There are, as is known, several methods for preparing serum to be used for therapeutic and diagnostic purposes, but the clotting process and the defibrination followed by centrifugation have widely been employed with noticeable efficiency in practical work. While by the former method clear and sterile serum is obtainable, simply allowing the blood to clot, the latter gives higher percentages in the gain of serum. It often occurs, however, by employing the latter method that the material is accidentally contaminated during the process of centrifugation and the serum obtained becomes quite unfit for use. In order to prevent the accidental contamination and to obtain a large possible amount of sterile seerum, several kinds of apparatus have atreacly been designed. As it will be of great interest to know which is the best method, which is the best apparatus and in what condition the serum gain is largest, I have made some experiments, employing the known methods and apparatuses, to determine, as a first step, the relation between the yield of serum and the amount of bleeding, slight starvation, atmospheric pressure, temperature, size and shape of glass jar, etc. The apparatuses used in my experiments are:
1) The apparatus used in the Pasteur Institute, Paris.
2) The bucket for blood clotting designed by the Laboratory of the Indian Civil Veterinary Department.
3) Latapie's apparatus.
The results obtained are as follows:
1) The yield of serum depends upon the individuality of animals and the total sum of the serum which is obtained from the usual clot at 2-day-intervals during 9 days amounts to 41-64% of the whole blood drawn.
2) The percentage of serum from the first bleeding (4000c.c.) was lower by 6.5% than that from the second which, being same in the quantity of blood, took place 2 days later.
No influence of an atmospheric pressure and slight starvation upon the yield of serum has been observed.
4) The adequate temperature for separation of serum is about 20°C.
5) The amount of serum obtained is proportional to the height of glass jar.
6) The compressive method employed in the Veterinary Laboratory at Buitenzorg (Java) is most applicable in horse and swine blood. According to the experiments, this method requires no complicated apparatus, shortens the time necessary for collec-ting serum, gives serum 4-13% more than that obtained from the usual clot, and the serum is guite free from hemoglobin. As for the bucket of Indian Civil Veterinary Laboratory, it seems to be more favourable to use it for the preparation of serum from cattle blood, though the serum is often found in an impure state, slightly mixed with red blood corpuscles.

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Aus den obigen Resultaten, können die Veränderungen der roten Blutkörperchen zufolge von Schwankung der Körpertemperatur während des Krankheitsverlaufes wie folgt erklärt und zusammengefasst werden.
1. Fieberform.
Die 10 Versuchspferde erkrankten nach verschiedenen Inkubationsstadien d. h. von 6 bis 21 Tagen nach Impfung des virsushaltigen Blutes und zeigten die alle eigentliche Körpertemperaturerhöhung. Wenngleich die Temperaturerhöhungen in ersten Anfällen meist so hoch wie 40.0°C oder darüber erreichen, doch sind in einigen Fällen (Nr. 4, Nr. 5 und Nr. 7) ganz niedrig geblieben. Der erste Anfall dauert meistens 4 bis 7 Tage lang, aber bisweilen 10 Tage lang bei einer geringeren wie in Nr. 5 und 3 Wochen lang bei einer heftigen Körpertemperaturerhöhung wie in Nr. 3. Das Auftreten des Zwischenstadiums nach dem ersten Anfall war verschiedenartig. In den meisten Fällen kehrte die Temperatur einmal zu normaler zurück und dann trat der zweite Anfall ein. Ganz interessant war es aber noch solche Fällen zu beobachten, wo sich die Temperatur, wie in Nr. 1, 2 Wochen lang um 38.0° bis 39.0°C anhielte oder ganz plötzlich auftretende auffallende Steigerung zeigte. Die Dauer des Zwischenstadium ist ganz verschieden. In Nr. 3, Nr. 4 und Nr. 6 ist das zweite Anfall jeden 2 Tage, und in Nr. 5 und 8 jeden 5 Tage wiederholt aufgetreten. In Nr. 7 ist aber die Dauer 7 Tage und in Nr. 2 17 Tage lang gewesen. Der zweite Anfall erscheint sich immer deutlich, und steigt Körpertemperatur häufig auf etwa 41.0°C und dauert von 5 bis 6 Tage lang. Aber kann man auch die Fälle beobachten, wo eine höhere Temperatur nur halben Tag wie am 16. in Nr. 5 dauerte oder sich ein unregelmässiges geringeres Fieber sich anhielte. Während einerseits z. B. in Nr. 4 und 8 der Anfall im Laufe des zweiten Zwischenstadiums einem vollkommenen fieberiosen Zustand zukam, beobachtete man anderseits die Fälle, wo sich Fieber in geringerem Grade anhielten und häufig zu dritten Anfällen übergegangen sind. Günstige Prognosen andeutend, sind die Anfälle grösstenteils aber allmählich undeutlicher und die Kranken ganz fieberlos geworden.
2. Die Zahlen-Veränderung der roten Blutkörperchen.
Im allgenimen fangen die rote Blutkörperchen an zu vermindern Während des Anfangsstadiums der Erkrankung und zwar meistens in den ersten 4 Tagen, bisweilen aber in den 9. bis 13. Krankheitstagen. Im Falle, wo sich eine tüchtige Körpertemperatursteigerung im Anfangsstadium erscheint, ist die Veränderung bemerkbar deutlich. Aber in anderen Fällen sind so geringe, ja sogar keine Veränderungen beobachtet worden. In Nr. 7 ist z. B. eine geringere Temperaturerhöhung während der ersten 3 Krankheitstage beobachtet worden, liess sich fast keine Veränderung an der Blutkörperchenzahl erwiesen und erst am 15. Krankheitstage d. h. am 2 Tage des zweiten Anfalls richtete sich die Rotenzahlkurve nach unten. Wie aus obigen ersichtlich, steht der Verminderungsgrad des roten Blutkörperchens immer im Verhältnis zu der Höhe der Körpertemperatur und der Dauer des Fieberanfalls. In dem ersten Anfall in Nr. 10 betrug die Temperatur um 40.5°C, aber wegen der Kurzen Dauer derselben, sind die einmal zu vermindernden Rote sogleich im Stillstande geblieben, und betrugen 5.50 Millionen. Es ist also zu beachten, dass die Verminderung der Rote nicht immer so lange fortdauert, d. h. in gewissem Zeitpunkte wo sich die Körpertemperatur senkt, ganz still steht und dann mit dem Verschwinden der Fiebersymptome allmählich wieder vermehrt. Die Zeitpunkte, wo keine Verminderung in den Roten bemerkbar ist, traten in dem Fall Nr. 9 schon erst am dritten, aber gewöhnlich an den 5. bis 14., seltens am 20. Krankheitstage auf.

