pneumonia, and the micrococcus in gonorrhoeal discharge-the staining is best carried out with a mixture of methyl-blue and vesuvin. ing out on a glass slide or cover-glass a very the micrococcus in the sputum of acute croupous thin film-the thinner the better-of the fluid (artificial or natural culture medium), blood, pus, or juice, and drying it rapidly by passing it several times over the flame of a spirit-lamp Weigert's double stain is very excellent for or gas-burner. The most successful prepara- many purposes; it is prepared thus: Saturated tions are obtained when the heating is carried watery solution of aniline, 100 ccm. This is on for such a time (one or two minutes) that made thus: Aniline oil, I part, dist. water, 3 the film, having become opaque at first, rapidly parts. Shake every half hour for four hours, turns transparent. Several drops of the ani- and decant the water as the oil settles to the line dye to be used are then poured over the bottom. Saturated alcoholic solution of fuchsin, specimen, and after remaining on it from five 11 ccm. Mix. The sections are well stained to 30 minutes or more are poured off. The in this mixture, then washed in distilled water; aniline dyes used are those that are commonly after this they are immersed for a few seconds known as having a great affinity for cell- in alcohol, and then transferred for one, two, nuclei (Hermann); they are also known as the or three hours to the following solution: Disneutral or basic, but not the acid ones. Methyl- tilled water, 100 ccm. Saturated alcoholic blue, methyl-violet, vesuvin, Bismarck-brown, solution of methyl-blue, 20 ccm. Formic acid, magenta, fuchsin, rosanilin, gentian-violet, 10 minims. After this, wash in alcohol, pass Spiller's purple, eosin, dahlia, purpurin, iodine- through clove oil, and mount in Canada-balsam green, are the aniline dyes commonly used. solution. The washing is carried out in all cases, except with tubercle-bacilli and the bacilli of glanders, with distilled water, then with alcohol; this latter as a rule is to wash out the dye from all parts except the micro-organisms, and it is therefore necessary not to carry the washing further than this, but to carry it as far as practicable. After this, wash again with distilled water, dry, and mount in a drop of Canadabalsam solution. Double staining is carried out with any two of the above dyes; as a rule a brown and blue or violet, or a red and blue, are preferred. Some of the violet and purple dyes have the peculiarity that in some-not in all instances they give to the preparation a double tint-some things appear blue, others more pink. The process of double staining is carried out either for each dye separately-i.e. we first apply one dye, after some minutes wash in distilled water, and then apply the second dye -or the two dyes to be used are mixed and then used like a single dye. In the case of tubercle-bacilli the staining is first done with a magenta mixture, then washed for a few seconds with a 10% solution of nitric acid, then for a few minutes with distilled water. After this the preparation is stained with methyl-blue in the ordinary way. Or, after Koch's method, the specimen is first stained in alkaline methyl-blue (mixed with a 10% solution of caustic potash) for 24 hours, dried for half an hour to one hour at 40° C., and then stained in a concentrated solution of vesuvin. Wash it next with water, then with alcohol, dry, and mount in Canada-balsam solution. In examining fresh or hardened tissues for micro-organisms it is necessary to make thin sections, which can be easily done with the aid of any of the microtomes in common use, amongst which Williams' microtome, and especially Dr. Roy's ether-spray freezing microtome, are no doubt the best and easiest to manipulate. As regards hardened material, it is necessary to remember that the hardening must be carried out properly, small bits-about a half to one cubic inch-of tissue being placed in alcohol, or better in Müller's fluid, and kept there; in the first instance, for two to five days; in the second, for from one to three weeks or more. Then small bits are cut out, of which it is desired to make sections. Those hardened in spirit must be soaked well in water to enable the material to freeze, then superficially dried with blotting-paper, and then used for cutting sections with Roy's microtome. Those hardened in Müller's fluid are at once superficially dried with blottingpaper and cut. When making sections with Williams' freezing microtome it is necessary to soak the material first in gum mucilage and then to freeze and to cut. Fresh tissues are at once cut with the freezing microtome, the sections placed in o'6% of saline solution, floated out and well spread out, and then stained by transferring them in this condition, i.e. well spread out, into a watch-glass containing the dye. The sections of hardened tissues are floated out in water, well spread out, and then transferred to the dye or dyes. It is necessary to prevent too much shrinking of the sections, especially those of fresh In leprosy, the specimen on the glass is tissues; for this reason it is advisable to float stained with magenta, then washed in distilled the sections in the saline solution or water, as water, then stained with methyl-blue, washed, the case may be, on a broad lifter or spatula, and mounted. With such organisms—as e.g. to spread them well out upon it, and to transfer A. Page's regulator.