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After the skin has been cut it is usually possible by a few superficial cuts with the scissor points to sever one or two of the small ventral branches of the external jugular vein. The blood is caught, as it drops, in a sterile Petri dish.

When sufficient blood is secured the guinea-pigs are placed on their backs and the skin cut closed with a few stitches (needle and thread). With the help of an assistant the cut edges are first brought together with forceps, and in this way the sewing operation can be made very rapid and easy. The flow of blood quickly ceases. Antiseptic is applied to the wound and the animals returned to their cages. The cuts heal over in the course of 3 or 4 days, and the guinea-pigs appear none the worse for the operation. One guinea-pig has been employed for this purpose, at frequent intervals, for 21/2 years. Some of the guinea-pigs have been bled at least 30 times. By operating on alternate sides of the neck the same animal may be bled at least once a week, almost indefinitely. though it is advisable to allow it to rest occasionally. From a large pig as much as 10-12 cc of blood may be drawn in this manner without apparent injury to the animal.

The blood is allowed to stand at room temperature for 20-30 minutes, or until the clot has completely formed. The clot is broken up with a glass rod or small shears, and the dish kept in the refrigerator for at least 5 or 6 hours, away from contact with moisture.

We have found it expedient to bleed for complement late in the afternoon preceding the day on which the serum is to be used.

The serum is drained from the Petri dish into centrifuge tubes and completely separated from suspended matter by centrifuging. The serum is then drawn off, diluted with two volumes of physiological saline solution, and immediately returned to the ice box. The titre of the fresh serum will as a rule be around 0.03, particularly when the sera of at least three or four guinea-pigs are pooled.

The following is an example of complement titration. Four units of known hemolytic rabbit serum are employed. The amount of complement used varies from 0.01 to 0.08 cc. Readings are made after 20 minutes incubation in a water bath held at 37°C.

Titration of Complement

Hemolysin Sheep's


Result 0.25 cc

No hemolysis


No hemolysin

No hemolysis Titre or unit is 0.03, and the amount to be used is 0.045 cc.

Tube number

NaCl sol.

diluted 1:2

1:50 unit 0.05

1.5 cc

0.01 CC 0.02


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0.08 “ 0.08 "


Bacterial Antigen At least three different strains of Bact. abortus of known antigenic value should be employed in the preparation of the antigen. The following method of antigen production is proving itself thoroughly satisfactory in our own hands.

Selected strains of Bact. abortus are transferred daily for two or three days on freshly prepared Fairchild peptone agar containing 0.5% meat extract, 1.0% peptone and 1.5% agar and having a hydrogen ion concentration of pH. 6.8 to pH. 6.9.* Large agar slants are then streaked with inoculum from the last tubes, and incubated at 37°C. for 48 hours. The growths are washed off with physiological saline solution, 5 cc to every tube (1 inch diameter). By using this amount of salt solution the emulsion will usually be such as to give the proper density† when diluted further with five volumes of the saline solution.

The undiluted emulsion is filtered into a glass-stoppered bottle through a small amount of absorbent cotton placed in the apex of a funnel, and shaken thoroughly for at least 10 minutes. The bottle is immersed to the neck in a water bath and heated at 6570°C. for 40 minutes. Temperature readings should be made on a tested thermometer placed in the bottle containing the suspension. A second thermometer may be kept in the bath as an added precaution. After cooling, 1.0% glycerine and 0.5% phenol are added, and the well-stoppered bottle stored in the refrigerator for

Antigen prepared and preserved in the above manner will retain its antigenic property for many months. Anti-complementary substances which appear occasionally may be destroyed by heating for 20-30 minutes at 56-58°C. It is advisable to dilute only as much antigen at one time as will be necessary for immediate use.

Titration of antigen requires, besides complement and hemolytic serum of known strength, a positive cow's serum which has a titre of at least 0.002, as is shown in the following sample titration.

future use.

* As Fairchild peptone is quite acid care must be exercised in the preparation of the agar. The acidity must be greatly reduced before addition of the agar, as it will exert a softening effect on the agar.

† The turbidity should be such as to match tube 1.75 on the McFarland nephelometer scale.

Titration of Antigen


Hemolysin Sheep's titre NaCl (dil, 1: 2) Anti- (dil. 1: 50) corpuscles 0.002 0.85% titre 0.03 gen

titre 0.05




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Titre of the antigen is 0.05 cc. In the complement fixation test we employ 4 units of antigen. As the binding power of the above antigen with complement is exhibited only in amounts of antigen greater than 0.5 cc, there is no danger from complement absorption with the required amount of the antigen.

In the above titration 5 times the titre of immune serum is used, namely 0.01 cc; one and a half units of complement and 5 units of hemolysin are employed.

The Complement Fixation Test Measured amounts of the sera under test are placed into tubes containing 1.5 cc of saline solution. The diluted sera are inactivated by heating in a water bath at 56-58°C. for 20-30 minutes. It should be made a rule to inactivate all sera, as traces of complement may, otherwise, remain even after several days of holding in the ice box.

