Bacteria: Chapter II - BACTERIA IN WATER

In entering upon a consideration of such a common article of use as water, we shall do well to describe in some detail the process by which we systematically investigate the bacteriology of a water, or, indeed, of any similar fluid suspected of bacterial pollution.

The collection of samples, though it appears simple enough, is sometimes a difficult and responsible undertaking. Complicated apparatus is rarely necessary, and fallacies will generally be avoided by observing two directions. In the first place, the sample should be chosen as representative as possible of the real substance or conditions we wish to examine. Some authorities advise that it is necessary to allow the tap to run for some minutes previous to collecting the sample; but if we desire to examine for lead chemically or for micro-organisms in the pipes biologically, then such a proceeding would be injudicious.

Hence we must use common sense in the selection and obtaining of a sample, following this one guide, namely, to collect as nearly as possible a sample of the exact water the quality of which it is desired to learn. In the second place, we must observe strict bacteriological cleanliness in all our manipulations. This means that we must use only sterilised vessels or flasks for collecting the sample, and in the manipulation required we must be extremely careful to avoid any pollution of air or any addition to the organisms of the water from unsterilised apparatus. A flask polluted in only the most infinitesimal degree will entirely vitiate all results.

Accompanying the sample should be a more or less full statement of its source. There can be no doubt that, in addition to a chemical and bacteriological report of a water, there should also be made a careful examination of its source. This may appear to take the bacteriologist far afield, and in point of fact, as regards distance, this may be so. But until he has seen for himself what "the gathering-ground" is like, and from what sources come the feeding streams, he cannot judge the water as fairly as he should be able to do.

The configuration of the gathering-ground, its subsoil, its geology, its rainfall, its relation to the slopes which it drains, the nature of its surface, the course of its feeders, and the absence or presence of cultivated areas, of roads, of houses, of farms, of human traffic, of cattle and sheep—all these points must be noted, and their influence, direct or indirect, upon the water carefully borne in mind.

When the sample has been duly collected, sealed, and a label affixed bearing the date, time, and conditions of collection and full address, it should be transmitted with the least possible delay to the laboratory. Frequently it is desirable to pack the bottles in a small ice case for transit. On receipt of such a sample of water the examination must be immediately proceeded with, in order to avoid, as far as possible, the fallacies arising from the rapid multiplication of germs. Even in almost pure water, at the ordinary temperature of a room, Frankland found organisms multiplied as follows:—

Another series of observations revealed the same sort of rapid increase of bacteria. On the date of collection the micro-organisms per cc. in a deep-well water (in April) were seven. After one day's standing at room temperature the number had reached twenty-one per cc. After three days under the same conditions it was 495,000 per cc. At blood-heat the increase would, of course, be much greater, as a higher temperature is more favourable to multiplication. But this would depend upon the degree of impurity in the water, a pure water decreasing in number on account of the exhaustion of the pabulum, whereas, for the first few days at all events, an organically polluted water would show an enormous increase in bacteria.

Furthermore, it is desirable to remember that organisms, in an ordinary water, do not continue to increase indefinitely. There is a limit to all things, even to numbers in bacteriology. Cramer, of Zurich, examined the water of the Lake after it had been standing for different periods, with the following results:—

The writer's own experience is entirely in agreement with this cessation of multiplication at or about the end of a week, and the later decline.

Method of Examination. At the outset of a systematic study of a water it is well to observe its physical characters. The colour, if any, should be noted. Suspended matter and deposit may indicate organic or inorganic pollution. If abundant or conspicuous, a microscopic examination of the sediment may be made. The reaction, whether acid, neutral, or alkaline, must be tested, and the exact temperature taken. Any and every fact will help us, perhaps not so much to determine the contents of the water as to interpret rightly the facts we deduce from the entire examination.

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. 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.

Moist Chamber in which Koch's Plates are Incubated

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.

Hot Air Steriliser
For the Sterilization of Glass Apparatus, etc.

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.

The Hanging Drop

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.

Drying Stage for Fixing Films

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.

Types of Liquefaction of Gelatine

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.

Wolfhügel's Counter

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.

Petri's Dish

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.

Apparatus for Filtering Water to Facilitate its Bacteriological Examination

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 Frankland 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

Bacteria of Typhoid Fever

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.

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, 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.

B. Coli Communis

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" 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.

The Comma-Shaped Bacilli of Cholera

The bacilli are actively motile, and possess at least one terminal flagellum. The organism is aërobic, and liquefies gelatine. It stains readily with the ordinary aniline dyes. It does not produce spores, though certain refractile bodies inside the protoplasm of the bacillus in old cultures have been regarded as such. The virulence of the bacillus is readily attenuated, and both the virulence and morphology appear to show in different localities and under different conditions of artificial cultivation a large variety of what are termed involution forms. Unless the organism is constantly being sub-cultured, it will die. Acid, even the .2 per cent. present in the gastric juice, readily kills it. Desiccation, 55° C. for ten minutes, and weak chemicals have the same effect. The bacilli, however, have comparatively high powers of resistance to cold. Unless examined by the microscope in a fresh and young stage, it is difficult to differentiate Koch's comma from many other curved bacilli.