-This consists of a tube filled with mercury, and immersed in the water surrounding the chamber of the incubator. In the upper part of the tube, above the mercurial column, is a fine open glass tube, having near the lower end a fine hole. When the temperature of the water rises, the mercurial column rises, and at a certain temperature rises above the lower open end of the small inner glass tube just mentioned. If this point is reached, then the burner at C receives only the amount of gas that passes through the fine lateral hole of that inner glass tube. If the temperature of the water falls, the mercury falls, and the lower end of the inner glass tube becomes again free, and now the burner at C receives a much greater supply of gas. If so, the temperature of the water again rises, the mercury rises, obstructs the lower end of the inner glass tube, the supply of gas is reduced to what can pass through the fine lateral hole, and consequently the temperature again falls, and so on. To adjust the regulator it is necessary when the thermometer indicates the required degree of temperature to push the outer large glass tube-and with it the inner tube-of the regulator so far down that the top of the mercurial column just obstructs the free end of the inner glass. The temperature then regulates itself for the reasons stated previously. These regulators are sufficient for all practical purposes when it is not a question of small differences in temperature, since they are tolerably constant within one or two centigrades. The trouble one experiences in the working of these and other similar regulators arises from the inconstancy of the main gas supply, this, as is well known, varying within wide limits. The stopcock, E, obviates this to a limited extent; when this is put at an angle of 45° only a limited amount of gas passes from the main supply tube to the regulator, and therefore the variations in pressure of gas are not felt to their full extent. A Sugg's regulator interposed between E and the main supply tap is very useful. B. Thermometer to indicate the temperature in the chamber. C. Gas burner. D. Chamber of incubator. E. Stopcock to regulate when required the supply of gas. F. Main supply. The upper, lower, right and left walls of the incubator are made of a double layer of tin; between the two is water. The front and back of the chamber are closed by a movable glass plate. them carefully into the dye, then into the dish with water used for washing off the excess of the dye, to transfer them, well spread out on the lifter, to alcohol, then after several minutes to oil of cloves, and finally on to a glass slide, on which they are mounted in the usual manner with Canada-balsam solution, the excess of clove-oil being previously drained off. have been sterile, twice boiling is found sufficient to destroy every impurity. But to make sure, the broth is placed in the incubator and kept there for 24 hours at 32°-38° C., and then boiled on the next day for half an hour in the usual way. The supposition is made that if by any chance after twice boiling the broth it should contain unchanged spores of bacilli PREPARATION OF CULTURE MATERIAL. Arti--the only organisms that will resist boiling. ficial cultivations of micro-organisms in suitable although they do not resist boiling for more nourishing media in the incubator (Fig. 1) at temps. varying between 30° and 40° C., are necessary in order to study more accurately the life-history of the septic as well as the pathogenic organisms. Moreover, large numbers of them become thereby available, and their exact relation to disease can be thus be tested more conveniently. ARTIFICIAL CULTIVATION MEDIA. A. Fluids. 1. Broth made from meat-pork, beef, rabbit, chicken. The connective tissue and fat are first cut out from the fresh meat-in the case of rabbit or chicken the whole animal without head or viscera is used-and then placed in water and boiled. Generally for each pound half an hour's good boiling is allowed. Each pound of meat ought to yield ultimately at least one pint of broth. When boiled, the broth is allowed to stand, the fat is skimmed off, and the broth well neutralised by adding carbonate of sodium. The fresher the meat the less acid (sarcolactic acid) is in the broth before neutralisation. The broth is then filtered through a filter, previously overheated into flasks previously sterilised. If the broth is not clear after once filtering it is filtered again. If not clear then, it is allowed to stand for several hours. A fine sediment is found at the bottom of the vessel, and from this the clear supernatant fluid is decanted into a. sterilised vessel. The broth, if not clear after the first filtering, can be cleared by boiling it with the white of an egg. The now clear fluid is filtered again. The flasks which receive the broth are well plugged with sterilised cotton-wool. In this state the flask is placed over a Bunsen burner (Fig. 2) on a wire netting and boiled for half an hour or more; during the boiling the cotton-wool plug is lifted out for half its length. The flask ought not to contain more broth than about 1⁄2 or 2/3 of its volume, to prevent the broth from rising too much and wetting the plug. When turning off the flame the plug is pushed down so as fully to plug the neck and mouth of the flask; a beaker with sterile cotton-wool cap is placed over the mouth of the flask (Fig. 3), and this is allowed to stand for one night. Next day the boiling is repeated for half an hour or more in the same manner as before. If the meat has been fresh and the vessels and cotton-woo I FIG. 2.