BIOLOGY
LIBRA**
G
A LABORATORY COURSE IN SERUM STUDY
THE MACMILLAN COMPANY
NEW YORK • BOSTON • CHICAGO • DALLAS
ATLANTA • SAN FRANCISCO
MACMILLAN & CO., LIMITED
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THE MACMILLAN CO. OF CANADA, LTD.
TORONTO
A LABORATORY COURSE
IN SERUM STUDY
BACTERIOLOGY 208
BEING A SERIES OF EXPERIMENTS AND DIAGNOSTIC
TESTS IN IMMUNOLOGY CARRIED OUT IN AN
OPTIONAL COURSE GIVEN TO MEDICAL AND
GRADUATE STUDENTS IN THE DEPART-
MENT OF BACTERIOLOGY, COLLEGE
OF PHYSICIANS AND SURGEONS
COLUMBIA UNIVERSITY
NEW YORK, BY THE
WRITERS
HANS ZINSSER, M.D.
\\
J. G. HOPKINS, M.D.
REUBEN OTTENBERG, M.D.
SECOND EDITION REVISED
THE MACMILLAN COMPANY
1921
AH rights reserved
- - . *..>
-
.
a
COPYRIGHT, 1916, 1921,
BT THE MACMILLAN COMPANY.
Sot up and f lectrotyped. Published February, 1916.
New Edition Revised. Published January, 1921.
PREFACE TO SECOND EDITION
THE present issue of the Laboratory Course in Serum Study
does not represent a new edition in that it has been found impos-
sible to completely revise the book at this time. There are, how-
ever, a number of important changes in protocols and technique
which have been made on the basis of experience hi giving the
course to medical and public health students. Most of these
changes are in the form of alterations hi the plan of experiments,
making them more easy to perform and more illustrative of under-
lying principles. In preparing these changes for the new printing,
we have been greatly aided by Dr. Samuel Cochran, Professor of
Bacteriology at the Shantung Christian University hi China, who
helped us hi giving this course at Columbia last year.
iva
INTRODUCTION
THE course here outlined is given by the authors at Colum-
bia University. The prerequisite theoretical knowledge is pre-
sented in a series of lectures based on the textbook " Infection
and Resistance/' by the senior author.
Immunity, like other branches of science, cannot be taught
without experiment and demonstration. For this reason we
have, for several years, supplemented our lecture course on
Infection and Resistance by an optional course on Serum Tech-
nique. Our purpose in this has been not so much to teach
beginners to carry out practical diagnostic tests as to allow the
student to carry out fundamental experiments, and, in drawing
conclusions from his results, to learn to reason from protocols
and in this way discover the basic principles for himself.
It has been our contention for a number of years that
thorough instruction in the phenomena of immunity consti-
tuted a logically necessary preparation for the clinic on infec-
tious diseases. For this reason our courses have been offered
as optionals to second and third year medical students. Con-
trary to ordinary belief, students at this stage of preparation
have found no difficulty in comprehending the work, and have,
we think, derived benefits in experimental methods and reason-
ing far beyond the actual gain in new facts. Though optional
now, these courses we hope may eventually become integral,
required parts of the regular medical curriculum — the lectures
and demonstrations correlated with — the laboratory course fol-
lowing — the course in Bacteriology. This, however, we realize
may have to await the lengthening of the medical course as
a whole. Meanwhile such a course can certainly be optionally
Vi INTRODUCTION
available for students who have the desire to take it — and
our experience at two medical schools has taught us that there
are always a good many who do.
The little volume has been compiled in the first place for
our own convenience from the protocols given to our students.
If incidentally it is of service to others in planning similar
courses, or to laboratory workers in repeating experiments,
we shall be pleased. The book as it stands is, of course, not
a manual of immunity. The course should follow or be syn-
chronous with lectures, reading, and demonstrations on the
principles of infection and immunity. In our own work the
course is offered to students who have had work in general
Bacteriology and have taken the lecture course on Infection
and Resistance, and reading is assigned in the textbook on
this subject.
Since the manual has grown out of the course after some
four years of experimentation and adaptation to classroom pos-
sibilities, it in no way represents an " ideal " formula but de-
scribes only work actually done with students. For this reason
we have often simplified the experiments in a way which would
be undesirable in actual research work. But we have not done
this at the expense of exactitude. Thus we have in many
cases adapted the technique to the possibilities of a single after-
noon with a class not yet fully trained. In order to facilitate
the giving of such a course by other teachers we have added
a time schedule of the whole course, and have given in each
exercise a list of the materials needed.
