Bacteria / Especially as they are related to the economy of nature, to industrial processes, and to the public health

by Sir George Newman

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.

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.

COMPARATIVE FEATURES OF BACILLUS TYPHOSUS AND B. COLI

B. TYPHOSUS	B. COLI
Morphology: Cylindrical bacillus 2.4 µ, unequal lengths; some filaments.	Shorter, thicker; filaments rare.
Flagella: Long, wavy, spiral, and very numerous; movement very active.	Shorter, stiffer, fewer; movement less active.
On Gelatine and Agar: Angular, irregular, raised colonies; slow growth; translucent; medium remains clear.	Even edge, homogeneous; much larger, quicker growth, and less translucent than B. typhosus; medium becomes turbid or coloured.
In Gelatine: In ordinary gelatine and in sugar gelatine no gas is produced.	Under the same circumstances abundant gas is produced.
Milk: Not curdled by the bacillus.	Milk is coagulated (within three days).
Indol: The production of indol in ordinary broth is nil.	Indol is present.
Potato: The "invisible growth," if potato is acid.	Thick, yellow growth.
Lactose: Fermentation very slight.	Fermentation marked.
25 per cent. Gelatine at 37° C.: Strongly and uniformly turbid (Klein).	Gelatine remains limpid and clear, but possesses thick pellicle.
Elsner's Iodised Potato Gelatine: Slow growth; small, very transparent colonies.	Very fast growth; larger, brown, less transparent colonies.
Widal's Test: Bacilli become motionless and clumped together when suspended in a drop of blood serum from a typhoid patient.	Bacilli remain actively motile.
[59]Broth containing 0.3 per cent. Phenol or Formalin (1:7000): No growth.	Grows well.
Thermal Death Point: 62° C. for five minutes (Klein).	66° C. for five minutes (Klein).
Vitality in Water and Sewage: Typhoid bacillus soon ceases to multiply and readily dies (Klein).	The B. coli retains for a much longer time its vitality and power of 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.

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"[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 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.

Bacillus Typhosus (Showing Flagella) × 1000	Bacillus Typhosus (Widal Reaction) (Agglutination by serum from typhoid patient) × 400
Bacillus Coli Communis (From agar culture, 48 hours growth) × 1000 By permission of the Scientific Press, Limited	Bacillus Mycoides (Spore formation. From agar culture) × 1000

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.