Its cultivation characters are not always distinctive. Microscopically the young colonies in gelatine appear as cream-coloured, irregularly round, and granular. Liquefaction sets in on the second day, producing a somewhat marked "pitting" of the medium, which soon becomes reduced to fluid. In the depth of gelatine the growth is very characteristic. An abundant, white, thick growth exactly follows the track of the needle, here and there often showing a break in continuity. Liquefaction sets in on the second day, and produces a distinctive "bubble" at the surface.

The liquefied gelatine does not fall from the sides of the tube, as in the Finkler-Prior comma of cholera nostras, but occurs inside the border where the gelatine joins the glass. In the course of a week or two all the gelatine may be reduced to fluid. On agar Koch's comma produces with rapidity a thick, greyish, irregular growth. On potato, especially if slightly alkaline, an abundant brownish layer is formed. Broth and peptone water are excellent media. In milk it rapidly multiplies, curdling the medium, with production of acid. Unlike Bacillus coli, it does not form gas, but, like B. coli, it produces large quantities of indol and a reduction of nitrates to nitrites. Hence the indol test may be applied by simply adding to the peptone culture several drops of strong sulphuric acid, when in the course of several hours, if not at once, there will be produced a pink colour, the "cholera red reaction."

Although it readily loses virulence, and its resistance is little, the comma bacillus retains its vitality for considerable periods in moist cultures, upon moist linen, or in moist soil. In cholera stools kept at ordinary room temperature the cholera bacillus will soon be outgrown by the putrefactive bacteria. The same is true of sewage water.

The lower animals do not suffer from any disease at all similar to Asiatic cholera, and hence it is impossible to fulfil the postulate of Koch dealing with animal inoculation. In this respect it is like typhoid. It is, however, provisionally accepted that Koch's bacillus is the cause of the disease. The four or five other bacteria which have from time to time been put forward as the cause of cholera have comparatively little evidence in their support. It is less from these, and more from several spirilla occurring in natural waters, that difficulties of diagnosis arise.

Some hold that, however many comma bacilli be introduced into the alimentary canal, they will not produce the disease unless there is some injury or disease of the wall of the intestine. It need hardly be added that cholera acts, like other pathogenic bacteria, by the production of toxins. Brieger separated cadaverin and putrescin and other bodies from cholera cultures, and other workers have separated a tox-albumen.

Methods of Diagnosis of Cholera:

1. The nature of the evacuations and the appearance of the mucous membrane of the intestine afford striking evidence in favour of a positive diagnosis. Nevertheless it is upon a minute examination of the flakes and pieces of detached epithelium that reliance must be placed. In these flakes will be found in cholera abundance of bacilli having the size, shape, and distribution of the specific comma of cholera. The size and shape have been already touched upon. The distribution is frequently in parallel lines, giving an appearance which Koch described as the "fish-in-stream arrangement." This distribution of comma bacilli in the flakes of watery stools is, when present, so characteristic of Asiatic cholera that it alone is sufficient for a definite diagnosis. But unfortunately it is not always present, and then search for other characters must be made.

2. The appearance of cultivation on gelatine, to which reference has been made, is of diagnostic value.

3. The "cholera red reaction." It is necessary that the culture be pure for successful reaction.

4. Isolation from water is, according to Dr. Klein, best accomplished as follows: A large volume of water (100–500 cc.) is placed in a sterile flask, and to it is added so much of a sterile stock fluid containing 10 per cent. peptone, 5 per cent. sodium chloride, as will make the total water in the flask contain 1 per cent. peptone and .5 per cent. salt. Then the flask is incubated at 37° C. If there have been cholera vibrios in the water, however few, it will be found after twenty-four hours' incubation that the top layer contains actively motile vibrios, which can now be isolated readily by gelatine-plate culture.

5. To demonstrate in a rapid manner the presence of cholera bacilli in evacuations, even when present in small numbers, a small quantity must be taken up by means of a platinum wire and placed in a solution containing 1 per cent. of pure peptone and .5 per cent. sodium chloride (Dunham). This is incubated as in the case of the water, and in twelve hours is filled with a turbid growth, which when examined by means of the hanging drop shows characteristic bacilli.

natural purification of water

We have already noticed that rivers purify themselves as they proceed. There are many excellent examples of this self-purification. The Seine as it runs through Paris becomes highly polluted with every sort of filthy contamination. But twenty or thirty miles below the city it is found to be even purer than above the city before it received the city sewage. In small rivers it is the same, provided the pollution is less in amount. Whilst authorities differ with regard to the mode of self-purification, all agree that in some way rivers receiving crude sewage are able in a marvellous degree to become pure again.

The conditions influencing this phenomenon are as follows:

(a) The Movement of the Water. It is probable, however, that any beneficial result accruing from this cause is due, not to any mechanical factor in the movement, but to the extra surface of water available for oxidation processes.

