At the beginning of the bacteriological work the water should be examined by means of the gelatine plate method. This consists in drawing up into a fine sterilised pipette a small quantity of the water and introducing it thereby into a test-tube of melted gelatine at a temperature below 40° C.13 It will depend upon the apparent quality of the water as to the exact quantity introduced into the gelatine; about .5 or .1 of a cubic centimetre is a common figure. The stopper is then quickly replaced in the test-tube, and the contents gently mixed more or less equally to distribute the one-tenth cubic centimetre throughout the melted gelatine. A sterilised sheet of glass (4 inches by 3) designated a Koch's plate is now taken and placed upon the stage of a levelling apparatus, which holds iced water in a glass jar under the stage. The gelatine is now poured out over the glass plate, and by means of a sterilised rod stroked into a thin, even film all over the glass. It is then covered with a bell-jar and left at rest to set. The level stage prevents the gelatine running over the edge of the plate; the iced water under the stage expedites the setting of the gelatine into a fixed film. When it is thus set the plate is placed upon a small stand in a moist chamber, and the whole apparatus removed to the room temperature incubator. A moist chamber is a glass dish, in which some filter paper, soaked with corrosive sublimate, is inserted, and the dish covered with a bell-jar. By this means the risks of pollution are minimized, and moisture maintained. In all cases at least two plates must be prepared of the same sample of water, and it is often advisable to make several. They may be made with different media for different purposes, and with different quantities of water, though the same method of procedure is adopted. In a highly polluted water extremely small quantities would be taken, and, vice versâ, in pure water a large quantity.

When we come to discuss the relation of disease organisms to water, particularly those causing typhoid fever, we shall learn that they are both scarce and intermittent. This point has been dwelt upon frequently by Dr. Klein, and it is clear that such a state of things greatly enhances the difficulties in detecting such bacteria, and he has proposed a simple procedure by which the difficulty of finding the Bacillus typhosus in a large body of water may be met.

One or two thousand cubic centimetres of the water under examination are passed through a sterilised Berkefeld filter by means of siphon action or an air-pump. The candle of the filter retains on its outer surface all, or nearly all, the particulate matter contained in the water. The matter thus retained on this outer surface is brushed by means of a sterile brush into 10 or 20 cc. of sterilised water. Thus we have all the organisms contained in two litres of the water reduced into 10 cc. of water. From this, so to speak, concentrated emulsion of the bacteria of the original water, phenol-gelatine plates or Eisner plates (both acid media) may be readily made. In this way we not only catch many bacteria which would evade us if we were content with the examination merely of a few drops of the water, but we eliminate by means of the acid those common water bacteria, like Bacillus fluorescens liquefaciens, which so greatly confuse the issue.

In the course of two or three days the film of gelatine on the plate becomes covered with colonies of germs, and the next step is to examine these quantitatively and qualitatively. We may here insert a simple scheme by which this may be most fully and easily accomplished:—

1. Naked-Eye Observation of the Colonies. By this means at the very outset certain facts may be obtained, viz., the size, elevation, configuration, margin, colour, grouping, number, and kinds of colonies, all of which facts are of importance, and assist in final diagnosis. Moreover, in the case of gelatine plates (it is otherwise in agar) one is able to observe whether or not there is present what is termed liquefaction of the gelatine. Some organisms produce in their development a peptonizing ferment which breaks down gelatine into a fluid condition. Many have not this power, and hence the characteristic is used as a diagnostic feature.

2. Microscopic Examination of Colonies, which confirms or corrects that which has been observed by the naked eye. Fortunately some micro-organisms when growing in colonies produce cultivation features which are peculiar to themselves (especially is this so when growing in test-tube cultures), and in the early stages of such growths a low power of the microscope or magnifying glass facilitates observation.

3. Make cover-glass preparations: (a) unstained—"the hanging drop"; (b) stained—single stains, like gentian-violet, methyl blue, fuchsin, carbol fuchsin, etc.; double stains—Gram's method, Ziehl-Neelsen's method, etc.

