. solution of the eosin, and 100 parts of 1/2 per-cent. solution of the methylene blue. Or the compound body formed by the eosin and methylene blue in this mixture may be purchased in the dry state and subsequently dissolved in absolute methylic alcohol. ​To use the stain, a few drops are poured on the blood film and covered to prevent evaporation and precipitation. In three minutes, or a little longer, the stain is rapidly poured off, and afterwards the film is washed in distilled water from five to ten seconds until it assumes a pink colour. It is then dried high over the flame, or in the air, and mounted in xylol balsam.
Leishman's stain.—Two solutions are prepared. A, a 1-per-cent. solution of medicinal methylene blue (Grübler) in distilled water, rendered alkaline by 0·5 per cent. sodium carbonate. Heat this to 65° C. for twelve hours, and allow it to stand at the room temperature for ten days. B, eosin, extra B.A. (Grübler), 1 in 1,000 of distilled water. Mix equal volumes of A and B and stand for six to twelve hours, stirring occasionally. Collect the flocculent precipitate in a filter, and wash with distilled water. Dry and powder the filtrate, which has a green metallic lustre and contains the active ingredient of the Romanowsky stain. (This powder has been placed on the market, and can be procured in condensed tablets called soloids.) Make a 0·15-per-cent. solution of the dye in methylic alcohol, and keep in a stoppered bottle.
To use the stain, drop three to four drops of the solution on the unfixed blood film. After about half a minute to one minute or longer add six to eight or more drops of distilled water and mix them by moving about the slide. A precipitate forms at once in the water in successful slides. After five minutes or longer wash off the stain with distilled water, leaving a few drops of the water on the film for over a minute. Dry without heat, and mount in xylol balsam.
Eosin-azur stain, sold by Burroughs and Wellcome in tabloid form, is an azur stain of somewhat similar composition to that of Leishman. One tabloid is dissolved in 10 c.c. of pure methyl alcohol and is used as above; it gives similar but more brilliant results. It may also be used as Giemsa's stain dissolved in 5 c.c. of a mixture of equal parts of glycerin and pure methyl alcohol.
Giemsa's stain.—Azur II. eosin 3·0 grm., azur II. 0·8 grm., dried, powdered, and dissolved in 250 grm. of glycerin, to which are subsequently added 250 grm. of methyl alcohol; heat to 40° C. Shake the mixture and stand for twenty-four hours, and then filter.
Fix the films in methyl alcohol (three minutes). The staining solution is prepared by adding 1 drop of stain to 1 c.c. of distilled water at 30° to 40°. Immerse the film in this for fifteen minutes, wash in stream of water, dry, and mount in balsam.
Laveran's method for staining and preserving permanent preparations. The following solutions are required:—
| Eosin, 1: 1,000 | . . . . | 4 | c.c. |
| Distilled water | . . . . | 10 | c.c.„ |
| Bleu borrel | . . . . . | 1 | c.c.„ |
Ross's thick-film method.—For the detection of small numbers of malaria parasites in the blood, and especially for the crescentic form, Ross recommends the adoption of thick films dehæmoglobinized in water. The parasites remain behind, clinging to the cell stroma. A drop of blood the size of a pin's head is dabbed on to a clean cover-glass so that an evenly spread smear results, about 5-7 mm. in diameter. After the dehæmoglobinized film has been dried in the air, it is stained in the ordinary way. The parasites are apt, however, to be greatly distorted by the action of the water.
Staining the flagellated body.—A sheet of thick blotting paper, having rows of oblong holes (1 in. by 2/3 in.) cut out in it, is prepared; it is slightly but sufficiently moistened with water, and laid smoothly on a sheet of window-glass.
A patient in whose blood the crescent form of the parasite abounds is selected. His finger is pricked and a droplet of blood, the size of a large pin's head, is expressed. A clean microscope slip is then breathed on once, and the droplet of blood immediately taken up by lightly touching it with the centre of the breathed-on surface of the slip. The blood is now rapidly and somewhat unevenly spread out with the needle so as to cover an area of about 3/4 in. by 1/2 in. The slip is immediately inverted over one of the blotting paper cells and pressed down sufficiently to secure thorough apposition of the slip to the paper, without, at the same time, bringing the blood into contact either with the moistened paper forming the wall, or with the glass forming the floor of what is now a very perfect moist chamber. The rest of the paper cells are rapidly covered with blood-charged slips prepared in the same way. In from a quarter to three-quarters of an hour the slips are removed and dried by gently warming them over the spirit-lamp blood surface away from the flame. When dry the films are fixed with absolute alcohol, a few drops being poured on each. After ten minutes the alcohol is dried off, and a few drops of weak acetic acid (10 to 20 per cent.) are laid on the films and left there long enough thoroughly to dissolve out all the hæmoglobin. The slides are then washed in water and dried. They may now be stained with various reagents. So far, I have obtained the best results from weak carbol-fuchsin (20 per
On examining with a one-twelfth immersion lens slides prepared with methylene blue, the nuclei of the white corpuscles are seen to be very deeply stained, the protoplasm of the white corpuscles is very lightly stained, whilst the parasites are stained an intermediate tint, and show up sharply enough in the faintly tinted red blood-corpuscles (Figs. 2, 16, 20). Contrast staining with eosin is uncertain in its results in methylene-blue preparations; even in practised hands good preparations are the exception. For ordinary purposes I do not recommend it; it is superfluous, troublesome, and unreliable.
