Eosin, if present in cultures containing tetanus spores, prevents the germination of these spores when its concentration (in glucose bouillon) reaches 0.2 per cent. When the concentration of the eosin sinks to 0.01 per cent., germination of the spores is no longer inhibited, but the vegetative bacilli developed from the spores execute a highly restrained form of multiplication. When the eosin concentration sinks to 0.001 per cent., vegetation and multiplication of the bacilli become more active, but no new spores are formed even after long periods of time. With glucose agar it is not until the concentration of the eosin in the cultures falls to .05 per cent. that sporulation again appears. At this concentration of the eosin, very few spores are formed ; but as the eosin sinks lower and lower, sporulation becomes more active, until with 0.001 per cent. it is essentially of normal degree. In concentrations of 0.003 per cent., eosin prevents perfect segmentation of the multiplying bacilli, with the result that, finally, long and convoluted threads of bacilli are produced. The spores which are formed in a medium containing 0.01 per cent. of eosin are often situated at the centre and not at one pole of the bacilli.
Eosin in a strength of 2 per cent. is capable of destroying the vegetative bacilli, if the contact is prolonged to fifteen minutes, and in strength of 0.1 per cent., in twenty-four hours. Placing this latter mixture of bacilli and eosin in the sunlight greatly hastens the bactericidal effect, and the bacilli are found to be incapable of growth at the end of several hours. Eosin in high concentrations is not capable of killing the tetanus spores, even after long exposure to sunlight (thirty hours).
The toxin production of tetanus bacilli grown in eosinized culture media diminishes as the concentration of the eosin increases. This effect is brought about partly by the restraining action of the dye on vegetation, and partly by its detoxicating action upon the poison.,
The toxin-producing power and the virulence of tetanus bacilli are not permanently modified by contact with eosin for a long period, or by successive cultivations in eosinized media.
Eosin is likewise capable of restraining the vegetation of tetanus spores in the animal body. In spore threads inserted beneath the skin of rats, and surrounded with eosin in solution, a very restricted vegetation takes place. If the injections of eosin are repeated. vegetation soon ceases and the vegetated bacilli degenerate and disappear.
The ungerminated tetanus spores remain alive in a latent condition indefinitely in the healed wound beneath the skin. These spores do not lose power to grow outside the body, or inside the body of animals under favorable conditions, or to produce toxin in a characteristic manner.