This third part of the investigation is obviously to prepare specimens for the microscope. "The hanging drop" is a simple plan for securing the organisms for microscopic examination in a more or less natural condition. A hollow ground slide, which is a slide with a shallow depression in it, is taken, and a small ring of vaseline placed round the edge of the depression. Upon the under side of a clean cover-glass is placed a drop of pure water, and this is inoculated with the smallest possible particle taken from one of the colonies of the gelatine plate on the end of a sterilised platinum wire. The cover-glass is then placed upon the ring of vaseline, and the drop hangs into the space of the depression. Thus is obtained a view of the organisms in a freely moving condition, if they happen to be motile bacteria. As a matter of practice the hollow slide may be dispensed with, and an ordinary slide used.

With regard to staining, it will be undesirable here to dwell at length upon the large number of methods which have been adopted. The "single stain" may be shortly mentioned. It is as follows: A clean cover-glass is taken (cleaned with nitric acid and alcohol, or bichromate of potash and alcohol), and a drop of pure sterilised water placed upon it. This is inoculated with the particle of a colony on the end of a platinum needle, and a scum is produced. The film is now "fixed" by slowly drying it over a flame. When the scum is thus dried, a drop of the selected stain (say gentian-violet) is placed over the scum and allowed to remain for varying periods: sarcinæ about thirty seconds; for many of the bacilli three or four minutes. It is then washed off with clean water, dried, and mounted in Canada balsam. The organisms will now appear under the microscope as violet in colour, and will thus be clearly seen.

The "double staining" is adopted when we desire to stain the organisms one colour and the tissue in which they are situated a contrast colour. Some of the details of these methods are mentioned in the Appendix.

4. Sub-culture. The plate method was really introduced by Koch in order to facilitate isolation of species. In a flask it is impossible to isolate individual species, but when the growth is spread over a comparatively large area, like a plate, it is possible to separate the colonies, and this being done by means of a platinum wire, the colonies may be replanted in fresh media; that is to say, a sub-culture may be made, each organism cultivated on its favourite soil, and its manner of life closely watched. We have already mentioned the chief media which are used in the laboratory, and in an investigation many of these would be used, and thus pure cultures would be obtained. Let us suppose that a water contains six kinds of bacteria. On the plate these six kinds would show themselves by their own peculiar growth. Each would then be isolated and placed in a separate tube, on a favourite medium, and at a suitable temperature. Thus each would be a pure culture; i. e., one and only one, species would be present in each of the six tubes. By this simple means an organism can be, we say, cultivated, in the same sort of way as in floriculture. From day to day we can observe the habits of each of our six species, and probably at an early stage of their separated existences we should be able to diagnose what species of bacteria we had found in the water. If not, further microscopic examination could be made, and, if necessary, secondary or tertiary sub-cultures.

5. Inoculation of Animals. It may be necessary to observe the action of supposed pathogenic organisms upon animals. This is obviously a last resource, and any abuse of such a process is strictly limited by law. As a matter of fact, an immense amount of bacteriological investigation can be carried on without inoculating animals; but, strictly speaking, as regards many of the pathogenic bacteria, this test is the most reliable of all. Nor would any responsible bacteriologist be justified in certifying a water as healthy for consumption by a large community if he was in doubt as to the disease-producing action of certain contained organisms.

By working through some such scheme as the above we are able to detect what quantity and species of organisms, saprophytic or parasitic, a water or similar fluid contains. For, observe what information we have gained. We have learned the form (whether bacillus, micrococcus, or spirillum), size, consistence, motility, method of grouping, and staining reactions of each micro-organism; the characters of its culture, colour, composition, presence or absence of liquefication or gas formation, its rate of growth, smell, or reaction; and lastly, when necessary, the effect that it has upon living tissues. Here, then, are ample data for arriving at a satisfactory conclusion respecting the qualitative estimation of the suspected water.

