TABLE I

Figures specify number of viable spores recovered from blood of treated and control rabbits at intervals indicated

1⁄2

I

1

* About half the number of spores in this tube were lost by the breaking of the tube. rabbits were all females of about the same age. At the time of the final injections No. 5 weighed 2130 gm.; No. 6, 2160 gm.; No. 55, 1900 gm.; No. 66, 1730 gm.

The number of spores were estimated by counts with a hemacytometer. Loops of blood were taken from these rabbits at half hourly, hourly, and eventually at less frequent intervals. The blood, taken from a slight prick in an ear vein, was at once mixed with sterile water in small vials, and as soon as possible made into separation cultures in Lindner roll-tubes (4). The last injection was made in the left ear and the blood to be tested was always taken from the right ear.

TABLE 2

Figures indicate number of viable spores in loop taken from tubes of serum or salt solution at intervals indicated

Table I shows the number of viable spores in the loops taken, as indicated by the number of colonies developing in these roll-tube separation cultures. The quantities of blood taken up in the loops unavoidably varied, and thus no doubt, influenced considerably the number of spores found at each test. The number of spores injected into the rabbits could not be exactly the same and they may have been for some time unequally distributed in the blood. Such experimental errors may account for a considerable variation in the number of spores found in the individual loops but can hardly explain the relatively large total count for rabbit No. 5. An individual peculiarity of this animal may be responsible for the larger number of spores recovered from its blood. After about 10 hours, viable spores gradually disappear from the blood stream and are no longer found after 43 hours. It is of significance that they disappear from the blood of the rabbits which had been previously treated with spores, no sooner than from the controls. No cytolysins, therefore, or other antibodies capable of destroying the spores have been developed in response to previous injections.

Each of the treated rabbits received an enormous number of spores during the period of injections and must have disposed of them in some way-how, has not been determined. Postmortems on animals 5 and 55 failed to show abnormal conditions of the viscera. Further, the lungs, liver, spleen and kidneys were preserved and later examined microscopically but failed to give evidence of any local accumulation of spores. The spores have hyaline walls and may not greatly differ from blood corpuscles in size or in appearance, when either of these are for any reason distorted. It seemed desirable, therefore, to postpone search after the fate of the injected spores until a form might be used with walls that could be easily identified.

A series of experiments was performed in vitro with the sera of the treated rabbits. Animals 5 and 55 were bled Aug. 22, nine days after their last injection. On Aug. 24, the action of their sera and that of a control rabbit No. 80 was tested on 8 and ♀ spores. The number of spores was roughly determined with a hemacytometer. One cc. of the spore mixture containing in the neighborhood of 12,000,000 spores was added to 0.5 cc. of the different sera and, together with controls in salt solution alone, the tubes were placed in the incubating oven. It is not impossible that rather more d than spores were added to each tube and this fact would explain the uniformly high counts for all tests of 8 spores in this series. The results are shown in Table 2 and merely confirm the facts brought out in Table 1-i. e., that the sera of the treated rabbits developed no antibodies for the destruction of spores.

Although no cytolysins were developed, there was found evidence of the formation of agglutinins in the blood of the treated animals. On Aug. 26, about 6,000,000 ♂ and ♀ spores, respectively, were added in 0.9 percent salt solution to various dilutions of the sera of the same rabbits (Nos. 5, 55 and 80), and controls were made with ở and ♀ spores alone in salt solution. The volume in each tube was 1.5 cc. They were incubated at 37° C. The spores in the salt solution, in settling, formed a uniform layer on the bottom of the tube. In the control serum (No. 80), at dilutions greater than 1:600, the spores also settled uniformly. In dilutions up to 1: 1,200, the sera of Nos. 5 and 55 caused a distinct clumping of both and spores, producing an irregular roughening of the edges of the spore mass at the bottom of the tubes. A slight but distinct reaction could be observed in the sera of Nos. 5 and 55 with♀ spores, and in serum No. 5 with ♂ spores at a dilution of 1:2,400. A similar clumping of spores was found in the control serum (No. 80) but only at the tested dilutions of 1:30, 1:150, and 1:300. At dilutions of 1:600, I: 1,200, and 1 : 2,400 no clumping of spores was observed in the control serum. The reaction seems comparable to the agglutination produced with bacteria. The tests failed to show any specific sexual reaction such as had been suggested in precipitin tests with the "press-saft" from the mycelium of this same mold (5).

It was not possible to carry on the work with the sera further at this time. They were inactivated at 56° C. for one half hour on Aug. 26, and later taken to Storrs, Conn., where they were preserved in an icebox. Pressure of other work prevented a repetition of the tests until Feb. 15, 1914. Despite the age of the sera the results were in general confirmatory of the previous tests. Fresh serum from another rabbit (No. 30) was used as a control. The spore mixture was estimated to contain somewhat over twice as many spores as were previously employed. Their greater number may in a measure account for the fact that in the higher dilutions of the control serum the spores, instead of forming a uniform layer on the bottom of the test tubes as had been the case in the control serum six months before, now settled together into a dense dot. Plate 2 shows spores in tubes of serum at a 1: 320 dilution, together with spores in salt solution alone. The tubes were slightly slanted after the spores were added and the slant reversed to show the character of the settled spores in the photograph. The roughening of the edges of the spore masses in sera Nos. 5 and 55 is a characteristic appearance. The spores in No. 55 were slightly disturbed in handling.

BLAKESLEE AND GORTNER: REACTION OF RABBITS TO INTRA-
VENOUS INJECTIONS OF MOULD SPORES

Five sixths natural size. Photograph showing male spores of Mucor V in tubes of the following fluids, reading from left to right: 0.9 per cent. salt solution, serum of rabbit No. 5, serum of rabbit No. 55, serum of control rabbit No. 30. The different sera were all at a 1:320 dilution.