TIME SCHEDULE OF LESSONS
SUBJECT OF LESSON
I. IMMUNIZATION OF ANIMALS.
II. BACTERICIDAL AND H.EMO-
LYTIC POWER OF NORMAL
SERUM.
III. H^MOLYSIS.
IV. QUANTITATIVE RELATIONS
OF AMBOCEPTOR AND
COMPLEMENT.
V. QUANTITATIVE RELATIONS
(Continued).
VI. REVERSIBILITY AND SPEED
OF UNION OF IMMUNE
BODY AND CELLS.
VII. PFEIFFER PHENOMENON.
WORK PRELIMINARY TO SUBSE-
QUENT LESSONS
Preparation of B. typhosus.
B. coli.
Staphylococcus au-
reus.
Vibrio choleras.
Sheep serum.
Ascitic serum.
Sheep red cells.
Cat red cells.
Horse serum.
Human serum.
Injection of rabbits.
Repetition of injections of Lesson I.
Repeat injections of Lesson I.
Commence collection of guinea pig
hearts in absolute alcohol for
Wassermann antigen (Lesson
XIX).
Repeat injections of Lesson I.
Repeat certain injections of Les-
son I.
Trial bleeding of animals which re-
ceived their last injections at 4th
session.
Bleeding of animals of Lesson I.
1 Arranged for two afternoons a week for a term of three and a half months.
vii
Vlll
TIME SCHEDULE OF LESSONS
SUBJECT OF LESSON
VIII. BACTERICIDAL TESTS IN
VITRO.
IX. AGGLUTININS.
X. AGGLUTINATION — MICRO-
SCOPIC METHOD. EF-
FECT OF SALT.
XI. ABSORPTION OF AGGLUTI-
NINS.
XII. IN Vivo EFFECT 6r H^EM-
AGGLUTINATIVE AND
H^MOLYTIC SERA.
XIII. ISOH^MOLYSIS AND IsO-
AGGLUTINATION.
XIV. PRECIPITINS.
XV. FORENSIC
TEST.
PRECIPITIN
XVI. BORDET-GENGOU PHENOM-
ENON.
XVII. ALEXIN FIXATION BY SPE-
CIFIC PRECIPITATES.
XVIII. FORENSIC COMPLEMENT
FIXATION.
XIX. WASSERMANN ANTIGEN :
PREPARATION AND Ti-
TRATION OF ANTIGEN.
XX. WASSERMANN REACTION :
TECHNIQUE OF TESTS.
XXI. COMPLEMENT FIXATION
WITH BACTERIAL EX-
TRACTS.
WORK PRELIMINARY TO SUBSE-
QUENT LESSONS
Titrate hemolytic sera made by
class.
Inoculate flasks with virulent diph-
theria strain for production of
diphtheria toxin (Lesson XXII).
Filter diphtheria toxin (Lesson
XXII).
Begin preparation of Wassermann
antigens for Lesson XIX.
Inject guinea pigs with diphtheria
toxin for determination of mini-
mal lethal dose (Lesson XXII).
Finish preparation of simple alco-
holic Wassermann antigen and
Noguchi antigen.
Inject guinea pigs with diphtheria
toxin-antitoxin mixtures for de-
termination of L dose (Lesson
XXII).
TIME SCHEDULE OF LESSONS
IX
SUBJECT OF LESSON
XXII. DIPHTHERIA TOXIN : Ti-
» TRATION OF AN UN-
KNOWN ANTITOXIN.
XXIII. TETANUS TOXIN; TET-
ANOLYSIN AND ANTI-
TETANOLYSIN J TET~
ANOSPASMIN.
XXIV. COBRA VENOM, KICIN,
SAPONIN.
XXV. ANTITRYPSIN.
XXVI. NORMAL OPSONINS.
XXVII. OPSONIC INDEX:
WRIGHT'S TECHNIQUE.
XXVIII. HEAT STABILITY OF
OPSONINS.
XXIX. TlTRATION OF XORMAL
OPSONINS BY DILU-
TION METHOD.