As to to the quantitative examination, that is fulfilled by counting the number of colonies which appear, say by the third and fourth day, upon the gelatine plates. Each colony has arisen, it is assumed, from one individual, so that if we count the colonies, though we do not thereby know how many organisms we have upon our plate, we do know approximately how many organisms there were when the plate was first poured out, which are the figures we require, and which can at once be multiplied and returned as so many organisms per cubic centimetre. There is, unfortunately, at present no exact standard to which all bacteriologists may refer.

Miquel and Crookshank have suggested standards which allow "very pure water" to contain up to 100 micro-organisms per cc. Pure water must not contain more than 1000, and water containing up to 100,000 bacteria per cc. is contaminated with surface water or sewage. Macé gives the following table:

Koch first laid emphasis on the quantity of bacteria present as an index of pollution, and whilst different authorities have all agreed that there is a necessary quantitative limit, it has been so far impossible to arrive at one settled standard of permissible impurity.

Besson adopts the standard suggested by Miquel, and, on the whole, French bacteriologists follow suit. They also agree with him, generally speaking, in not placing much emphasis upon the numerical estimation of bacteria in water. In Germany and England it is the custom to adopt a stricter limit. Koch in 1893 fixed 100 bacteria per cc. as the maximum number of bacteria which should be present in a properly filtered water. Hence the following has been recognised more or less as the standard:

The personal view of the writer after some experience of water examination would favour a standard of "under 500" being a potable water, if the 500 were of a nature indicating neither sewage pollution nor disease. Miquel holds that not more than ten different species of bacteria should be present in a drinking water, and such is a useful standard. The presence of rapidly liquefying bacteria associated with sewage or surface pollution would, even though present in fewer numbers than a standard, condemn a water. Thus it will be seen that it is impossible to judge alone by the numbers unless they are obviously enormously high.

When we are counting colonies upon a Koch's plate, Wolfhügel's counter may be used. This is a thin plate of glass a size larger than Koch's plates, and upon it are scratched squares, each square being divided into nine smaller squares. The Wolfhügel plate is superimposed upon the Koch's plate, and the colonies counted in one little square or set of squares and multiplied.

By using flat, shallow, circular glass dishes, generally known as Petri's dishes, instead of Koch's plates, much manipulation and time is saved, and, on the whole, less risk of pollution occurs. Moreover, these are easily carried about and transferred from place to place. When counting colonies in a Petri's dish it is sufficient to divide the circle into eight equal divisions, and counting the colonies in the average divisions, multiply and reduce to the common denominator of one cc. For example, if the colonies of the plate appear to be distributed fairly uniformly we count those in one of the divisions. They reach, we will suppose, the figure of 60; 60 × 8=480 micro-organisms in the amount taken from the suspected water and added to the melted gelatine from which the plate was made. This amount was .25 cc. Therefore we estimate the number of micro-organisms in the suspected water as 60 × 8=480 × 4= 1920 m.-o. per cc., which is over standard by about 1500. A water might then be condemned upon its quantitative examination alone or qualitative alone, or both. If the quantity were even that of an artesian well, say 4–10 m.-o. per cc., but those four or ten were all Bacillus typhosus, it would clearly be condemned on its quality, though quantitatively it was an almost pure water. If, on the contrary, the water contained 10,000 m.-o. per cc., and none of them disease-producing, it would still be condemned on the ground that so large a number of organisms indicated some kind of organic pollution to supply pabulum for so many organisms to live in one cc. of the water. It is not the number per se which condemns. The large number condemns because it indicates probable pollution with surface water or sewage in order to supply pabulum for so many bacteria per cc.

