THE GERMICIDAL PROPERTIES OF NUCLEINS. 
A Preliminary Contribution from the Laboratory of Hygiene 
of Michigan University. 
BY 
VICTOR C. VAUGHAN, M.D., F. G. NOVY, M.D., 
AND 
CHARLES T. McCLINTOCK,- 
OF ANN ARBOR, MICH. 
FROM 
THE MEDICAL NEWS, 
May 2o, 1893. [Reprinted from The Medical News, May 20, 1893.] 
T\HE GERMICIDAL PROPERTIES OF NUCLEINS. 
A Preliminary Contribution from the Laboratory of Hygiene 
of Michigan University. 
By VICTOR C. VAUGHAN, M.D., FREDK. G. NOVY, M.D., 
AND 
CHARLES T. McCLINTOCK, 
OF ANN ARBOR, MICH. 
In studying the subject of immunity we have been 
led to test the action of certain constituents of the 
cell upon the life and growth of microorganisms, 
because it has seemed to us that the cells of the 
animal body must be concerned in the production 
of immunity, and in a way not explained by the 
phagocytic theory of Metschnikoff. 
Immunity may be natural or acquired. Natural 
immunity may be peculiar to the race or to the in- 
dividual. An example of racial immunity is that of 
the domestic fowl to anthrax. The chick, even at 
the moment when it comes from the egg, is immune 
to the most virulent cultures of the bacillus anthracis. 
It is true that this animal may be made susceptible 
to this disease, but this is an artificially induced 
susceptibility, and the immunity is natural to every 
period of life. Another example of racial immunity 
is that of the frog to the same disease, and here 
again an artificial susceptibility may be induced. 
Racial immunity must be inherent in the parent cell. The natural immunity which is peculiar to the 
individual usually comes with adult life. The 
young are susceptible to a given disease, but adults 
of the same species lose this susceptibility, and be- 
come immune. Examples of this are common. The 
young rat is susceptible to anthrax, while the adult 
is naturally immune, but can be rendered suscep- 
tible by reducing the vital strength. The child is 
highly susceptible to scarlet-fever and diphtheria, 
while the adult, though not wholly immune to these 
diseases, loses much in susceptibility, and is likely 
to become infected only when much reduced in 
vitality, or when exposed to the prolonged influ- 
ence of the infecting agent. The only reasonable 
explanation of this immunity is that it is inherent 
in the parent cell, and comes as naturally as the 
changes in form and voice at puberty, or as the 
growth of the beard in early manhood. The evolu- 
tion of the condition of immunity in these cases is 
due to natural development of the functions of cer- 
tain cells of the body. 
In selecting the constituent of the cell upon which 
we could begin our studies, the nucleins, constitut- 
ing as they do the most complex part and possess- 
ing marked physiologic properties, naturally sug- 
gested themselves. We will not in this paper dis- 
cuss the chemistry of the nucleins any farther than 
to say that they consist of nucleic acids combined 
with a complex proteid base. It is more than prob- 
able that nucleins from diverse sources differ in 
both their acids and bases. The nucleins are not 
digested by hydrochloric acid and pepsin, and this 
affords in many cases a means for their isolation. 
2 3 
Nuclein from dog's testes. The testicles of two 
small dogs were stripped of their investing mem- 
branes, and were digested for some days (until the 
supernatant fluid failed to respond to the biuret test 
for peptones) at 40° C., with pepsin and 0.2 per 
cent, hydrochloric acid. The undigested portion 
was collected on filter-paper, and washed first with 
dilute hydrochloric acid, then with alcohol. Finally, 
it was dissolved in a 0.5 per cent, solution of potas- 
sium hydrate, and filtered through a Chamberland 
filter without pressure. 
This solution is clear, golden-yellow, and feebly 
alkaline. On the addition of a drop of nitric acid 
a white precipitate forms, and dissolves colorless in 
the cold on the further addition of the acid. The 
nuclein solution does not give the biuret reaction, 
but does respond to the Millon test. 
With this solution the following experiments have 
been made in order to test its germicidal effect. 
The solution of testicular nuclein was diluted 
with seven volumes of physiologic salt-solution. 
This reduced the alkali to 0.0625 per cent., an 
amount too small to have itself any germicidal effect. 
One tube of this solution was inoculated with 
a loop of a beef-tea culture of the staphylococcus 
pyogenes aureus, and plates were made after 5 min., 
4 hrs., 6 hrs., 23 hrs., and 54 hrs. The number of 
colonies on each plate was as follows: 
Time.... 5 min. 4 hrs. 6 hrs. 23 hrs. 54 hrs. 
No. of colonies 10 o o o o 
Another tube was inoculated with a loop of a 
beef-tea culture of bacillus venenosus, and plates 
were made, with the following results : 4 
Time.... 5 min. 4 hrs. 6 hrs. 
