THE CULTIVATION OF AMOEBAE IN PURE CULTURE UPON AUTOLYZED TISSUES

Since the purpose of this paper is to record the cultivation of amoebae upon autolyzed tissue without bacterial association, the morphological characteristics, life cycle, means of differentiating species, and pathogenicity of the protozoa have been omitted. These subjects will be considered in later publications. The result of this study proves that some species of amoebae from liver abscesses and the human intestine can be cultivated upon various autolyzed tissues of man and some of the lower animals without a symbiotic microorganism. Their cultivation from liver abscesses upon such bacteria-free autolyzed tissue indicates that their multiplication in these lesions depends upon some product or products in the process of dissociation of the liver cells. That such a process exists in amoebic liver abscesses cannot be questioned when histological and biochemical studies are made of such lesions, and this explains not only why the multiplication of the parasites in the organ occurs, but suggests the probable origin of the lesion. It has long been known that tissue kept for several days in a perfectly aseptic condition and at body heat, or preferably at slightly higher temperature undergoes softening and final disintegration of its cells. Wells and others who have made a thorough study of this phenomenon find that different enzymic actions take place in this process: thus in the liver they find that soluble nitrogen compounds are greatly increased, the nucleoproteids are altered by nuclease, the purin bases are freed and in their turn acted upon by the guanase and adenase, the fats are split and fatty acids set free, the glycogen gives rise to glucose and undergoes further splitting. lecithin is cleaved, and allied bodies appear, and there is a marked appearance of cholin and cholesterin. Similar changes varying only in degree occur in the process of autolysis of other tissues. Furthermore, Duval in his experiments upon the cultivation of Bacillus leprae found that the initial multiplication was accomplished when the specific organism was in symbiosis with an associated bacterium capable of hydrolyzing the leprous tissue. In later experiments he noted that the products of split proteins supply what is actually required for the growth of this particular obligate cell parasite, and that while this end is reached with bacteria through their proteolytic action equally good results can be obtained with tissue free from contaminating microörganisms provided that it is allowed to autolyze. The separation of amoebae cultivated from the human intestine from their bacterial symbiont, and their development upon various autolyzed tissues indicate that it is not the bacterium that is essential for the life of these protozoa, but the action of the living bacteria upon the protein contained in the media. This would explain the failure of many investigators to cultivate amoebae with dead bacteria or bacterial filtrates. Mention has been made that the autolyzed tissue used in the cultivation experiments gave a distinct acid reaction. The multiplication of amoebae upon a medium with such a reaction appears contradictory to the findings of Musgrave and Clegg, Walker, and others, who have emphasized the necessity of an alkaline medium for the successful cultivation of amoebae with a bacterial symbiont, though in accord with what is known to be the reaction of the contents from amoebic liver abscesses and of the bloody stools in intestinal amoebiosis. The fact should not be lost sight of that, in the cultivation of amoebae, these authors lay stress upon the selectiveness of amoebae for a special microörganism. A comparison of their work with our own results indicates that bacteria known to possess strong hydrolyzing properties, e. g., Vibrio cholerae, Bacillus coli, Bacillus subtilis, Vibrio proteus (Finkler-Prior), etc., furnish the best symbionts to the amoebae. It is well known that these bacteria growing upon gelatin or blood serum liquify the medium and alter its reaction to a marked degree of acidity. This acidity, for the most part, results from dissociation of the protein molecule contained in the media, for Wiener has shown that autolysis does not begin until the normal alkalinity of the tissues has been neutralized by the production of organic acids. Since experiments show that the development of the amoebae is scant or completely arrested if the autolyzed tissue is smeared upon an acid agar base or upon agar with an alkalinity higher than 1 per cent., the limit within which multiplication can occur must be small. It is possible that this explains why amoebae are so few in the center of liver abscesses where the acidity is very high and so plentiful in or near its walls where the normal tissue juices furnish a controlling influence over the acidity of the autolyzing tissue. It explains also why a neutral or a 1 to 1.5 per cent. alkaline medium is essential for the cultivation of amoebae with a bacterial symbiont. Here the necessity for a medium with an alkaline reaction seems necessary, as bacteria, especially those having high hydrolyzing properties, develop a marked acidity in the medium. If the medium possesses an initial acidity the limit is either quickly reached or already present and no multiplication occurs. On the other hand, a medium with a reaction too alkaline either inhibits the growth of symbiotic microörganisms, or neutralizes the acid products as rapidly as they are formed by the associated bacterium. Whether the amoebae cultivated from liver abscesses and from the intestinal canal upon diverse autolyzed tissues are able to produce lesions similar to those from which they were isolated, or whether they are non-pathogenic species which are accidental contaminators to those responsible for frank lesions, remains still to be determined. Experiments bearing upon these points, as well as on many others made possible by this work, are now under way.