This report gives an outline of the results of observations on over 1,100 tissue cultures made during the fall, winter, and spring of 1913 to 1914. The work has been resumed in the fall of 1914 and will be continued along the same and allied lines, confirming the above results with other strains of the same organisms and with other bacteria and bacterial products. Tests should be made with tissue and plasma from other animals, and the known pathogenicity of the organism for the animal and tissue used should always be borne in mind. We hope to be able to grow on these cultures some of the more strictly parasitic bacteria not developing on ordinary media, as other strict parasites have been grown by other workers, viz., poliomyelitis virus by Levaditi (4), vaccinia by Steinhardt, Israeli, and Lambert (5), and rabies by Moon (6). By comparative studies with various types of cells and various natural and artificial media, clearer ideas as to the exact part of cell plasma in antibody production, by elaborating on the methods of Carrel and Ingebrigtsen (7), Lüdke (8), Przygode (9), and others, should be possible.
After this work was begun a reference was found to the use of some pathogenic bacteria in tissue cultures by Pheiler and Lentz (10), but no publication of the results of these observations has been observed.
The results here reported may be summarized as follows:
Bactericidal Action of Chicken Plasma.—On Bacillus typhosus, very strong—never grows in plasma alone; on Bacillus prodigiosus, very strong—never grows in plasma alone; on Bacterium pseudodiphtheriticum, strong—slight growth in cover-glass preparations; on Bacterium diphtheriticum, moderately strong; on Bacillus coli verus, slight; on Micrococcus aureus, very slight or none.
A few pseudodiphtheria bacilli and more diphtheria bacilli survived in plasma stored in the cold for four days. The presence of growing tissue overcomes the bactericidal influence of plasma on diphtheria bacilli and in some instances on pseudodiphtheria bacilli.
Bacterium diphtheriticum grows in plasma without tissue only if inoculations are very heavy; and very heavy inoculations of all organisms will probably overcome the bactericidal action of plasma, as it is undoubtedly a quantitative reaction. The bactericidal influence of plasma is overcome by exposure to incubator temperature for twenty-four to forty-eight hours. Bacterium diphtheriticum in light or moderate inoculations grows in tissue cultures only in clusters around the tissue fragments, and never in plasma away from tissue. The growth of this organism has a decided inhibitory influence on tissue activity and growth, especially marked with nervous tissue, but this action may be overcome by the addition of antitoxin to the plasma. Cultures inhibited by diphtheria growth have a tendency to resume growth later, probably due to antitoxin production.
Bacterium pseudodiphtheriticum is distinctly less active in tissue cultures than is Bacterium diphtheriticum and never develops in plasma without tissue. The presence of diphtheria toxin in tissue cultures causes this organism to behave as does Bacterium diphtheriticum. Without toxin it has little or no direct influence on tissue growth except in massive doses.
Bacillus prodigiosus fails to develop, as a rule, in tissue cultures except where inoculated from hypertonic media, and then it has no decided influence on tissue growth.
Micrococcus aureus grows freely in these cultures with or without tissue, and inhibits tissue growth markedly, except as noted with splenic tissue.
Bacillus coli verus always grows freely with or without tissue fragments and is uninfluenced by splenic tissue growth. In heavy inoculations it lessens tissue growth.
Bacillus typhosus, except with extremely heavy inoculations, fails absolutely to grow in these cultures with or without plasma, unless the bactericidal action of the plasma has been destroyed by incubation. When this is the case it develops freely with especial affinity for the tissue cells either for support or nourishment. It appears to have no toxic action on the tissue cells. Note the sharp differentiation between typhoid and coli verus organisms.
Diphtheria toxin has a quantitatively inhibiting action on all tissue growth and on heart tissue pulsations, the action being greatest on nervous tissue and least on heart tissue growth. Tissues affected by toxin tend to recovery if not killed. Antitoxin counteracts the action of toxin.
Splenic tissue has little or no effect on the growth of Bacillus coli verus, but has a decided bactericidal action on Bacterium diphtheriticum and Micrococcus aureus, probably due to lymphatic cells and cell products, as seen by the area of cell migration coinciding with the bacteria-free area, by the similar action of splenic extract on cultures, and by the failure of such action in cultures of very early splenic tissue showing no lymphatic cells.
Nervous Tissue Growth