On examining successful slides prepared by any of the Romanowsky methods, the red blood-cells will be found to be stained pale pink or greenish; the polynuclear leucocytes will show nuclear network ruby-red, the margins of the nuclei being sharply defined, whilst the protoplasm is unstained, except such fine eosinophil granules as it may contain, which are red; the mononuclears and lymphocytes have sharply defined ruby-red nuclei and faint blue protoplasm; the coarse-grained eosinophiles have a less deeply stained ruby-red nucleus and pale pink granules; the basophiles have dark purple-black granules and ruby-red nucleus; nucleated red cells have almost a black and sharply defined nucleus; the blood-plates are deep ruby-red with spiky margins and sometimes a pale blue peripheral zone. The body of the malarial parasite is stained blue and the chromatin of the nuclei ruby-red; and, in deeply stained preparations of the tertian parasite, the hæmoglobin of the including red blood-corpuscles will be dotted over with certain fine or coarse red granules known as Schüffner's dots.
​In preparations intended to display the flagellated body most of the slides will show numbers of spheres and several or many well-stained flagellated bodies (Figs. 3, 4, 7). Very few crescents remain untransformed. If the slips are removed and dried in from five to ten minutes after being placed on the blotting-paper cells, only crescents, ovals, and spheres will be found; if they are left for three-quarters of an hour to an hour, free microgametes and residual masses may also be found, the latter sometimes enclosed in phagocytes. Occasionally, flagellated bodies are found partially included in phagocytes. Staining with carbol-fuchsin, as above described, makes, when successful, beautiful preparations, but they do not differentiate the chromatin of the nuclear elements. To show this, some form of the Romanowsky method, preferably Leishman's or Giemsa's, must be employed.
Cultivation of the Malaria Parasite
The cultivation in vitro of the malaria parasite, long and unsuccessfully attempted, was at last (1911) effected by Bass, whose results have been fully confirmed. His method is, briefly, as follows: 10 c.c. of aseptically drawn blood from the median basilic vein of an untreated malaria patient are immediately transferred to a large test-tube containing 1/10 c.c. of a 50-per-cent. solution of Merck's dextrose, The tube is fitted with a cap through which runs a glass rod, and with this the blood is gently defibrinated. The mixture is distributed into culture tubes so as to form columns of about 2 in., and then incubated at 40° C. After a time it settles into three layers—an upper (1/2 in.) of serum, an intermediate (1/15 to 1/20 in.) of red and white corpuscles, and a bottom of red corpuscles. It is only in the red corpuscles of the very thin intermediate layer that the parasites grow and multiply. They are absent from the serous or top layer, and they die in the bottom layer. By drawing off with a fine pipette a little of the middle layer the progress of the culture can be watched and subcultures started. To succeed with the latter, ​however, the leucocytes have to be removed by centrifugation. Strict asepsis and the avoidance of airbubbles in drawing off and defibrinating the blood are indispensable. The sexual cycle has not been cultivated.
Important results are likely to accrue from Bass's discovery, particularly in the determination of species of parasite, in their morphology, in diagnosis, in the action of quinine, and also in settling disputed points in regard to the latency of malarial infections.
* ↑ Bleu borrel is methylene blue and silver oxide. It is prepared as follows: In a 150-c.c. glass measure several crystals of nitrate of silver are placed, and the measure is then filled with a concentrated solution of soda. The black precipitate of silver oxide is washed several times in distilled water, then a saturated solution of methylene blue (Höchst) is poured into the oxide and left in contact for fifteen days.
* ↑ When it is not intended to preserve the slides a cover-glass may be dispensed with, and the immersion lens used with only the cedar oil between it and the film. For purposes of diagnosis this suffices, and much time and material are saved. Indeed, stained films retain their colour longest if balsam and cover-glass are not applied.