It should always be remembered that a chemical report and a bacteriological report should assist each other. The former is able to tell us the quantity of salts and condition of the organic matter present; the latter the number and quality of micro-organisms. Neither can take the place of the other and, generally speaking, both are more or less useless until we can learn, by inspection and investigation of the source of the water, the origin of the organic matter or contamination. Hence a water report should contain not only a record of physical characters, of chemical constituents, and of the presence or absence of micro-organisms, injurious and otherwise, but it should also contain information obtained by personal investigation of the source. Only thus can a reasonable opinion be expected. Moreover, it is generally only possible to form an accurate judgment of a water from watching its history, that is, not from one examination only, but from a series of observations. A water yielding a steady standard of bacterial contents is a much more satisfactory water, from every point of view, than one which is unstable, one month possessing 500 bacteria per cc. and another month 5000. It is obvious that rainfall and drought, soil and trade effluents, will have their influence in materially affecting the bacterial condition of a water.

It is perhaps scarcely necessary to add that we have not in the above account of the examination of water included all, or nearly all, the various methods adopted for acquiring a knowledge of the bacterial contents of the water. Many of these are of too detailed and technical a nature to enter into here. Three points, however, we may touch upon. In the first place, as we have said, the particulate matter out of a large body of water should be concentrated in a small quantity. Accordingly it has become the custom to pass 2000 or 3000 cc. of the suspected water through a Berkefeld filter. When this has been accomplished, by means of a Berkefeld Filter Berkefeld Filter
In Position for Filtration of Water to be Examined. sterile brush the particulate matter on the candle of the filter is brushed off into 10 or 15 cc. of sterilised water. This simple arrangement is analogous to the use of gravity or centrifugal methods of securing the solid matter in milk. The smaller quantity of water is then readily examined, and scanty germs more readily detected. A second point elaborating the scheme of water examination is the choice of media for sub-culturing. Mere examination on gelatine is not sufficient. Even in making the primary plate cultivations it is well to vary the media—agar, carbol-gelatine, Elsner, etc. But when colonies have appeared upon these plates it is important to sub-culture with accuracy and good judgment upon all or any media—gelatine, agar, broth, potato, milk, blood serum, glucose agar, glycerine agar, etc.—that will reveal the real characters of the bacteria present. A method proposed by Professor Sheridan Delépine is to place some of the suspected water in sterilised test-tubes without further treatment, and incubate at 37° C. for twelve or eighteen hours, and then plate out and estimate the number of bacteria as in the ordinary course. "In polluted water, containing an excess of organic matter," he says, "an extremely rapid multiplication of bacteria is observed. In unpolluted water, containing only water bacteria and a very small amount of organic matter, very little or no multiplication takes place, and the growth of the water bacteria liquefying gelatine is checked to a remarkable extent." Thirdly, by none of these methods should we be able to isolate anaërobic bacteria, and to furnish a complete report these also must receive careful attention.

The Bacteriology of Water. In many natural waters there will be found varied contents even in regard to flora alone: algæ, diatoms, spirogyræ, desmids, and all sorts of vegetable detritus. Many of these organisms are held responsible for divers disagreeable tastes and odours. The colour of a water may also be due to similar causes. Dr. Garrett, of Cheltenham, has recorded the occurrence of redness of water owing to a growth of Crenothrix polyspora, and many other similar cases make it evident that not unfrequently great changes may be produced in water by contained microscopic vegetation.

With the exception of water from springs and deep wells, all unfiltered natural waters contain numbers of bacteria. The actual number roughly depends upon the amount of organic pabulum present, and upon certain physical conditions of the water. As we have already seen, bacteria multiply with enormous rapidity. In some species multiplication does not appear to depend on the presence of much organic matter, and, indeed, some can live and multiply in sterilised water: Micrococcus aquatilis and Bacillus erythrosporus. Again, others depend not upon the quantity of organic matter, but upon its quality. And frequently in a water of a high degree of organic pollution it will be found that bacteria have been restrained in their development by the competition of other species monopolising the pabulum. Probably at least one hundred different species of non-pathogenic organisms have been isolated from water. Some species are constantly occurring, and are present in almost all natural waters. Amongst such are B. liquefaciens, B. fluorescens liq., B. fluorescens non-liquefaciens, B. termo, B. aquatilis, B. ubiquitus, and not a few micrococci, etc. Percy Frankland14 collected water from various quarters at various times and seasons, and some of his results may here be added:

Again, another example:

"During the summer months these waters are purest as regards micro-organisms, this being due to the fact that during dry weather these rivers are mainly composed of spring water, whilst at other seasons they receive the washings of much cultivated land."—Frankland.

