In view of its possible relation to the epidemiology of plague, a knowledge of the biology of the common rat-flea of England, Ceratophyllus fasciatus Bosc, is much to be desired, and therefore, at the request of Professor Niittall, the present investigation was undertaken. The observations recorded below are mainly the result of experimental work with fleas conducted in the Quick Laboratory, Cambridge; but before proceeding to a description of the experiments it is considered advisable to give a short summary of the life-cycle of the rat-flea.

It is shown that provided the total number of individuals exposed to a constant environment which is inducing change within them be constant, and the number of units of change which must take place within any given individual in order to cause a given event be also constant, then the number of these events is a quantitative measure of the extent of the change in all of the individuals taken together.

From this it follows that the results of Madsen and Nyman and of Chick may legitimately be regarded as proving that the process which underlies disinfection obeys the time-relations and other characteristics of a mono-molecular chemical reaction.

Interesting results were obtained with cultures in ammonia agar, from them it is evident that the nitroso-bacterium grows readily through this medium whether inoculated on the centre of a plate or when covered with the agar jelly itself. Further, the presence of 5 to 10% of beef broth in the ammonia agar allows of better development.

Potassium phosphate agar containing bouillon gelatine but no free ammonia, on being inoculated with cultures of nitroso-bacteria, also developed nitrification; the organic matter was broken down, ammonia formed, and nitrites built up.

Nitrification in ordinary bouillon agar plates shows that organic matter does not necessarily form a barrier to the process; and further, that two of the cultures had only been fed with inorganic salts for three and four years respectively prior to the inoculation into the plates, is good evidence that gradual additions of organic matter are not absolutely necessary.

Calcium sulphate blocks allowed me to work with a larger amount of very active culture: from these it was found that urine was a very good culture medium indeed. Also that peptone water diluted with water, or peptone beef broth, milk, or blood serum diluted with ammonium sulphate could be used as culture media; sometimes these checked nitrification for a time but it afterwards became well established.

The experiments with undiluted urine were wholly successful and stand as a further proof of nitrification in presence of organic matter.

From these results it is clear that the nitroso-bacterium is a very powerful nitrite-forming micro-organism, that it is not readily destroyed, and that a certain amount of organic matter is essential to its greatest development, but for this a large amount of culture must be used.

In conclusion I have to offer my deepest thanks to Sir William Power for the great help that he has given me and for the kindness that he has always shown; also to others who have most kindly advised me my grateful thanks are due.

It is well known that typhoid bailli, freshly isolated from enteric fever patients, vary in their capacity to undergo agglutination. The following series of experiments was carried out with the object of discovering whether these differences in agglutinability are dependent on the stage of the disease at whih the bacillus is isolated, or on the body substance from which it is obtaind.

Whether the vaccine virus is filterable or not has been the subject of experiment and discussion for many years and since Negri's results published in 1905, from which he deduced that the virus was filterable, numerous experiments dealing with the subject have been made by many workers.

Considerable difference of opinion has existed as to whether the incidence of cancer is at all affected by the habits of life of different classes of the community. There is of course no doubt that in certain occupations particular forms of malignant disease are exceptionally prevalent, the case of chimney sweeps being notorious; but the more general question as to the incidence on large groups of occupations has not been so definitely determined. The present research was suggested by the apparently contradictory results obtained by Heron from his study of London statistics, and by Maynard from an analysis of United States data.

Both the sensitation of typhoid bacilli by serum and their ingestion by leucocytes when sensitised, have been shown by Ledingham (1912) to take place in accordance with the laws regulating an adsorption process, the removal of opsonin from a serum by bacteria, and of sensitised bacteria from an emulsion by leucocytes following very satisfactorily the adsorption equation y=kxn, where y= the amount adsorbed, x= the amount left free and k and n are constants.

(1) If B. coli be subcultured into another sample of the same medium when growing at full pace, it will continue to grow at the same pace.

(2) If the maximum rate of growth be interrupted by a short application of cold, growth will recommence without lag on the temperature being raised. If the cold be long continued, lag will tend to reappear.

(3) Differences in the size of inoculum have practically no effect on lag in the case of large inoculums, in the case of small ones, on the other hand, diminution of the seeding has the effect of lengthening lag, and this lengthening effect is more marked the smaller the seedings become.

(4) Lowering the temperature lengthens the lag. The effect is very similar to the effect on growth.

(5) The older a parent culture (within limits) the longer the lag.

(6) The length of lag varies with the medium even if adaptation has been arranged for beforehand.

(7) Heat-stable products in B. coli cultures on peptone water have, in the case of overnight cultures, but little effect on lag.

(8) After washing the bacteria for two hours with saline in order to remove possible inhibiting agents, it was found that the lag, on subculture, still occurred and was indeed slightly longer.

(9) If a peptone water culture of B. coli be centrifuged, it is found that the few bacteria remaining in the supernatant commence to grow again at a quick rate but not without a period of lag.

Of the large number of experiments recorded by Penfold (1914) in connexion with the question of lag in bacterial growth, a certain proportion have been carried out in such detail as to render them eminently suitable for mathematical analysis. In all work of this kind, which is intended to throw light on the numerical aspect of bacterial growth, it is desirable to take observations at frequent and, if possible, regular intervals, during the period of lag, and to count a sufficiently large number of colonies on the plates. To secure this last and most important desideratum, orientating experiments must be performed, from the results of which one is enabled to calculate what dilution of the culture at any given period of growth will yield a tolerably large and accurately countable plate population.

