ing water, whilst the spores of anthrax in their normal state will withstand such temperatures for a considerable length of time. In order to turn these properties to practical account, portions (1 c.c. or 3 c.c.) of the water supposed to contain anthrax spores are mixed with a little sterile broth (1 c.c.), and heated for periods of two or five minutes to 50° C., to 70° C., or to 90° C., after which treatment they are submitted to ordinary plate cultivation. As an illustration of the manner in which this method works the following example may be cited:-Thames water, purposely infected with anthrax spores, and containing upwards of 100,000 water bacteria in 1 c.c., had this number reduced after heating as above for five minutes to 50° C. to from thirty-five to thirty-nine per c.c., amongst which several of the colonies on the plate were recognisable as those of anthrax. Again, on the same day, other portions of the same water were heated to 70° C. for two minutes, after which only from ten to thirty colonies per c.c. made their appearance, amongst which from four to ten were recognisable as anthrax. Other portions of the same water were heated on the same day to 90° C. for two minutes, with the result that only from seven to ten colonies per c.c. appeared, of which from three to six were found to be anthrax. By this simple method the water is deprived of its rapidly liquefying forms, which so quickly render the gelatine-plate fluid and necessitate its being thrown away before the more slowly developing anthrax colonies have been able to make their appearance. Such a water, swarming originally with water bacteria, may thus be easily examined, and the anthrax colonies identified by gelatine-plate cultures. The longer the bacteriological examination of water is practised the more evident does it become that in searching for pathogenic organisms special methods must be devised and adopted according to the nature of the particular microbe of which we are in quest, and that only under the most exceptional circumstances is there any possibility of such pathogenic bacteria being found in the course of ordinary plate cultivations made with natural waters, the colonies of the common waterbacteria almost invariably so predominating as to exclude all others present only in small numbers. Such special methods have, as pointed out above, already been employed, more especially for typhoid and cholera bacilli, as well as for anthrax spores; in all cases these methods should be so arranged as to permit of the examination of larger volumes of water, as in this manner the chance of discovery is correspondingly increased. NOTE. In examining water for the typhoid bacillus it is advisable to pass a considerable volume, 250 c.c. or upwards, through a sterile porcelain or infusorial earth filter (see p. 172), and then to transfer the deposit on the surface of the cylinder by means of a sterile brush into a small quantity of sterile water; the latter, which then contains the bacteria from the large original volume of water, should be treated by phenol-broth culture or one of the other methods of typhoid detection. To assist the diagnosis of the typhoid bacillus in the presence of the B. coli communis, Schild (Zeitschrift f. Hygiene, vol. xvi. 1894, p. 373) recommends the use of broth to which formalin' has been added. Formalin may be procured from Schering, in Berlin, and consists of a concentrated (40 per cent.) aqueous solution of formaldehyde. It should be added by means of a sterile pipette to ordinary neutral broth, of course after the sterilisation of the latter, as heating volatilizes the formaldehyde. The proportion recommended by Schild is 1 : 7000, and he states that, whereas the B. coli communis will flourish in this formalin-broth, rendering it turbid in from 8-24 hours, the typhoid bacillus refuses to grow and the liquid remains clear. This formalin-broth must only be used when freshly prepared, as the formaldehyde volatilizes on being kept. Schild states that in adding the formalin to the sterile broth he has only rarely been troubled with contaminations, and then they were traced to moulds which were easily recognisable. CHAPTER VIII THE VITALITY OF PARTICULAR PATHOGENIC BACTERIA IN DIFFERENT WATERS THE difficulties which, in the last chapter, we have seen attend what may be called the analytical method of investigation into the fate of pathogenic bacteria gaining access to natural waters, early led to supplementary researches by what may be called the synthetic method, in which the pathogenic organisms were purposely introduced on an experimental scale into different kinds of water, which were then kept under observation and examined from time to time for the bacteria in question. Although at first sight it might appear an easy task to thus synthetically determine the vitality of organisms in water, requiring only their introduction into various waters and the subsequent estimation of their numbers. at suitable intervals of time, as a matter of fact, however, the number of problems requiring solution in this connection is continually increasing; for as our knowledge of the physiology and morphology of bacteria becomes more extended from day to day, new factors arise which have to be reckoned with in these investigations. In a subsequent chapter we shall refer to the action of light on micro-organisms. Whilst we have already learnt how great is the effect of temperature upon the vitality of bacteria, and have seen that the composition of the water into which they are introduced is of cardinal importance, we have so far omitted from the difficulties besetting the investigator perhaps the most troublesome of all, viz. the individual characteristics possessed by micro-organisms, and not only exhibited by different varieties, but even by the different individuals in one and the same cultivation, rendering it essential that in all experiments the previous history, as far as possible, of the organisms under observation. should be recorded. In the following tabulated series of researches on the behaviour of pathogenic bacteria in various waters we have endeavoured to give in all cases as fully as possible the essential details of the experiments as described by the authors themselves, together with the references to the original memoirs in which they are recorded. The following is a list of the micro-organisms subsequently dealt with:- 1. Bacillus typhosus-abdominalis (Typhoid Bacillus) 2. Spirillum cholerae asiaticae 3. Bacillus anthracis 4. Bacillus tuberculosis 5. Staphylococcus pyogenes-aureus 8. Micrococcus tetragenus 9. Bacillus of mouse septicemia 1 10. Bacillus of rabbit septicemia 12. Bacillus of swine plague The mode of procedure usually adopted by the various investigators is to ascertain at different intervals of time, generally by means of plate-cultures, whether the organism is still present in the water in a vital condition. By this means information is also obtained as to the decrease or increase which has taken place in the numbers present during residence in the water. This method, reasonable enough as it may appear, is not without certain objections, for in the first place the quantity of water which can be thus examined is relatively very small, and it is highly probable, therefore, that if only a few individual microbes were present in the water they might very easily escape detection altogether; whilst, secondly, the bacteria may, during their residence in the water, have their vitality so far enfeebled that they are unable to grow on a gelatineplate, which often proves a somewhat adverse medium for the development of micro-organisms in a weakly condition. Straus and Dubarry, and Percy Frankland in his later experiments on anthrax in water, have made a practice in particular cases of finally adding nutritive broth to the various samples of water. In this manner, if there are only a few microbes still remaining alive in the water, they will, on the addition of the broth, undergo abundant multiplication, and their presence can then be easily revealed by subsequent plate-cultures. Although in this manner we lose the means of estimating the actual numbers in which the particular pathogenic bacteria under observation are present in the water, we acquire more exact information as to the real duration of their vitality. In addition to the question of vitality, it is of course also of the highest importance to ascertain whether the bacteria have retained their virulence or not, but this can only be done in the case of those bacteria which produce powerfully pathogenic effects on animals. Reference to the experiments which have been carried out in this direction will also be found embodied in the following tabular records of these investigations on the vitality of pathogenic bacteria in water. |