Prausnitz has shown that water differs, as would be expected, according to the locality in the stream at which examination is made. His investigations were made from the river Isar before and after it receives the drainage of Munich:

Professor Percy Frankland also points out how the river Dee affords another example, even more perfect, of pollution and restoration repeated several times until the river becomes almost bacterially pure.

We cannot here enter more fully into the many conditions of a water which affect its bacterial content than to say that it varies considerably with its source, at different seasons, and under different climatic conditions. An enormous increase will occur if the sediment is disturbed, and conversely sedimentation and subsidence during storage will greatly diminish the numbers of bacteria. Sand filtration, plus a "nitrifying layer," will remove more than 90 per cent. of the bacteria. Sea-water contains comparatively few bacteria, and the deeper the water and the farther it is from shore so much less will be the bacterial pollution.

THE CHIEF DISEASE ORGANISMS FOUND IN WATER

We will now consider several of the more important disease-producing bacteria found in water.

Bacillus Typhosus (Eberth-Gaffky). In 1880–81 Eberth announced the discovery of this bacillus in cases of clinical enteric fever. In 1884 it was first cultivated outside the body by Gaffky. Since then other organisms have been held responsible for the causation of enteric (or typhoid) fever. In 1885 the B. coli communis was recognised, and it has been a matter of great debate amongst bacteriologists as to how far these two organisms are the same species, and the typhoid germ merely a higher evolution of the B. coli. The differentiating signs between them will be referred to shortly. Bacteriologists generally regard the Eberth-Gaffky bacillus as the specific cause of the disease, though complete proof is still wanting.

Microscopic Characters (in pure culture). Rods, 2–4 µ long, .5 µ broad, having round ends. Sometimes threads are observable, being 10 µ in length. In the field of the microscope the bacilli differ in length from each other, but are all the same thickness approximately. Round and oval cells constantly occur even in pure culture, and many of these shorter forms of typhoid are identical in morphology with some of the many forms of Bacillus coli. There are no spores. Motility is marked; indeed, in young culture it is the most active pathogenic germ we know. The small forms dart about with extreme rapidity; the longer forms move in a vermicular manner. Its powers of movement are due to some five to twenty flagella of varying length, some of them being much longer than the bacillus itself, though, owing to the swelling of the bacillus under flagellum-staining methods, it is difficult to gauge this exactly. The flagella are terminal and lateral, and are elastic and wavy. Their active contraction produces an evident current in the field of the microscope.

Cultures. This organism may be isolated from ulcerated Peyer's patches in the intestine, from the liver, the spleen, and the mesenteric glands. Owing to the mixture of bacteria found elsewhere, it is generally best to isolate it from the spleen. The whole spleen is removed, and a portion of its capsule seared with a hot iron to destroy superficial organisms. With a sterilised knife a small cut is made into the substance of the organ, and by means of a sterilised platinum wire a little of the pulp is removed and traced over the surface of agar. Agar reveals a growth in about twenty-four hours at 37° C., which is the favourite temperature. A greyish, moist, irregular growth appears, but it is invariably attached to the track of the inoculating needle. On gelatine the growth is much the same, but its irregular edge is, if anything, more apparent. There is no liquefaction and no gas formation. On plates of gelatine the colonies appear large and spreading, with jagged edges. The whole colony appears raised and almost limpet-shaped, with delicate lines passing over its surface. There is an appearance under a low power of transparent iridescence. The growth on potato is termed "invisible," and is of the nature of a potato-coloured pellicle, which looks moist, and may at a late stage become a light brown in colour, particularly if the potato is alkaline. Milk is a favourable medium, and is rendered slightly acid. No coagulation takes place. Broth is rendered turbid.

