Experiments on the Agglutination of Watery Extracts of B. typhosus

Extract

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.