(b) The Pressure of the Water. It is believed that the volume of water pressing down upon any given area beneath it weakens the vitality of certain microbes. In support of this theory, it is urged that the number of bacteria capable of developing is less the greater the depth from the surface. Yet it must be remembered that mud at the bottom of a river, or at the bottom of the sea, is teeming with living organisms.

(c) Light. We have seen how prejudicial is light to the growth of organisms in culture media. This is so, though to a less extent, in water. Arloing held that sunlight could not pierce a layer of water an inch in thickness and still act inimically on micro-organisms. But Buchner found that the sun's rays could pass through fifteen or twenty inches and yet be bactericidal. This evidence appears contradictory. On the whole, however, authorities agree that the influence of the sun's rays upon water is distinctly bactericidal and causes a marked diminution in the quantity of organisms after acting for some hours. Especially will this be so when the water is spread over a wide area and is therefore shallow and stationary, or moving but slowly.

(d) Vegetation in Water. Pettenkofer, in his observations upon the Iser below Munich, has shown how algæ bring about a marked reduction in the organic matters present in water.

(e) Dilution. There can be no doubt in anyone's mind that the pollutions passing into a flowing river are very soon diluted with the large quantities of comparatively pure water always forthcoming. But this, whilst it would lower the percentage of impurity, cannot remove impurities.

(f) Sedimentation. Whilst Pettenkofer attributes self-purification to oxygenation and vegetation, most authorities are now agreed that it is largely brought about by the subsidence of impure matters, and by their subsequent disintegration at the bottom of the river. Sedimentation obviously is greatest in still waters. Hence lake water contains as a rule very few bacteria. "The improvement in water during subsidence is the more rapid and pronounced the greater the amount of suspended matter initially present" (Frankland). Tils has pointed out that the number of micro-organisms was invariably smaller in the water collected from the reservoir than in that taken from the source supplying the latter. Percy Frankland has demonstrated the same effect of sedimentation by storage as follows:

The large reservoir would of course necessitate a prolonged subsidence, and hence a greater diminution than in the small reservoirs. Many like examples might be cited, but a typical one such as the above will suffice.

(g) Oxidation. Many experiments and observations have been made to prove that large quantities of oxygen are used up daily in oxygenising the Thames water. Oxygenated water will come up with the tide and down with the fresh water from above London. There will also be oxygen absorption going on upon the surface of the water, and from these three sources enough oxygen is obtained to oxidise impurities and produce what is really an effluent. In many smaller streams the opportunity for oxidation is afforded by weirs and falls.

Probably all these factors play a part in the self-purification of rivers, but we may take it that oxidation, dilution, and sedimentation are three of the principal agencies.

We may here digress to refer in passing to the facts obtainable from Sir Edward Frankland's report on Metropolitan water supply in 1894, as they will afford a connecting link between self purification and artificial purification. Judged by the relatively low proportion of carbon to nitrogen, the organic matter present in the water was, as usual, found to be chiefly, if not entirely, of vegetable origin.

An immense destruction of bacteria was found to be effected by storage in subsidence reservoirs. The bacterial quality of the water might differ widely from its chemical qualities. These three facts are of primary importance in the interpretation of water reports, and it will be well to bear them in mind. Sir E. Frankland also refers to the physical conditions affecting microbial life in river waters. The importance of changes of temperature, the effect of sunlight, and rate of flow had been referred to in previous reports. Respecting the relative proportion of these factors, he adds:

"The number of microbes in Thames water is determined mainly by the flow of the river, or, in other words, by the rainfall, and but slightly, if at all, by either the presence or absence of sunshine, or a high or low temperature. With regard to the effect of sunshine, the interesting researches of Dr. Marshall Ward leave no doubt that this agent is a powerful germicide, but it is probable that the germicidal effect is greatly diminished, if not entirely prevented, when the solar rays have to pass through a comparatively thin stratum of water before they reach the living organisms."

From which it is clear that evidence favours the effect of sedimentation and dilution. These two factors in conjunction with filtration are, practically speaking, the methods of artificial water purification, with which we are now in a position to deal.

artificial purification of water

Sedimentation and Precipitation. Naturally, we see this factor in action in lakes or reservoirs. For example, the water supply of Glasgow is the untreated overflow from Loch Katrine. Purification has been brought about by means of subsidence of impurities. Nothing further is needed. Artificially, we find it is this factor which is the mechancial purifier of biological impurity in such methods as Clark's process. By this mode "temporary hardness," or that due to soluble bicarbonate of lime, is converted into insoluble normal carbonate of lime by the addition of a suitable quantity of lime-water. Carbonates of lime and magnesia are soluble in water containing free carbonic acid, but when fresh lime is added to such water it combines with the free CO2 to form the insoluble carbonate, which falls as a sediment:

CaCO3 + CO2 + CaH2O2 (lime-water) = 2 CaCO3 + H2O.