Micro-pathology. Typhoid fever is an infiltration and coagulation, necrosis, and ulceration of the Peyer's patches in the small intestine of man. The mesenteric glands show the same features, except that there is no ulceration. The spleen is enlarged, and contains the germs of the disease in almost a pure culture. The bacillus is present in the intestinal contents and excreta, particularly so when the Peyer's glands have commenced ulceration. In the blood of the general circulation the bacillus is not demonstrable, except in very rare instances. Typhoid fever is not, like anthrax, a blood disease.

COMPARATIVE FEATURES OF BACILLUS TYPHOSUS AND B. COLI

The two species, Bacillus typhosus and B. coli, agree in possessing the following characters: no spores, no liquefaction of gelatine; both grow well on phenolated gelatine, and in Parietti's broth; both act similarly upon animals, though typhoid fever is not a specific disease of animals.

The Bacillus typhosus, though a somewhat susceptible bacillus, can when dried retain its vitality for weeks. In sewage it is very difficult indeed to detect, and is soon crowded out. Dr. Andrews and Mr. Parry Laws, in their bacterial researches into sewage for the London County Council,15 found that when they examined specially infected typhoid sewage it was only with extreme difficulty they isolated Eberth's bacillus. In ordinary sewage it is clear such difficulty would be greatly enhanced.

We have pointed out elsewhere the relation between soil and typhoid. In water, even though we know it is a vehicle of the disease, the Bacillus typhosus has been only very rarely detected. The difficulties in separating the bacillus from waters (like that at Maidstone, for example), which appear definitely to have been the vehicle of the disease, are manifold. To begin with, the enormous dilution must be borne in mind, a comparatively small amount of contamination being introduced into large quantities of water. Secondly, the huge group of the B. coli species considerably complicates the issues, for it copiously accompanies the typhoid, and is always able to outgrow it. Further, we must bear in mind a point that is systematically neglected, namely, that the bacteriological examination of a water which is suspected of having conveyed the disease is from a variety of circumstances conducted too late to detect the causal bacteria. The incubation period of typhoid we may take at fourteen days. Let us suppose a town water supply is polluted with some typhoid excreta on the 1st of January. Until the 14th of January there may be no knowledge whatever of the state of affairs. Two or three days are required for notification of cases. Several more days elapse generally before bacteriological evidence is demanded. Hence arises the anomalous position of the bacteriologist who sets to work to examine a water suspected of typhoid pollution three weeks previously. There can be no doubt that these difficulties are very real ones. The solution to the problem will be found in Dr. Klein's dictum that "a water in which sewage organisms have been detected in large numbers should be regarded with suspicion"16 as the vehicle of typhoid, even though no typhoid bacilli were discoverable. The chief of these sewage bacteria are believed to be Proteus vulgaris, B. coli, P. zenkeri, and B. enteritidis, and they are all nearly related to B. typhosus. The presence of the B. coli in limited numbers is not sufficient to indicate sewage pollution, seeing that it is so widely distributed. But in large numbers, and in company with the other named species, it is almost certain evidence of sewage-polluted water.

It may occur to the general reader that, as the typhoid bacillus is not extremely rare, drinking water may frequently act as a vehicle to carry the disease to man. But, to appreciate the position, it is desirable to bear in mind the following facts: the typhoid bacillus is only found in the human excrement of patients suffering from the disease; it is short-lived; in ordinary waters there exist organisms which can exert an influence in diminishing its vitality; exposure to direct sunlight destroys it; and it has a tendency to be carried down-stream, or in still waters settle at the bottom by subsidence. Even when all the conditions are fulfilled, it must not be forgotten that a certain definite dose of the bacillus is required to be taken, and that by a susceptible person. Into these latter questions of how bacteria produce disease we shall have an opportunity of inquiring at a later stage.