-A Bunsen Burner with Rose for Boiling than half an hour-the spores would germinate 2. Peptone and Sugar Solution. Beef peptone (Savory and Moore's) is dissolved in distilled water, over a burner, to the amount of about 2%; to the solution is added cane sugar about 1%, so that every 100 ccm. of the fluid contains two grammes of peptone and one gramme of sugar. When dissolved it is well neutralised and then filtered (the vessels being of course also in this, as in all other cases, sterilised by overheating) into flasks and treated in the same manner as the broth. The same fluid can be used without addition of the canesugar. 3. Buchner's Fluid. 10 parts of Liebig's extract, and 8 parts of peptone, in 1,000 parts of water. B. Solids. The solid media have the great advantage over the fluids that in the former artificial cultures can be carried out more easily; as, owing to the resistance the solid basis offers to the growth of the organisms, they remain more limited to the spot or spots on which they are sown, and therefore can be watched more easily; besides, an accidental contamination, i.e. a growth appearing at a spot at which no sowing was made, can be recognised at once. These advantages are perhaps of the greatest use when it is intended to grow the organisms on a surface exposed to the influence of air-of course protected from contamination with other organisms. 4. Hydrocele Fluid (Koch). A new or well 1. Slices of Boiled Potato or Boiled White of sterilised (by overheating) trocar and cannula Egg or Paste (Fokker, Schröter, Cohn, Werare used for the tapping; to the cannula is nich). Although these are of great use in the fixed an indiarubber tube that has been soak- study of hyphomycetes, and especially of piging in strong carbolic acid solution for 48 ment-bacteria, they are not generally used in hours. The distal end of the tube is intro- the case of other bacteria and pathogenic orduced carefully and rapidly into the neck of a ganisms. The progress of the growth of a sterilised flask plugged with sterile cotton-particular organism sown out at a particular wool, and the fluid thus allowed to flow into the flask to about 2% of its volume. This is then exposed in a water- or sand-bath to 58° 62° C. for three to five hours on five or six consecutive days. Placed then into the incubator at 32-38° C. for from one to three weeks, the fluid remains limpid. 5. Blood Serum (Koch). A glass cannula and indiarubber tubing are soaked for 48 hours in strong carbolic acid; the cannula is tied into the carotid artery of a healthy sheep, and the arterial blood, after opening the clip at the proximal end of the artery, is allowed to flow into a sterile flask, the distal end having been introduced into the neck of the flask as above. After letting it stand for 12 to 24 hours the clear serum is taken off by means of a large sterilised glass pipette or glass syphon; this is carefully introduced between the cotton-wool plug and glass neck, and then discharged into a sterile plugged flask or large test-tube, the pipette or syphon being also here carefully introduced between cotton-wool plug and glass. This stock serum is then heated for successive days in the same manner as the hydrocele fluid. Of less common use are: 6. Pasteur's Fluid. In 100 parts of distilled water are dissolved 10 parts of pure cane-sugar, I part ammonium tartrate, and the ash of I part of yeast. 7. Cohn's Fluid. 100 ccm. of distilled water, I gramme of ammonium tartrate, no sugar, and instead of the ash of yeast are substituted (A. Mayer) o'5 gramme of potassium phosphate, 0'5 gramme of crystallised magnesium sulphate, 0.5 gramme of (tribasic) calcium phosphate. These two fluids are treated in the same manner as the broth and · peptone solutions. spot or line on the surface of these substances can be easily watched with the unaided eye. These substances when quite fresh are placed on flat glass dishes; these are covered with a well-fitting bell-glass, the space being kept moist by a piece of moist blotting-paper placed in the dish. 2. Gelatine (Brefeld, Grawitz, Koch). This is used advantageously as a mixture with broth, peptone, beef-extract, blood-serum, or hydrocele fluid. Koch, who introduced this mixture, used it for the cultivation of bacteria on solids, to be exposed to the air; the proportion of gelatine in the mixture was 2 to 3%. But this mixture, although solid at ordinary temp., does not keep solid in the incubator, not even at 20° C. I have found that at least 75% of gelatine must be contained in the mixture to keep it solid at 20° to 25° C. Above this not even 11% gelatine will keep solid. The finest (gold label) gelatine, in thin tablets, is cut up in small strips; these are soaked in distilled water (1 in 6) overnight, and then dissolved over a water-bath, well neutralised with carbonate of sodium, and filtered hot. If not clear, it is boiled with white of egg, and passed hot through sterilised fine calico. Then this fluid is mixed with half its bulk of broth, peptone solution, or beef-extract solution, so that there is I part of gelatine in 9 parts of fluid, or II% of gelatine. This mixture is boiled repeatedly and treated like broth, as described above. The mixture can, when cast solid, be liquefied by melting it on the water-bath, can be easily decanted into sterilised plugged test-tubes, and can then be used as a good solid nourishing material