Solvent action of Sarcina lutea on egg-albumen-agar. This print

FIG. 4.

FIG. 5.

was made by placing the Petri dish on the photographic paper and illuminating it for the proper time.

FIG. 6. Solvent action of Bact. lactis acidi on calcium carbonate suspended in the agar.

CRABILL AND REED: CONVENIENT METHODS FOR DEMONSTRATING
THE BIOCHEMICAL ACTIVITY OF MICROORGANISMS

REACTION OF RABBITS TO INTRAVENOUS INJECTIONS OF MOULD SPORES1

A. F. BLAKESLEE AND ROSS AIKEN GORTNER

(Conn. Agric. College)

(Univ. of Minnesota)

(WITH PLATE 2)

The work outlined in this paper was undertaken in connection with experiments having as their object a determination of the chemical differences which may exist between the two sexual races in the fungous group commonly known as the mucors. In these forms the majority of the species are diœcious, having separate male and female races, which may be propagated independently by means of vegetative spores.

As is well known the repeated injection of red blood corpuscles and of certain other cells is capable of producing in the blood of rabbits cytolytic antibodies that will dissolve these cells when they are subsequently mixed with the serum of the treated animal. It was thought that a similar cytolysin might be developed for mould spores, and that the action might be found to be sexually specific.

Although agglutinins apparently are formed, no cytolysins for fungus spores could be induced by intravenous injections. Despite the largely negative character of the results obtained, they seem to be sufficiently controlled to show positively that rabbits are incapable of producing cytolysins for the spores of the mucor tested. The fact that the cell wall is highly resistant throughout the fungi renders it extremely improbable that cytolysins can be developed for spores of any other fungus form.

Preliminary tests showed that spores of Cunninghamella echinulata, when injected intravenously, kill a rabbit within a week's time (four instances). Postmortem examination demonstrated the presence of germinated spores in the lungs. This mould is a tropical form growing readily at temperatures above 31° C., and its growth in the rabbits may have caused death by mechanically interfering with the functions of the organs infected.

Most species of the mucors will not grow at temperatures over 30° C. Among the forms that grow only at relatively low temperatures, "Mucor V"-a form similar to M. hiemalis, if not identical with this species gives an especially strong sexual reaction. Its spores are relatively small (about 8 × 3.5μ) and can offer little interference to the circulation. No strong toxins, moreover, are developed by this mould such as have been found in the allied form Rhizopus nigricans (1, 2). Altogether, the species seemed especially favorable and has been used in the present investigation.

The mold was grown, generally on agar, in shallow pie-tins protected with paper covers. Water was poured over the mature culture and filtered through linen which allowed the spores to pass while keeping back fragments of the aerial mycelium which it was feared might block the capillaries. The spore-water was centrifuged and the resulting compacted mass of spores was mixed with 0.9 percent salt solution and used at once for the injections. Centrifuged spores were dried in a vacuum desiccator and in a few instances were used later when fresh spores were not available. The injections were all made in an ear vein, the usual aseptic precautions being observed. The dose varied from 3 to 4 c.c. The sporewater was always very dark with spores and, although counts were not made each time, the individual injections can be considered to average about 500,000,000 spores.

Rabbit No. 5, beginning April 2, 1913, received at intervals of about 4 days, 28 preliminary injections of the spores of the race (3) of "Mucor V." Rabbit No. 55, beginning April 17, similarly received 27 preliminary injections of the ♀ spores of the same species. On August 13, five days after the last injection, rabbits Nos. 5 and 55 received approximately 800,000,000 spores, respectively, of the andraces; and at the same time two control rabbits, Nos. 6 and 66, previously untreated, were similarly injected with like doses of the ♂ and spores, respectively. This made the 29th injection for rabbit No. 5, and the 28th for No. 55. The four