As the carbonate falls to the bottom of the tank it carries down with it the organic particles. Hence sedimentation is brought about by means of chemical precipitation. It is obviously a mechanical process as regards its action upon bacteria. Nevertheless its action is well-nigh perfect, and 300 or 400 m.-o. per cc. are reduced to 4 or 5 per cc. We shall refer to this same action when we come to speak of bacterial purification of sewage. 

Alum has been frequently used to purify waters which contain much suspended matter. Five or six grains of alum are added to each gallon of water, with some calcium carbonate by preference. Precipitation occurs, and with it sedimentation of the bacteria, as before. But, as Babes has pointed out, alum itself acts inimically on germs; in such treatment, therefore, we get sedimentation and germicidal action combined.

As a matter of actual practice, however, sedimentation alone is rarely sufficient to purify water. It is true that the collection of water in large reservoirs permits subsidence of suspended matters, and affords time for the action of light and the competitive suicidal behaviour of the common water bacteria. Yet, after all, filtration is the most important and most reliable method.

Sand Filtration, as a means of purifying water, has been practised since the early part of the present century. But it was not till 1885 that Percy Frankland first demonstrated the great difference in bacterial content between a water unfiltered and a water which had passed through a sand filter. Previous to this time the criterion of efficiency in water purification had been a chemical one only, and the presence or absence of bacteria in any appreciable quantity was described, not in mathematical terms, but in indefinite descriptive words, like "turbid," "cloudy," etc. It is needless to say that this difference in estimation was due to the introduction by Koch of the gelatine-plate method of examination. As a result of Percy Frankland's work, he formulated the following conclusions as regards the chief factors influencing the number of microbes passing through the filter.

It depends upon:

(1) The Storage Capacity for Unfiltered Water. This, of course, has reference to the advantages, which we have noticed above, of securing a large collection of water previous to filtration for subsidence, etc.

(2) The Thickness of Fine Sand through which Filtration is Carried on. An argument needing no further support, for it is clear, other things being equal, the more sand water passes through the greater the opportunity of leaving its impurities behind.

(3) Rate of Filtration. The slower filtration will be generally the more complete in its results.

(4) Renewal of Filter-Beds. After a certain time the filter-bed becomes worn out and inefficient; at such times renewal is necessary. Not only may the age of the filter act prejudicially, but the extra pressure required will tend to force through it bacteria which ought to have remained in the filter.

In 1893, Koch brought out his monograph upon Water Filtration and Cholera, and his work had a deservedly great influence upon the whole question. He shows how the careful filtration of water supplied to Altona from the Elbe saved the town from the epidemic of cholera which came upon Hamburg as a result of drinking unfiltered water, although Altona is situated several miles below Hamburg, and its drinking water is taken from the river after it has received the sewage of Hamburg. Now, from his experience of water filtration, Koch arrived at several important conclusions. In the first place, he maintained that the portion of the filter-bed which really removed micro-organisms effectively was the slimy organic layer upon the surface.

This layer is produced by a deposit from the still unpurified water lying immediately above it. The most vital part of the filter-bed is this organic layer, which, after formation, should not be disturbed until it requires removal owing to its impermeability. A filter-bed, as is well known, consists of say three feet of sand and one foot of coarse gravel. The water to be filtered is collected into large reservoirs, where subsidence by gravitation occurs. Thence it is led by suitable channels to the surface of the filter-bed. Having passed through the three or four feet of the bed, it is collected in a storage reservoir and awaits distribution. The action of the whole process is both mechanical and chemical. Mechanically by subsidence, much suspended matter is left behind in the reservoir.

Again, mechanically, much of that which remained suspended in the water when it reached the filter-bed is waylaid in the substance of the sand and gravel of the filter-bed. Chemically also the action is twofold. Oxidation of the organic matter occurs to some extent as the water passes through the sand. Until recently this chemical action and the double mechanical action were believed to be the complete process, and its efficiency was tested by chemical oxidation and alteration, and absence of the suspended matter.

Now, however, it is recognised that the second portion of the chemical action is vastly the more important, indeed, the only vital, part of the process. This is the chemical effect of the layer of scum and mud on the surface of the sand at the top of the filter-bed. The mechanical part of this layer is, of course, the holding back of the particulate matter which has not subsided in the reservoir; the vital action consists in what is termed nitrification of unoxidised substance, which is accomplished in this layer of organic matter.

We shall deal at some length with the principles of nitrification when we come to speak of soil. But we may say here that by nitrification is understood a process of oxidation of elementary compounds of nitrogen, by which these latter are built up into stable bodies which can do little harm in drinking water. From what has been said it will be seen that the action of a filter-bed is of a complicated nature.