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1. As the medium which I adopted in this experiment contains no beef protein, partial reaction due to meat does not occur.
2. Rabbits which received successive injections of peptone do not show any development of specific precipitins in their serum.
3. It is possible to obtain precipitins which react only with the protein of B. chauvoei, by treating rabbits with liver-peptone culture of this organism.
4. The negative results in my experiments probably depend upon the other disease which is apparently analogous with the black-leg from biological points of view.
5. The precipitin reaction is serviceable for differentiation of B. chauvoei from B. oedematis maligni, B. tetani, B. welchii and the bacillus of chicken diarrhoea (Nakamura).

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A. E. Evans (1918) demonstrated that B. abortus and B. melitensis are hardly distinguishable either by morphological, biochemical or serological reactions. Later, Meyer & Shaw, Feusier & Meyer, Zeller, Khaled and Skaric tried to differentiate them again and confirmed that they are closely allied to each other in morphological and biological characters, but in serologica reaction they are not quite identical. Feusier & Meyer stated that they can generally be divided into four groups if an absorption test is applied.
Khaled noticed, furthermore, that, while they are in a fair agreement with each other as to the absorbing power for agglutinins from the anti-melitensis serum, the anti-abortus serum absorbed by B. melitensis losts its power to agglutinate it, but agglutinins for B. abortus still remain. For this reason, he suggested that the B. melitensis should be listed under the subdivision of the B. abortus.
Skaric suggested that the anti-serum prepared with one of these organisms has not only a single agglutinin, but also a compound agglutinin.
Thus it has been generally recognized that both organisms are identical with regard to their biological characters, but when the absorption test is applied, the results do not agree to the same effect. With 20 strains of B. abortus which were isolated from the infected materials of cattle in this country and 2 strains of B. melitensis and B. abortus from hog which were brought here from England by Dr. Nakamura, I have carried out some experiments with the view of establishing all what was stated by those investigators cited above. The results obtained are summarised as follows:-
1. It is difficult to differentiate B. abortus from B. melitensis morphologically as biochemically.
2. Either of the anti-serums prepared with these organisms have an identical agglutination titer for the both living organisms.
3. If an antigen heated at 60° or 100°C. for an hour is used, then the anti-abortus serum agglutinates B. abortus more powerfully than B. melitensis. It seems to be most probable that, when the anti-abortus serum prepared with the heated antigen is the anti-melitensis serum does not show any difference in serological reaction.
4. The absorption test indicates that the agglutinins seemto be present in the immune serum in the following ratio.
A number of experiments show that the agglutinogens for AM and A are stable against heat and those for M and A' are so labile that they lost to a great extent their antigenic power by heating. It may be seen morever, that B. melitensis has two kinds of agglutinogen which differ from those of B. abortus.
5. In the complement fixation test there is no difference between these two organisms but the antigenic power of B. abortus appears more powerful.
6. That the abortus antigen has always stronger antigenic power than the melitensis antigen, will be an aid in the differentiation of both organisms.
7. No difference between these two organisms could be found in the precipitin reaction.
8. No difference was found between B. abortus isolated from hog and that obtained from cattle.
9. From the foregoing, B. melitensis may he regarded as a heterogenetic strain of B. abortus.

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