Chapter II: Bacteria in Water (1)

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.[11] 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:--

No. of Germs
Hours. per cc.

0 1,073
6 6,028
24 7,262
48 48,100

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:--

Hours and Days of No. of Micro-organisms
Examination. per cc.[12]

0 hours 143
24 " 12,457
3 days 328,543
8 " 233,452
17 " 17,436
70 " 2,500

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.[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.

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.

For the Sterilizatin of Glass Apparatus, etc.]

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:

Very pure water 0- 10 bacteria per cc.
Very good water 20- 100 " "
Good water 100- 200 " "
Passable (mediocre) water 200- 500 " "
Bad water 500- 1,000 " "
Very bad water 1,000-10,000 and over "

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:

0- 100 bacteria per cc. =a good potable water,
100- 500 " " =a suspicious water.
500-1000 or more " =a water which should have
further filtration before
being used for drinking
purposes.

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.

In Position for Filtration of Water to be Examined.]

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 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 Frankland[14] collected water from various quarters at various times and seasons, and some of his results may here be added:

RIVER THAMES WATER COLLECTED AT HAMPTON

Number of Micro-organisms Obtained from 1 cc. of Water.

----------------------+--------------+--------------+--------------
MONTH. | +1886.+ | +1887.+ | +1888.+
----------------------+--------------+--------------+--------------
| | |
January | 45,000 | 30,800 | 92,000
February | 15,800 | 6,700 | 40,000
March | 11,415 | 30,900 | 66,000
April | 12,250 | 52,100 | 13,000
May | 4,800 | 2,100 | 1,900
June | 8,300 | 2,200 | 3,500
July | 3,000 | 2,500 | 1,070
August | 6,100 | 7,200 | 3,000
September | 8,400 | 16,700 | 1,740
October | 8,600 | 6,700 | 1,130
November | 56,000 | 81,000 | 11,700
December | 63,000 | 19,000 | 10,600
----------------------+--------------+--------------+--------------

Again, another example:

RIVER LEA WATER COLLECTED AT CHINGFORD

Number of Micro-organisms Obtained from 1 cc. of Water.

----------------------+--------------+--------------+--------------
MONTH. | +1886.+ | +1887.+ | +1888.+
----------------------+--------------+--------------+--------------
| | |
January | 39,300 | 37,700 | 31,000
February | 20,600 | 7,900 | 26,000
March | 9,025 | 24,000 | 63,000
April | 7,300 | 1,330 | 84,000
May | 2,950 | 2,200 | 1,124
June | 4,700 | 12,200 | 7,000
July | 5,400 | 12,300 | 2,190
August | 4,300 | 5,300 | 2,000
September | 3,700 | 9,200 | 1,670
October | 6,400 | 7,600 | 2,310
November | 12,700 | 27,000 | 57,500
December | 121,000 | 11,000 | 4,400
----------------------+--------------+--------------+--------------

"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:

No. of Colonies
per cc.

Above Munich 531
Near entrance of principal sewer 227,369
13 kilometres from Munich 9,111
22 " " " 4,796
33 " " " 2,378

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

B. TYPHOSUS B. COLI

_Morphology:_ Cylindrical bacillus Shorter, thicker; filaments rare.
2.4 µ, unequal lengths; some
filaments.

_Flagella:_ Long, wavy, spiral, Shorter, stiffer, fewer; movement
and very numerous; movement very less active.
active.

_On Gelatine and Agar:_ Angular, Even edge, homogeneous; much
irregular, raised colonies; slow larger, quicker growth, and less
growth; translucent; medium translucent than _B. typhosus_;
remains clear. medium becomes turbid or
coloured.

_In Gelatine:_ In ordinary Under the same circumstances
gelatine and in sugar gelatine abundant gas is produced.
no gas is produced.

_Milk:_ Not curdled by the Milk is coagulated (within three
bacillus. days).

_Indol:_ The production of indol Indol is present.
in ordinary broth is _nil_.

_Potato:_ The "invisible growth," Thick, yellow growth.
if potato is acid.

_Lactose:_ Fermentation very slight. Fermentation marked.

_25 per cent. Gelatine at 37° C.:_ Gelatine remains limpid and clear,
Strongly and uniformly turbid but possesses thick pellicle.
(Klein).

_Elsner's Iodised Potato Gelatine:_ Very fast growth; larger, brown,
Slow growth; small, very less transparent colonies.
transparent colonies.

_Widal's Test:_ Bacilli become Bacilli remain actively motile.
motionless and clumped together
when suspended in a drop of blood
serum from a typhoid patient.

_Broth containing 0.3 per cent. Phenol Grows well.
or Formalin_ (1:7000): No growth.

_Thermal Death Point:_ 62° C. for five 66° C. for five minutes (Klein).
minutes (Klein).

_Vitality in Water and Sewage:_ The _B. coli_ retains for a much longer
Typhoid bacillus soon ceases to time its vitality and power of
multiply and readily dies (Klein). self-multiplication (Klein).

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.

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.

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(Agglutination by serum from typhoid patient)

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(From agar culture, 48 hours growth)

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(Spore formation. From agar culture)

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_By permission of the Scientific Press, Limited_]

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