There is (1) subsidence of the grosser particles of impurity in the water; (2) mechanical obstruction to impurities in the interstices of the scum, sand, and gravel in the filter; (3) oxidation of organic matter by the oxygen held in the pores of the sand and gravel; (4) nitrification in the vital scum layer, which is accomplished by micro-organisms themselves. This latter is now considered to be incomparably the most important part of the filter. That being so, its removal, except when absolutely necessary, is to be avoided as detrimental to the efficiency of the filter. New filters have obviously but little of this action. Hence it is wise to allow a new filter-bed to act for a short period (say twenty-four to forty-eight hours) before the filtered water is used for domestic purposes, in order to allow the organic layer to be formed. This must also be borne in mind after a filter-bed has been cleaned.

To maintain this nitrifying action of a filter in efficiency, Koch suggested, in the second place, that the rate of filtration must not exceed four inches per hour. At the Altona water-works this rate of filtration was maintained, and the number of organisms always remained below 100 per cc., which, as we have seen, is the standard. Thirdly, it is important that periodic bacteriological examinations should be made. Koch's emphasis upon this point is well known, and the cumulative experience of bacteriologists only further supports such a course being taken. If it be true that efficient sand filtration is a safeguard against pathogenic germs like typhoid and cholera, then there can be but one criterion of efficiency, viz., their absence in the filtered water, which can only be ascertained by regular examination. But it is not alone for pathogenic germs that filtration is proposed. Filtered water containing more than 100 micro-organisms of any kind per cc. is below the standard in purity, and should on no account be distributed for drinking purposes.

In this country chemical analysis, with a more or less cursory microscopic examination, has been almost invariably accepted as reliable indication of the condition of the water. But such an examination is not really any more a fair test of the working of the filter than it is of the actual condition of the water. It is true, the quantity of organic matter can be estimated and the condition in which it exists in combination obtained; but it cannot tell us what a bacteriological examination can tell us, viz., the quantity and quality of living micro-organisms present in the water. Upon this fact, after all, an accurate conclusion depends. There is abundant evidence to show that no valuable opinion can be passed upon a water except by both a chemical and a bacteriological examination, and further by a personal investigation, outside the laboratory, of the origin of the water and its liabilities to pollution.

So convinced was Koch of the efficiency of sand filtration as protection against disease-producing germs that he advocated an adaptation of this plan in places where it was found that a well yielded infected water. Such pollution in a well may be due to various causes; surface-polluted water oozing into the well is probably the commonest, but decaying animal or vegetable matter might also raise the number of micro-organisms present almost indefinitely. Koch's proposal for such a polluted well was to fill it up with gravel to its highest water level, and above that, up to the surface of the ground, with fine sand. Before the well is filled up in this manner it must, of course, be fitted with a pipe passing to the bottom and connected with a pump.

This simple procedure of filling up a well with gravel and sand interposes an effectual filter-bed between the subsoil water and any foul surface water percolating downwards. Such an arrangement yields as good, if not better, results than an ordinary filter-bed, on account of there being practically no disturbance of the bed nor injury done to it by frost.

The effect of the remedies we have been discussing upon the number of bacteria is demonstrated in the results which Sir Edward Frankland arrived at in his investigation of London waters.

The teaching of these figures could, with great ease, be reproduced again and again if such was necessary; but these will suffice to show that sand filtration, when carefully carried out, offers a more or less absolute barrier to the passage of bacteria, whether non-pathogenic or pathogenic.

Domestic Purification of Water. Something may here be said, from a bacteriological point of view, relative to what is called domestic purification. There is but one perfectly reliable method of sterilising water for household use, viz., boiling. As we have seen, moist heat at the boiling point maintained for five minutes will kill all bacteria and their spores. The only disadvantages to this process are the labour entailed and the "flat" taste of the water. Nevertheless in epidemics due to bad water it is desirable to revert to this simple and effectual purification.

Water companies and those responsible for water supply appear to hold the opinion that so long as there is sand filtration or subsidence reservoirs it is unnecessary to consider the gathering-ground or transit. But, as we have seen, a frost may completely dislocate the efficient action of a filter, and times of flood may prevent proper sedimentation; then our dependence for pure water is wholly upon the gathering-ground and source. Hence we find water contaminated at its source by polluted wells, by sewage-infected rivers and streams, by drainage of manured fields, by innumerable excremental pollutions over the areas of the gathering-grounds, and in transit by careless laying, poor construction, bad jointing, and close proximity of water-and drain-pipes. In the third place, we may get a water infected at the periphery, in the house itself. Such cases are generally due to one of two causes: filthy cisterns or suction. Cisterns per se are more or less indispensable where a constant service does not exist, but they should be inspected from time to time and maintained in a cleanly condition. Suction into the tap has been recently emphasised by Dr. Vivian Poore as a cause of pollution. It is liable to occur whenever a tap is left turned on, and a vacuum is produced in the supply-pipe by intermission of the water supply, so that foul gas or liquid is sucked back into the house-pipe.

One more point requires our attention. It has relation to bacterially polluted water when it has gained entrance to the body. It has been known for some time past that not all waters polluted with disease germs produce disease. As we have before said, this may depend upon the infective agent, its quantity and quality; the body being able in many cases to resist a small dose of poison. It is, however, necessary to infection, especially in water-borne disease, that the tissues shall be in some degree disordered.