The experiments detailed above have already been discussed in the sections in which they have been recorded. The following is a brief summary of the results obtained.

(1) In many ways a clear watery extract of B. typhosus behaves like an ordinary emulsion of the bacilli. This is true in a general sense in respect of agglutination by serum, and the property of agglutinability by weak salts, which some emulsions of B. typhosus possess, is also exhibited by a watery extract of the same strain.

(2) The optimum hydrogen-ion concentration for the agglutination of an emulsion is also approximately the optimum for the agglutination of a watery extract made from the same emulsion. The washed bacilli and the original emulsion show a second optimum. Agglutination appears later at this secondary optimum which is in stronger acid. There is a zone of absent or deficient agglutination in solutions of intermediate strength. The lower [H·] of the two is the characteristic optimum for B. typhosus, i.e. about 3·6×10−5. The second optimum [H·], i.e. 1·1 × 10−3 to 2·2 × 10−3, is approximately the same as the optimum for the agglutination of emulsions of many different bacteria, e.g. some strains of B. coli, staphylococcus, etc., and moreover is about the optimum for the agglutination of an emulsion of B. typhosus after it has been heated to 100°C.

These two optima appear to indicate the presence of two acid agglutinable substances in an emulsion of B. typhosus, (a) which is characteristic of this bacillus and is present in a watery extract (this substance is rendered inagglutinable by heating to 80° C. for 30 minutes or to 100° C. for five minutes, or longer, e.g. one hour), and (b) which is present in the bodies of the bacilli after repeated washing and which is not characteristic of B. typhosus, but has characters shared by similar substances in other bacilli; it resists heating to 100° C. for five minutes and is apparently little, if at all, affected by heating to 80° C. for half an hour.

(3) The substance agglutinable by serum and that agglutinable by acid No. 5, i.e. [H·] 3·6 × 10−5, are both present in a watery extract and can both be completely removed from the bacillary bodies by washing with distilled water. The serum-agglutinable substance is however less easily completely removed. This apparent difference is perhaps due to the fact that serum is a more delicate reagent for testing for the presence of agglutinable substance. If an extract is diluted it will give a precipitate in higher dilution with serum than with acid and the bacillary bodies when repeatedly washed often react with serum after the acid agglutination in solution 5 or 6 has ceased to appear. These substances agglutinable by acid and by serum are however closely associated and are both precipitated by acid No. 5 or 6.

The identity of the two substances has however not been proved and the following considerations are very strongly opposed to this assumption, (a) a considerable number of strains of B. typhosus are agglutinable by serum and not by acid; (b) strains have been described which are agglutinable by acid No. 5 or 6 but not by serum; (c) the diminished agglutinability by serum exhibited by some strains which are inagglutinable by acid is apparently due to a total diminution of the “soluble” or extractible substance; (d) the substance agglutinable by acid, tube 5 or 6, i.e. [H·] 3·6 × 10−3, is rendered inagglutinable by heating to 100° C. for five minutes, whereas Porges and Prantschoff (1906) have shown that the serum agglutinable substance is still active after being boiled.

It is not possible at present to say for certain whether the substances agglutinable by serum and acid are identical or distinct. If they are identical and there is only a single substance, its agglutination properties vary independently in different strains of B. typhosus.

(4) The importance for agglutination by serum of the substances in “solution” in the liquid part of a bacterial emulsion, and the impossibility of distinguishing between agglutination and the precipitation of bacterial extracts by serum, were first pointed out by Kraus and Paltauf, and by Kraus and v. Pirquet.

Their view is confirmed and extended to the agglutination by acids Nos. 5 and 6 by the result of some of the experiments described above. The facts adduced in support of the view that agglutination is essentially the formation of a coagulum in the liquid part of an emulsion are the following:

(a) The property of agglutination may be completely removed from B. typhosus by washing with water; the resulting washed bacterial bodies no longer agglutinate with specific serum or in acid solution Nos. 5 to 7.

(b) The washings of the bacteria or watery extracts are agglutinable by specific serum or by acid solution No. 5 or 6.

(c) The watery extract even in very small quantities when added to the washed bacilli, indifferent bacilli or other finely divided particles, confers on them the property of being agglutinated by specific serum or acid solution 5 or 6.

(d) The extract reacts with highly diluted serum, e.g. 1/27,000, and may be considerably diluted, e.g. 1/96, without entirely losing its agglutinable properties with serum or acid.

(e) In order to demonstrate the presence of agglutinable substance in very low concentration (especially if highly diluted serum or if acid is used) it is necessary to add some indifferent substance, e.g. B. coli, kieselguhr, globulin, etc. in order to increase the bulk of the precipitate.

(f) The extract or successive extracts appear to contain all the characteristic agglutinable substance of the original emulsion. This is shown by the delicacy of the reaction, and the small amount of extract required to agglutinate an indicator, e.g. B. coli. The demonstration of the reaction with highly diluted serum 1/27,000 and much diluted extract 1/96 appears to be an advance on the work of previous writers. Nicolle did not use indifferent particles with high dilutions of serum or of extract, and the only other use of an indicator under such conditions appears to be the use of conglutinin by Bordet and Gay, and Streng, and of mid-piece by Dean.

(g) The microscopic demonstration of the coagulum formed in the process of agglutination has been made easy by taking a watery extract containing very few bacilli, i.e. ordinary emulsion from which most of the bacilli have been removed by centrifuging. After adding dilute specific serum or acid the formation of the coagulum may be watched in a microscope cell with dark ground illumination or by adding dilute fuchsin or eosin to the microscope cell.