We must now mention several of the special media and tests used in the separation of Bacillus typhosus and B. coli.

1. The Indol Reaction. Indol and skatol are amongst the final products of digestion in the lower intestine. They are formed by the growth, or fermentation set up by the growth, of certain organisms. Indol may be recognised on account of the fact that with nitrous acid it produces a dull red colour. The method of testing is as follows. The suspected organism is grown in pure culture in broth, and incubated for forty-eight hours at 37° C. Two cc. of a 4 per cent. solution of potassium nitrite are added to 100 cc. of distilled water, and about 1 cc. of this is added to the test-tube of broth culture. Now a few drops of concentrated sulphuric acid (unless quite pure, hydrochloric should be used) are run down the side of the tube. A pale pink to dull red colour appears almost at once, and may be accentuated by placing the culture in the blood-heat incubator for half an hour. Much dextrose (derived from the meat of the broth) inhibits the reaction. Bacillus typhosus does not produce indol, and therefore does not react to the test; B. coli and the bacillus of Asiatic cholera do produce indol, and react accordingly. It should be pointed out, however, that the bacillus of cholera also produces nitrites. Hence the addition of acid only to a peptone culture of cholera yields the "red reaction" of indol.

2. Carbolised Gelatine. To ordinary gelatine .05 per cent. of phenol is added. This inhibits many common water bacteria.

3. "_Shake Cultures._" To 10 cc. of melted gelatine a small quantity of the suspected organism is added. The test-tube is then shaken and incubated at 22° C. If the organism is Bacillus coli, the next day reveals a large number of gas-bubbles.

4. Elsner's Medium. This special potassium-iodide-potato-gelatine medium is used for the examination of typhoid excreta. It is made as follows: 500 grams of potato gratings are added to 1000 cc. of water; stand in cool place for twelve hours, and filter through muslin; add 150 grams of gelatine; sterilise and add enough deci-normal caustic soda until only faintly acid; add white of egg; sterilise and filter. Before use add half a gram of potassium iodide to every 50 cc. Upon this acid medium common water bacteria will not grow, but Bacillus typhosus and B. coli flourish.

5. Parietti's Formula consists of—phenol, five grams; hydrochloric acid, four grams; distilled water, 100 cc. To 10 cc. of broth 0.1–0.3 cc. of this solution is added. The tube is then incubated in order to see if it is sterile. If that is so, a few drops of the suspected water are added, and the tube reincubated at 37° C. for twenty-four hours. If the water contains the B. typhosus or B. coli, the tube will show a turbid growth.

6. Widal's Reaction. Mix a loopful of blood from a patient suspected of typhoid fever with a loopful of young typhoid broth culture in a hanging drop on a hollow ground slide. Cover with a cover glass and examine under 1/6-inch objective. If the patient is really suffering from typhoid, there will appear in the hanging drop two marked characteristics, viz., agglutination and immotility. This aggregation, together with loss of motility, is believed to be due to the inhibitory action of certain bacillary products in the blood of patients suffering from the disease. The test may be applied in various ways, and its successful issue depends upon one or two small points in technique into which we cannot enter here, but which the reader will find dealt with in the appendix.

7. Flagella-staining. Special methods must be adopted for staining the flagella of Bacillus typhosus and B. coli. The cover glasses should be absolutely clean, the cultures young (say eighteen hours old), and a diluted emulsion with distilled water must be made in a watch-glass in order to get bacilli discrete and isolated enough. Van Ermengem's Method is as follows:—Place a loopful of the emulsion on a clean cover glass and dry it in the air, fixing it lastly by passing it once or twice through the flame of a Bunsen burner. Place films for thirty minutes in a solution of one part boric acid (2 per cent.) and two parts of tannin (15.25 per cent.), which also contains four or five drops of glacial acetic acid to every 100 cc. of the mixture. Wash in distilled water and alcohol. Then place for five to ten seconds in a 25.5 per cent. solution of silver nitrate. Immediately thereafter, and without washing, treat the cover glass to the following solution for two or three seconds: gallic acid, five grams; tannin, three grams; fused potassium acetate, ten grams; distilled water, 350 cc. After this place in a fresh capsule of silver nitrate until the film begins to turn black. Wash in distilled water, dry, and mount. The process contracts the bacilli somewhat, but the flagella stain well.