The perverted action of the stomach influences the acid secretion of the gastric juice, through which bacilli might then pass uninjured. Particularly must this be so in the bacillus of cholera, which is readily killed by the normal acid reaction of the stomach.

Hence, in this disease at least, it is the opinion of bacteriologists that the condition of the mucous membrane of the stomach is of primary importance. Metschnikoff has indeed demonstrated the presence of the bacillus of cholera in the intestinal excretion of apparently healthy persons, which shows that they were protected by the resistance of their tissues to the bacilli. Further light has been thrown on this question by the researches of MacFadyen, who has pointed out that suspensions of cholera bacilli in water passed through the stomach untouched, and were thus able to exert their evil influence in other parts of the alimentary canal.

When, however, cholera bacilli were suspended in milk, none appeared to escape the germicidal action of the gastric juice. The explanation of this is probably the simple one that the stomach reacted with its secretion of gastric juice only to food (milk), but simply passed the water on into the lower and more absorptive parts of the alimentary canal. Such a condition of affairs clearly increases the danger of water-borne germs.

the bacteriology of sewage and sewage-polluted waters

It will not be needful to insist upon the obvious fact that bacteria abound in sewage. Such a large quantity of organic matter, in which decomposition is constantly taking place, will afford an almost ideal nidus for micro-organic life. There is indeed but one reason why such a medium is not absolutely ideal from the microbe's point of view, and that reason is, that in sewage the vast number of bacteria present make the struggle for existence exceptionally keen. Not only are the numbers incredibly large, but we also find a very extensive representation of species, including both saprophytes and parasites, non-pathogenic and pathogenic. Not infrequently it is from pollution by sewage that drinking water is contaminated with disease.

A patient, we will say, suffers from typhoid fever. The specific organism has its habitat largely, though not exclusively, in the alimentary canal. It passes out in the excreta, and though sometimes partially disinfected, may escape without hindrance into the drains, and thus to the sewer or cesspool. How often, by means of direct connection or by percolation, sewage, from sewers or cesspools, gains access to drinking water, the history of typhoid outbreaks in this country only too fully records.

It is impossible to lay down any exact standard of the chemical and bacteriological quality of sewage. The quality will differ according to the size of the community, the inclusion or otherwise of trade-waste effluents, the addition of rain-water, and other like physical conditions. Moreover, sewage itself when, so to speak, fully formed is liable to undergo rapid changes owing to fermentation and the competition of micro-organisms. It is clear that these latter are the chief agents in bringing about the change, because, if sewage be placed in hermetically sealed flasks and sterilised by heat, it is found that no change occurs. From facts such as the above it will be apparent that no exact standard of chemical or bacterial contents is possible.

Respecting the chemical condition we may shortly say that the chief characteristic of sewage is its enormous amount of contained organic matter in suspension or solution; respecting the bacterial content we may say that the chief species of the very numerous organisms are those commonly concerned in fermentative putrefaction. London crude sewage contains on an average about four millions of micro-organisms per cc. Many of these are "liquefying" bacteria; that is to say, they have the power of liquefying gelatine, which is generally one of the features of putrefactive species. In considering the quality of the bacteria present in sewage, a still wider field of research opens before us. For though we can say that, roughly, all sewage will contain probably between four and eight millions of bacteria, we cannot even lay down a rough standard respecting the kinds of bacteria present more than we have done already in stating that a very large number indeed out of the total will belong to putrefactive species.

We may, however, make a provisional list of normal sewage bacteria as follows

1. Bacillus coli communis and all its varieties and allies.

2. Proteus vulgaris and the various protean species.

3. B. enteritidis sporogenes (Klein).

4. Liquefying bacteria, e. g., Bacillus fluorescens liquefaciens, B. subtilis, B. mesentericus.

5. Non-liquefying bacteria.

6. Sarcinæ, yeasts, and moulds.

We have not included in the above inventory any pathogenic bacteria. Doubtless such species (e. g., typhoid) not infrequently find their way into sewage. But they are not normal habitants, and though they struggle for survival, the keenness of competition among the dense crowds of saprophytes makes existence almost impossible for them. Nor can they expect much sympathy from us in the difficulties of life which fortunately confront them in sewage.

Of those we have named as normally present it is unnecessary to speak in detail, with the exception of the newly discovered anaërobe, Bacillus enteritidis sporogenes of Klein. This bacillus is credited to be a causal agent in diarrhœa, and has been isolated by Dr. Klein from the intestinal contents of children suffering from severe diarrhœa, and from adults having cholera nostras. It has been readily detected in sewage from various localities, and also in sewage effluents, after sedimentation, precipitation, and filtration. Its biological characters are shortly as follows: It is in thickness somewhat like the bacillus of symptomatic anthrax, thicker and shorter than the bacillus of malignant œdema, and standing therefore between the latter and anthrax itself. It is motile and possesses flagella, but has no threads. It readily forms spores, which develop as a rule near the ends of the rods and are thicker than the bacilli. It is stained by Gram's method. In various media (particularly milk) it produces gas rapidly. It is an anaërobe, and is cultivated in Buchner's tubes. A recent epidemic of diarrhœa affecting 144 patients in St. Bartholomew's Hospital was traced to milk in which B. enteritidis was present.

