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.