THE IN VITRO REACTION OF LIMULUS AMEBOCYTES TO BACTERIA

1. For effective in vitro study of the defense mechanisms against bacterial infection of Limulus polyphemus an unsiliconized and a siliconized culture system for blood cell maintenance were developed. The former contained viable but partially degranulated amebocytes for at least 36 days, whilst the latter system kept the cells in an intact state resembling the in vivo for up to 30 days. Replenishment of the culture medium (undiluted Limulus serum) and use of antibiotics were found unnecessary. No cell multiplication was noted.2. The reactions of the blood elements to certain nonpathogenic and pathogenic marine bacteria (and to their toxin) were studied. When a pathogenic Vibrio was added to the siliconized system of granular, discoid cells, persistent gelation of the medium regularly accompanied bacterial growth, the gel containing pathologically altered amebocytes and granules. While gelation is characteristic for shed normal Limulus blood, intact cultures—both uninfectcd as well as those which overcome the nonpathogenic Alkaligenes-like bacterium—often showed either absence or ephemeral presence of gel in the medium. Such cultures also contained normal granules and showed large clumps of agglutinated dead bacteria consistently, suggesting involvement of the gelation process in immobilization and, perhaps, killing of the invading organism. The culture systems were active against several strains of marine bacteria. Experiments with a nonpathogenic Alkaligenes-like bacterium showed that only the intact, granular cultures showed rapid (6 hours at 20-25° C.) and permanent elimination of large numbers (inoculum sizes up to several million), the unsiliconized extended cell and whole blood homogenate systems merely exhibiting different levels of evanescent activity.3. The successful infection of the intact system, by very small numbers (10-100), of pathogenic Vibrio clearly established its in vitro pathogenicity. Its thermostable toxin caused prompt changes in the morphology of the intact amebocytes in culture, even at a 10-3 dilution, and a phase microscopic study of the action of the Vibrio itself on the cells revealed rapid cellular alterations culminating in cytolysis.