The Bacillus coli communis occupies such an important place in all bacteriological investigation that a few words descriptive of it are necessary in this place. The "colon bacillus," as it is termed, appears to be almost ubiquitous in distribution. The idea once held that it belonged exclusively to the alimentary canal or sewage is now discarded. It is one of the most widely distributed organisms in nature, though, as its name implies, its habitat is in the intestinal tract of man and animals. It is an aërobic, non-sporulating, non-liquefying bacillus, about .4 µ in thickness, and twice that measurement in length; hence it often appears oval or egg-shaped. Its motility is in varying degree, occasionally being as active as B. typhosus, but generally much less so. It possesses lateral flagella. On gelatine plates at 20° C. B. coli produces non-liquefying, greyish-white, round colonies; in a stroke culture on the same medium, a luxuriant greyish band, much broader and less restricted to the track of the needle than B. typhosus. In depth of medium or "shake" cultures there is an abundant formation of bubbles of gas (methane or carbon dioxide) in the medium. On potato it produces a light yellow, greasy growth, which must be distinguished from the growth of B. fluorescens liquefaciens, B. pyocyaneus, and several other species on the same medium. If the potato is old or alkaline, the yellow colour may not appear. Milk is curdled solid in from twenty-four to forty-eight hours, and a large amount of lactic acid produced. In broth it produces a uniform turbidity, with later on some sediment and a slight pellicle. It gives the reaction to indol.

It is now the practice to speak of the family of Bacillus coli rather than the individual. The family is a very large one, and shows throughout but few common characters. The morphology readily changes in response to medium, temperature, age, etc. Fermentation of sugar, coagulation of milk, or indeed the indol reaction cannot always be used as final tests as to whether or not the organism is B. coli, for unfortunately some members of the family do not show each of these three features. Most varieties, however, appear to show some motility, a small number of flagella, a typical growth on potato, and develop more rapidly on all media than B. typhosus. These characters, plus one or more of the three features above named, are diagnostic data upon which reliance may be placed.

Cholera. This word is used to cover more a group of diseases rather than one specific well-restricted disease. In recent years it has become customary to speak of Asiatic cholera and British cholera, as if indeed they were two quite different diseases. But, as a matter of fact, we know too little as yet concerning either form to dogmatise on the matter. Until 1884 practically nothing was known about the etiology of cholera. In that year, however, Koch greatly added to our knowledge by isolating a spirillum from the intestine and in the dejecta of persons suffering from the disease.

Cholera has its home in the delta of the Ganges. From this endemic area it spreads in epidemics to various parts of the world, often following lines of communication. It is a disease which is characterised by acute intestinal irritation, manifesting itself by profuse diarrhœa and general systemic collapse, with cramps, cardiac depression, and subnormal temperature. The incubation period varies from only a few hours to several days. In the intestine, and setting up its pathological condition, are the specific bacteria; in the general circulation their toxic products, bringing about the systemic changes. Cholera is generally conveyed by means of water.

The spirillum of Asiatic cholera (Koch, 1884) generally appears, in the body and in artificial culture, broken into elements known as "commas." These are curved rods with round ends, showing an almost equal diameter throughout, and sometimes united in pairs or even a chain (spirillum). The latter rarely occur in the intestine, but may be seen in fluid cultures. The common site for Koch's comma is in the intestinal wall, crowding the lumina of the intestinal glands, situated between the epithelium and the basement membrane, abundant in the detached flakes of mucous membrane, and free in the contents of the intestine. They do not occur in the blood, nor are they distributed in the organs of the body.