Complement and antigen of known strength are then added, as is shown in the following tables, and the tubes incubated at 37°C. in a water bath for one hour in order to allow complete binding of complement in the presence of positive sera. Hemolysin and sheep's corpuscles* are then added, and after thorough mixing of the contents the tubes are returned to the water bath for a period of 50-60 minutes.

A known negative and a known positive serum should be set up as controls in every fixation test, in the same manner as the sera on which tests are sought.

Hemolysin and corpuscles may be combined in the proper proportion, before being added to the serum-complement-antigen mixture, thus necessitating only one pipetting operation instead of two.

Titre 0.05

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The Fixation Test
(1) Unknown Cow's Serum
Comple Anti-

Hemolysin Sheep's
Serum Naci ment gen


cells Tube used sol. Titre Titre


Final No. CC. CC. 0.03 cc. 0.05 cc.


Readings 0.2 1.5 0.045 none Incu

0.25 0.5

Incu- Complete hemolysis 0.1 0.2 bation

bated 3 0.05

for i hr.

50-60 4 0.02

at 37° C.


at 37° C.
(Cow is a non-reactor, according to test)
(2) Known Negative Serum
I 0.2


Complete hemolysis 2 0.1


bated 3 0.05

for i hr.

50-60 4 0.02

at 37° C.


at 37° C.
(3) Known Positive Serum
0.2 1.5 0.045

none Incu-

Incu- Complete hemolysis 2 0.1 0.2 bation

bated No 3 0.05

for 1 hr.

50-60 4 0.02

at 37° C.

minutes 5 none

at 37° C. Complete hemolysis O

0.4 7

none 3 none


0.5 Incu

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The method employed here allows adequate checks on all reagents used.

In all tests (4 tubes) tube No. I should be hemolyzed, since antigen is absent and hence the complement cannot be deviated. If laking does not take place an error in technique has occurred or an anti-complementary substance is interfering. Few cows' sera have been anti-complementary, however.

If the given animal is a reactor, tubes 2, 3 and 4 will show no hemolysis. Negative sera do not fix or deviate complement, and therefore all four tubes should be completely hemolyzed when such sera are employed.

In the table for known positive serum eight tubes are set up. Tube No. I serves as control in each individual serum test. Tubes 5, 6, 7 and 8 are additional checks. If 5 and 6 fail to hemolyze the antigen has become anti-complementary and should be discarded. Numbers 7 and 8 are useful chiefly as further checks on accuracy in manipulation.

The Agglutination Method. Serological pipettes are employed
for the transfer of serum from the blood sample to the aggluti-
nation tubes. This is necessitated, both in abortion and in white

diarrhea diagnosis, by the large numbers of daily tests. For the same reason, no account is taken of the difference in the total volume of fluid in tubes i and 2, and in the corresponding slightly unequal dilution of serum in the two tubes.

Results of routine tests are recorded as “+”, "?”, and “O”, largely as a matter of convenience. Furthermore, this method of reporting is desirable as a matter of accuracy, and of justice to the owner. Animals should never be pronounced positive unless they are unquestionably so, particularly in herds in which there are no other positive animals.

Experience has shown that the time and method of incubation employed here are highly desirable. Though final readings are not made before the end of 3 days, the sharpness of the reactions is full compensation for the time allowed. Incubation at temperatures above 37°C. would probably considerably shorten the period, but few laboratories are supplied with high temperature incubators and furthermore reactions would as a rule not be so sharp.

The Complement Fixation Test. Here again the serum is transferred directly from the blood tube to the different dilution tubes, with serological pipettes, and no provision is made to have the same volume of total fluid in all of the tubes. As it is well known that, within reasonable limits, the serum reactions depend on amount of agents in solution, and not on their dilution, the saving of time effected by this method is fully justified. Experience over quite a number of years has shown the results to be consistent.

All along, the routine fixation results have been recorded in terms of hemolysis, rather than fixation. The symbols "+", "?", and “O” are used to designate the individual tube reactions. It may appear to many persons more logical to report the results in terms of fixation, and of course this may be done. It is true that to the lay mind our system may be somewhat confusing, as complete hemolysis in the four tubes is recorded as "O" in each, though the fixation test is negative, and on the same page positive agglutination is indicated by “+”. For our own permanent record, however, the method now used appears to us the more desirable of the two.


COMPLEMENT FIXATION TESTS These tests have been employed for the past ten years as a basic part of the investigation of infectious abortion at the Storrs Experiment Station. During this period at least ten thousand bovine blood samples have been subjected to the tests.

No method of disease diagnosis has yet been devised which is absolutely free from criticism. Even the most recent improvements in the methods of conducting the agglutination test for typhoid fever in man are still far from perfect. The writers believe

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