for the cultivation of organisms up to 25° C. The above gelatine solution without admixture can be boiled once or twice, and thus made sterile and kept as a stock. This can be used as an addition to blood-serum or hydrocele fluid; the mixture must be sterilised in the same way as serum or hydrocele fluid alone, i.e. exposed for five to seven days to 58° 62° C. Of course, whatever the proportions are in which the two are mixed, the mixture does not keep solid above 25° C. But by exposing it for from several days to several weeks to the heat of the incubator, the mixture can by evaporation be rendered practically solid for higher temps. also. 3. More satisfactory, because capable of remaining solid at any temp., is solid serum of blood, solid hydrocele fluid, and Agar-Agar (Koch). The serum of blood, and the hydrocele fluid, can be made solid by heating the above sterile serum or hydrocele fluid respectively (see page 175) gradually up to 68°-70° C. In the course of an hour or two the material becomes solid, losing very slightly its limpidity, but is sufficiently transparent for all practical purposes. By heating it rapidly, or heating it above 70°, it becomes solid, granular, and opaque. Of course, once thus made solid it cannot be liquefied again, and therefore must be already contained in the vessels (test-tubes and small flasks) in which the growth of organisms is to be carried on. Or blood serum and hydrocele fluid can be rendered solid by exposing the sterilised material, in sterile plugged test-tubes, to a moderate heat-e.g. in the incubator at 32° to 38° C.-for several weeks. Through evaporation the material is rendered solid. Thus treated it retains its limpidity in a perfect manner. quite limpid after this time it may safely be considered as sterile. Amongst all the solid media, I have found this mixture of Agar-Agar and peptone sugar solution to be the best in many respects. It is beautifully limpid and solid, and an excellent nourishing material. Agar-Agar alone, as recommended by Koch, without admixture of peptone and sugar is not so satisfactory as a culture medium. VESSELS AND INSTRUMENTS USED IN CULTIVATIONS. All vessels (flasks, test-tubes, beakers, filters, calico), to be used are first thoroughly sterilised by overheating. In the case of flasks and test-tubes, this can be done by exposing them thoroughly in all parts to the open flame of a large Fletcher's burner; while thoroughly heated the mouth is plugged with a good long plug (1 to 2 inches) of sterile cotton-wool, this being pushed in by means of overheated forceps. The plug in all cases must not be loose, but also not too firm-an error in the latter direction being of course preferable to one in the former. The cotton-wool plug may, if long enough, be single; or, if short ones be used, double. Or the flasks and test-tubes are placed in an air chamber (see Fig. 4) heated by a large Fletcher's burner for several hours, up to between 130° and 150° C. In the case of small flasks and test-tubes this process is of course much more convenient, since a large number can be heated simultaneously. Beakers and glass filters to be used merely for a temporary operation are placed over a wire net on a tripod and heated by the flame of a Bunsen's burner. In the case of test-tubes which are to receive cultivation-fluids, I generally expose them, after having been cleaned and dried, in the air-chamber for several hours (three to six) to 130°-150° C.: while hot they are taken out seriatim, plugged with the sterile cotton-wool, and replaced in the air-chamber, and heated again for several hours. All this, and other operations to be described, may appear complicated, but in these matters one cannot be too scrupulous. Long experience in these matters has taught me that, although in some instances less scrupulous care has not been followed by bad results, still I have had also many unpleasant failures, owing to slight laxity in these matters. Several weeks' work may be annihilated by a single omission. Sometimes one is perhaps in a slight hurry, and does not think the want of an additional heating of the test-tube or cotton-wool or an additional boiling of the fluid will be followed by any bad consequences. But nature does not take into account our convenience, and failure is our reward. Agar-Agar, or Japan isinglass is very difficult to obtain, [McKesson & Robbins, New York, we are informed, keep a supply] it is sold in the shape of very thin, shrivelled, transparent lamellæ, or narrow bands. It is soaked over night in distilled water (one to five or six), and then dissolved on the water-bath; well neutralised with carbonate of sodium, and filtered and mixed with a third of its bulk of broth, peptone, or beef-extract solution. I use as a rule peptone and sugar solution, as described above. Well boiled on two or three successive days, each time for 30 minutes to an hour, in sterile flasks, a sterile material is obtained, which is quite transparent, and remains solid up to 45°-50° C., i.e. a temp. much higher than is ever used for the cultivation of micro-organisms. It becomes liquid at higher temps., and in case of necessity can be again subjected to boiling. Before considering it as perfectly sterile it ought to be kept like all The cotton-wool used for plugging flasks and other materials for from several days to several test-tubes is prepared by pulling up loosely a weeks in the incubator at 32°-38° C. If quantity of good cotton-wool and exposing it |