Sewer Air. Though not of material importance as regards bacterial treatment of sewage, this subject calls for some remark. For long it has been known that air polluted by sewage emanations is capable of giving rise to various degrees of ill-health. These chiefly affect two parts of the body; one is the throat, and the other is the alimentary canal. Irritation and inflammation may be set up in both by sewer air. Such conditions are in all probability produced by a lowering of the resistance and vitality of the tissues, and not by either a conveyance of bacteria in sewer air or any stimulating effect upon bacteria exercised by sewer air. What evidence we have is against such factors. (See p. 105.)

Several series of investigations have been made into the bacteriology of sewer air, amongst others by Uffelmann, Haldane, Laws, and Andrewes. From their labours we may formulate four simple conclusions:

1. The air of sewers contains very few micro-organisms indeed, sometimes not more than two organisms per litre (Haldane), and generally fewer than the outside air (Laws and Andrewes).

2. There is no relationship between the microbes contained in sewer air and those contained in sewage. Indeed, there is a marked difference which forms a contrast as striking as it is at first sight unexpected. The organisms isolated from sewer air are those commonly present in the open air. Micrococci and moulds predominate, whereas in sewage bacilli are most numerous. Liquefying bacteria, too, which are common in sewage, are extremely rare in sewer air. Bacillus coli communis, which occurs in sewage from 20,000 to 200,000 per cc., is altogether absent from sewer air.

3. Pathogenic organisms and those nearly allied to them are found in sewage, but absent in sewer air. Uffelmann isolated the Staphylococcus pyogenes aureus (one of the organisms of suppuration), but such a species is exceptional in sewer air. Hence, though sewer air is popularly held responsible for conveying diphtheria and all sorts of other virulent bacteria, there is up to the present no evidence of a substantial nature in support of such views. Sewer air neither conducts pathogenic organisms nor stimulates the virulence of such.

4. Lastly, only when there is splashing in the sewage, or when bubbles are bursting (Frankland), is it possible for sewage to part with its contained bacteria to the air of the sewer.

Whilst we cannot here enter more fully into an account of the bacteria found in sewage or of their functions, it is necessary to remark upon one distinguishing feature. A very large number of sewage bacteria are decomposing and denitrifying, that is to say, breakers down, by means of putrefaction, of organic compounds. The knowledge of this fact has recently been applied, in conjunction with oxidation, to the biological treatment of sewage. As this illustrates in a marked degree some of the facts we have dwelt upon in considering the bacteriology of soil, and as it is likely that the future will witness a still wider application of these same facts, it will be necessary to refer in some detail to the matter.

Hitherto there has been adopted one of four methods of treatment of sewage. In the first place, in towns situated on the coast the sewage has, by means of a conduit, been carried out to sea. It is clear that such a course, which is in itself open to criticism, is applicable to but few towns. In the second place, methods of chemical treatment have been practised. This has generally been of the nature of a "precipitation" process. Six to twelve grains of quicklime have been added to each gallon of sewage. The process is simple and cheap, but it does not remove the organic matter in solution.

On the one hand, it does not produce a valuable manure; on the other, it fails to purify the effluent. A dozen other methods have been tried, but all based on the addition of chemical substances to precipitate or change the organic matter of the sewage. Electrolysis, too, has been proposed. The third mode adopted in the past has been that known as intermittent downward filtration. This may be defined as "the concentration of sewage at short intervals on an area of specially chosen porous ground, as small as will absorb and cleanse it, not excluding vegetation, but making the product of secondary importance" (Metropolitan Sewage Commission).

The action is mechanical and biological, that is to say, due in part to nitrification by bacteria in the upper layers of soil. The fourth plan is that of irrigation, or "the distribution of sewage over a large surface of ordinary agricultural ground, having in view a maximum growth of vegetation (consistently with due purification) for the amount of sewage supplied." Like the former, there is biological influence at work here, though in a less degree. About one acre is required for every hundred persons in the population. These two latter modes are much to be preferred to chemical treatment, yet on account of space and management, as well as on account of the non-removal of the "sludge," their success has not been all that could be desired. Until comparatively recent times the above methods of treating sewage were the only ones available.

In 1881 it appears that M. Louis Mouras, of Vesoul (Haute Saône), published an account of a hermetically sealed, inodorous, and automatically discharging cesspool, in which sewage was anaërobically broken down by "the mysterious agents of fermentation." This is the first record we have of the newly applied treatment of sewage by simply allowing Nature to fulfil her function by means of bacteria. We shall most easily arrive at an appreciation of the recent developments of the process in England by describing the so called septic tank and cultivation beds.

