Juke i8, 1903J 



NATURE 



153 



jection of killed cultures of the bacteria in question. 

 The typical examples are the vaccines employed in 

 cholera, plague, and typhoid fever for prophylactic 

 purposes. The killed cultures of the several organisms 

 are injected directly into the healthy individual in cal- 

 culated doses, and the method is generally described as 

 one of protective inoculation. In all these cases the im- 

 munising value of the vaccine appears to lie essentially 

 in the dead bodies of the bacteria it contains. The 

 active immunisation that occurs depends upon a solu- 

 tion of the dead bacteria by the blood and tissues, and 

 a consequent liberation of any immunising substances 

 peculiar to the cells. The properties developed by the 

 blood of the treated individuals are antibacterial and 

 not antitoxic, or if so only to a small degree. If one 

 assumes that the properties of the blood in such in- 

 stances are purely of a bacteriolytic character, there 

 would be no protection necessarily afforded against 

 any poisonous substances that might be present in the 

 bacterial cells, and liberated from them in the process 

 of their dissolution or in the course of the disease. 

 Whatever the point of view, the conviction is now an 

 established one that in a number of infective diseases 

 it is the direct study of the specific cellular agents that 

 will be most likely to lead to results of therapeutic 

 value. The important conclusion has been arrived at 

 that there are two kinds of bacterial poisons — soluble 

 toxins, which are secreted by the bacteria, and cellular 

 toxins, which are contained within their body sub- 

 stance. The toxins may be either extra- or intracellu- 

 lar. The diphtheria and tetanus poisons, already re- 

 ferred to, are examples of the first group, and are to 

 be met with in the nutrient fluids in which the 

 organisms are cultivated. The typhoid and plague 

 toxins are examples of the second group, and are prac- 

 tically absent from the culture fluids in which the 

 specific organisms are grown. The poisonous prin- 

 ciples are contained within the bodies of the microbes. 

 The dead bodies of typhoid bacilli, although destitute of 

 all infective properties, are yet toxic when introduced 

 into animals in virtue of the intracellular toxins they 

 contain — the animals succumb to an intoxication. 



In the case of many diseases formerly regarded as 

 purely infective in character, it has now become ap- 

 parent that, in addition to the infective, the poisonous 

 properties of the invading bacterial cells have to be 

 taken into account. Any therapeutic endeavours of a 

 curative character, it appears to the writer, ought 

 therefore to be based on the presumption that every 

 infection implies, sooner or later, an intoxication. 



The number of infective organisms in connection 

 with which research has failed to demonstrate soluble 

 toxins of possible clinical importance is considerable. 

 The presumption in such cases is that the missing 

 toxins are intracellular, and that if antitoxic principles 

 of treatment are to be devised they must be based on 

 a knowledge of the nature and properteis of these 

 cellular poisons. A vital question consequently for 

 bacteriologists at the present moment is the relation 

 of intracellular toxins to immunity. The study of the 

 intracellular constituents of bacteria has, it will be 

 obvious, assumed great importance on account of the 

 issues involved. It is interesting to note, by way of 

 parenthesis, how generally biological research is being 

 attracted to the direct study of the cell, and how widely 

 it is being recognised that the processes of life, whether 

 of a natural or a morbid character, are in their essentials 

 of an intracellular nature. In this respect the re- 

 searches of Buchner were of wide biological signifi- 

 cance. They were suggestive of much more than a 

 theory of a cell-free alcoholic fermentation of sugars. 

 And in the bacteriological field the original investiga- 

 tions of Koch, and the preparation by him of tuberculin 

 from the tubercle bacillus, drew general attention to 



NO. 1755, "^OL. 68] 



the important results that might be obtained from the 

 study of the bacterial cell and its constituents. Various 

 methods are employed with this object in view. The 

 killed cultures of bacteria may be used, and their 

 physiological effects determined by injection into 

 animals, or by chemical means extracts may be pre- 

 pared from the organisms and their effects similarly 

 tested, or mechanical methods may be adopted in oider 

 to obtain the fresh intracellular juices. 



In the investigations carried out by the writer, in 

 conjunction with Mr. Rowland, during the past four 

 years, mechanical methods were selected as the best 

 adapted for the general purpose in view, viz. the 

 study of the intracellular toxins and ferments of bacteria 

 and other cells. The first essential was the elaboration 

 of a method to obtain the plasma of such minute cells 

 as the bacteria. The aim was to procure the fresh cell 

 juices and to avoid their possible modification by heat 

 or by chemical agents. For this purpose the cells were 

 mechanically triturated by a cold grinding process. 

 The necessary cold was most conveniently obtained by 

 the use of liquid air. It was found that the cells could 

 be mechanically broken up when in the brittle condi- 

 tion produced by immersion in liquid air, without any 

 admixture of sand or other foreign substances. A 

 number of bacteria and other cells have been triturated 

 in this fashion, and their fresh intracellular constituents- 

 obtained. The results In the case of the typhoid 

 bacillus will serve to illustrate the general line of re- 

 search being followed. The typhoid organisms were 

 grown on ordinary beef broth agar, and after careful 

 washing with distilled water were disintegrated in a 

 mechanical contrivance at the temperature of liquid 

 air ( - 180° C). The disintegrated mass was freed 

 from insoluble suspended particles by centrifugalisa- 

 tion, and an opalescent fluid, representing the cell- 

 juices of the organism, resulted. The typhoid cell- 

 juices obtained by this method, on inoculation \nX.o 

 animals in small doses, invariably proved toxic or fatal. 

 It was therefore concluded that the typhoid bacillus 

 contains within itself an intracellular toxin and that It Is 

 possible to extract It from the organism. 



The typhoid cell-juices were further tested for im- 

 munising and other properties, and were administered 

 subcutaneously to suitable animals. The experiments 

 showed that the serum of the monkey, after injection 

 of the typhoid cell-juices, possessed antibacterial and 

 antitoxic properties, inasmuch as the serum protected 

 experimental animals against the typhoid bacilli, and 

 also against the intracellular toxin obtained from them. 

 The serum was found to possess curative and preven- 

 tive properties as regards the typhoid bacillus and the 

 intracellular toxin extracted from the same organism. 

 The research thus afforded proof that in the case ot 

 one species of pathogenic bacterium, the intracellular 

 juices of the organism, when injected into a suitable 

 animal, give rise to the production of a serum which 

 is both bactericidal to the organism Itself and anti- 

 toxic as regards a toxin contained in its substance. 



The results already obtained are such as to en- 

 courage further inquiry as to the possibility of their 

 practical application In the case of typhoid fever, as 

 well as to determine in how far such properties of the 

 cell-juice are shared by other pathogenic microbes. The 

 particular method employed has proved of value in the 

 study of a distinct class of toxins and ferments brought 

 to light by recent research which are contained and 

 operate within the cell and bacterium, in contradistinc- 

 tion to the well-known class of toxins which are extra- 

 cellular, i.e. extruded during life from the cell to the 

 surrounding medium. The importance attached to the 

 intracellular group of bq^terial poisons is evidenced 

 by the increasing attention that is now being paid to 

 their study. Allan Macfad^en. 