No. of colonies 210 8 9 
Another portion of the testicular nuclein solution 
was diluted with four parts of the physiologic salt- 
solution. This reduced the alkalinity to o. 10 per 
cent, (water containing 0.5 per cefit. of potassium 
hydrate failed to destroy this culture of the aureus 
after twenty-four hours’ exposure). To a tube of 
this a loop of a beef-tea culture of the aureus was 
added. The plates showed the following: 
Time.... 5 min. 1 hr. 2 hrs. 14 hrs. 23 hrs. 
No. of colonies 250 0000 
In another, in which the same dilution and the 
same germ were used, the number of colonies was 
as follows: 
Time . Immediately. 20 min. 1 hr. 2 hrs. 17 hrs. 24 hrs. 
No. of colonies 680 00000 
In still another experiment, in which the same 
dilution of the testicular nuclein was used with the 
anthrax-bacillus (without spores), the colonies num- 
bered as follows : 
Time . Immediately. 20 min. 1 hr. 2 hrs. 17 hrs. 24 hrs. 
No. of colonies 45 o 00 o o 
With nuclein obtained from the testes of a rat 
and diluted with physiologic salt-solution until the 
amount of alkali was reduced to 0.06 per cent., one 
experiment with aureus gave the following results: 
Time . . Immediately. 5 min. 1 hr. 2 hrs. 27 hrs. 
No. of colonies mo 0000 
Nuclein from thyroid glana. We took the thyroid 
gland of a rabbit, killed by drawing the blood from 
the carotid, cut the gland into fine pieces, extracted with alcohol and ether, then placed the extract in 
0.2 per cent, hydrochloric acid with pepsin, and 
kept it in the incubator at 40 C. for two days, having 
decanted and renewed the digestive fluid several 
times. The slight glanular residue which remained 
undigested was collected upon a filter, and washed 
with o. 2 per cent, hydrochloric acid until the wash- 
ings failed to give the biuret reaction. After this treat- 
ment there appeared on the filter glistening scales 
which, under the microscope, showed bundles of 
radiating needles. These proved to be fat, and 
were dissolved by washing with alcohol and ether. 
The residue now on the paper was exceedingly 
small. This was dissolved in 5 c.c. of a 0.25 per 
cent, potassium hydrate solution, diluted with an 
equal volume of physiologic salt-solution, and with 
this the experiments were made. 
This solution gave a faint opalescence on the 
addition of nitric acid. It did not color on heat- 
ing with nitric acid, but did become markedly yel- 
low on the further addition of ammonia. It failed 
to respond to the biuret test. 
This solution was inoculated with a loop of a 
beef-tea culture of the aureus, and the plates showed 
the following number of colonies : 
Time . . . Immediately, io min. i hr. 18 hrs. 
No. of colonies 805 830 256 o 
Nuclein from yeast-cells. Yeast-cells, after having 
been washed with water by decantation, were ex- 
tracted with dilute alkali. The alkaline solution 
was precipitated with dilute acid, and this process 
repeated a number of times. The solution, as kept 
for use, was made by dissolving the nuclein in o. 25 
per cent, alkali. That the nuclein in this solution 
5 6 
is not free from albuminous bodies is shown by the 
fact that it responds promptly to the biuret, xantho- 
proteic, and Millon tests. However, this solution 
has stood in an ordinary glass-stoppered bottle, 
which is frequently opened for nearly five months, 
and remains germ-free. 
The amount of nuclein (impure) in this solution 
is nine milligrams per cubic centimeter. With this 
solution the following experiments were made : 
Two parts of the yeast nuclein solution were 
diluted with three parts of the physiologic salt- 
solution, and this was inoculated with a loop of a 
beef-tea solution of the aureus. Plates made showed 
the following results : 
Time.... 5 min. 1 hr. 2 hrs. 14 hrs. 23 hrs. 
No. of colonies mo o o o o 
The same experiment repeated gave the follow- 
ing: 
Time .... 5 min. 1 hr. 2 hrs. 14 hrs. 23 hrs. 
No. of colonies 1490 20 o o o 
In one, with the staphylococcus pyogenes albus, 
the following are the figures : 
Time. .Immediately. 20 min. 1 hr. 2 hrs. 17 hrs. 24 hrs. 
No. of colonies 680 o 0 0 o o 
In one, with the bacillus anthracis : 
Time . Immediately. 20 min. 1 hr. 2 hrs. 17 hrs. 24 hrs. 
No. of colonies 45 o 00 o o 
During the past year we have also tested the 
effects of injections of nuclein upon the progress of 
certain infectious diseases. Moreover, we have 
some evidence that the germicidal constituent of 
blood-serum belongs to the nucleins. This work, 
however, we wish to confirm before publishing. The Medical News. 
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