A Plan of Septic Tank and Filter-Beds
As Used at Exeter

The septic tank is a large underground vault of cemented brick, having a capacity of thousands of gallons, according to the population. That at Exeter has a capacity of 53,800 gallons, and takes the average sewage of 1500 inhabitants in twenty-four hours. Near the entrance is a submerged wall, seven feet from the entrance and twelve inches below the surface of the liquid in the full tank. Within this are caught, by gravity, gravel and such-like deposits.

The remaining solid matter of the sewage becomes deposited in the tank itself. Both in the sediment at the bottom of the tank and in the thick scum on the surface the organic compounds are broken down and made soluble. In the former position this is accomplished by anaërobic bacteria, in the latter on the surface by aërobic bacteria. It need hardly be added that these are denitrifying and putrefactive bacteria, and that those at the bottom of the tank perform greater service than those at the top.

When the liquid sewage passes out of the tank it differs from the crude sewage which enters the tank in the following particulars: (a) The gravel and particulate débris have been removed; (b) the organic solids in suspension are so greatly diminished that they are almost absent; (c) there is an increase of organic matter in solution; (d) the sewage is darker in colour and more opalescent; (e) compounds like albuminoid ammonia, urea, etc., have been more or less completely broken down, and reappear in elementary conditions, like ammonia, methane, carbon dioxide, and sulphuretted hydrogen. These latter bodies may be in solution or may have escaped as gas.

The cultivation beds are four or five filters, to which the sewage from the tank flows in such a manner as to produce a weir. By an automatic arrangement the fluid is distributed to each filter in turn. When the second filter is full the first is discharged, and remains empty during the time that the third and fourth are being filled. Each filter is thus full, say, about six hours, and has from ten to twelve hours' rest. These filter-beds (at Exeter) have an area of eighty square yards and a depth of five feet; collecting drains are laid on the bottom of the filters, joining main collectors, the latter terminating in discharging wells. The filtrant is broken furnace clinker or broken coke.

The changes occurring in these filters are of the nature of oxidation, with the result that the proportion of the oxidised nitrogen increases (as nitrites and nitrates), the ammonia becomes less, and the total solids and organic nitrogen almost disappear. It will thus be seen that the work of these filters is not merely a straining action. It is true that particulate matter in the effluent from the tank is caught on the surface by the film (resulting from previous effluents), but the real work of the bed is nitrification, an oxidation of ammonia into nitrites and nitrates.

This change obviously begins when the tank effluent flows over the "weir" on to the filter-beds, and the oxygen thus obtained by the effluent is carried down in solution into the coke-breeze. Upon the surface of the filtrant are oxidising bacteria. When the effluent is on the bed they oxidise its contained products; when the bed is empty and "resting" they oxidise carbon. An advantage arising from the periodical emptying and filling of the filter is that the products of decomposition which would eventually inhibit the action of the aërobic bacteria are washed away, and pass into the nearest stream, where they become absolutely innocuous.

The "filter" is more correctly termed a cultivation bed, for its purpose is to furnish a very large surface upon which the nitrifying organisms present, as we have seen, in all soils, may flourish, and thus feeding upon the organic matter of the sewage, may perform their function of oxidation.

It is not possible to lay down exact limits as to where denitrification ends and oxidation begins. To a certain extent, and in varying degree, they overlap each other. But roughly we may say that in the tank there is a breaking down (denitrification and decomposition) and in the filter-beds a building up (nitrification). The case is precisely parallel to similar changes occurring in soil, and which we have dealt with elsewhere. The advantage indeed of this biological treatment of sewage is that it exactly follows the processes of nature, in contradistinction to the mechanical and chemical methods hitherto adopted.

At Sutton and some other places the same principles are applied,—that is to say, bacterial filtration,—but there is no tank. A metal screen in some measure takes its place, and holds back solid matter from being carried on to the beds. The filtrant is burnt clay, and it is forked over occasionally to let in oxygen. The crude sewage is run over the top of the burnt ballast, where it is left for two or three hours. It is then slowly run off on to a finer filter, where it also stays two hours. Thence the effluent is run into the stream.

Filter-Beds
As Used at Sutton

It must be admitted that the bacterial treatment of sewage, though exhibiting such excellent results where it has been given a fair trial, is still in a probationary stage. It appears to stand on reason. The sludge of previous methods is avoided. The sewage is entirely broken down, and the effluent is a comparatively pure one, yet taking back nitrogen, as nitrate, to the soil. The whole change, indeed, in the opinion of Dr. Dupré, is more effective and radical than in chemical treatment.

Further, it has been tested as regards its action upon the pathogenic bacilli—those of tubercle and typhoid—with the result that these infective bacteria have been completely destroyed. It appears that such destruction of infective germs occurs in the tank, and depends in degree upon the rapidity with which sewage is passed through the tank. The cultivation beds also have an inimical effect upon infective bacteria. Hence the final effluent is practically germ-free as regards pathogenic organisms.