XXX. TlTRATION OF IMMUNE
OPSONINS BY DILU-
TION METHOD.
XXXI. PREPARATION OF BAC-
TERIAL VACCINES.
XXXII. ANAPHYLAXIS.
WORK PRELIMINARY TO SUBSE-
QUENT LESSONS
Inject guinea pigs with serum for
Lesson XXXII.
TO BE PREPARED OR OBTAINED BY INSTRUCTOR
BEFORE COURSE.
BEFORE LESSON I.
BEFORE LESSON IL
BEFORE LESSONS III, IV, V, AND
VI.
BEFORE LESSON VII.
BEFORE LESSON VIIL
BEFORE LESSON IX.
BEFORE LESSON X.
BEFORE LESSON XI.
BEFORE LESSON XII.
Haemolytic (antisheep) serum.
Immunize a guinea pig to cholera
spirillum.
Inoculate students against typhoid.
One day before lesson plant agar
slants of B. typhosus, B. coli, Sta-
phylococcus aureus, Vibrio cho-
lerae.
Obtain sheep serum, ascitic fluid,
horse serum, sheep's blood, two
cats, ten rabbits, one guinea pig.
One day before, inoculate broth cul-
ture of B. typhosus.
Fresh dog serum.
Washed guinea pig cells.
Washed human cells.
Sheep's blood (washed).
Fresh guinea pig serum.
Diluted haemolytic serum.
Plant culture of virulent cholera
vibrios.
Guinea pig immunized to cholera.
24 hours before, plant broth culture
of B. typhosus.
Obtain sterile blood from normal
rabbit.
24 hour broth culture typhoid.
Virulent diphtheria strain.
24 hour broth culture B. typhosus.
Slant cultures of B. typhosus and
B. coli.
Two cats, one rabbit.
Xll
TO BE PREPARED OR OBTAINED BY INSTRUCTOR
BEFORE LESSON XIII.
BEFORE LESSON XV
BEFORE LESSON XVI.
BEFORE LESSON XVIII.
BEFORE LESSON XIX.
BEFORE LESSON XXIII.
BEFORE LESSON XXIV.
BEFORE LESSON XXV.
BEFORE LESSON XXVI.
BEFORE LESSON XXVII.
BEFORE LESSON XXVIII.
BEFORE LESSON XXIX.
Beef hearts.
Absolute alcohol for Wassermann
antigens.
Prepare blood stains to be tested.
Typhoid slants.
Obtain standard diphtheria anti-
toxin.
Horse meat. Give to class at
previous exercise.
Syphilitic and normal human sera.
Tetanus toxin and antitoxin. Test
haemolytic power of toxin.
Guinea pig brains.
Guinea pigs.
Cobra venom. Saponin, ricin, leci-
thin.
Guinea pig's blood. Sheep blood.
Prepare alkaline casein solution and
trypsin solution.
Obtain blood from carcinoma and
other patients.
Inject guinea pigs with aleuronat.
Slants of Staphylococcus aureus.
Guinea pig serum.
Inoculate slants with Staphylococ-
cus aureus.
Glass tubing for making Wright's
capsules and pipettes.
Rubber nipples.
Triangular files.
Citrate solution.
Staphylococcus immune guinea pig
serum.
Staphylococcus emulsions.
Guinea pigs for preliminary work
for Lesson XXXII.
Horse serum.
Sheep serum.
Guinea pig serum.
Same materials as for Lessons
XXVII and XXVIII.
TO BE PREPARED OR OBTAINED BY INSTRUCTOR
Xlll
BEFORE LESSON XXX.
BEFORE LESSON XXXI.
BEFORE LESSON XXXII.
BEFORE LESSON XXXIII.
Serum of rabbit immunized against
staphylococcus.
Inject guinea pig with aleuronat. *
Serum of normal rabbit.
Other materials same as for three
preceding lessons.
Agar slants of Staphylococcus au-
reus.
Sterile tubes with glass beads.
Hsemocytometers.
Methylene blue solution.
Wright's stain or Jenner's stain.
Hopkins centrifuge tube.
Horse serum.
Sheep serum.
Immune anti-horse serum injected
into guinea pigs day before lesson.
Horse serum.
A LABORATORY COURSE IN SERUM STUDY
2 LABORATORY COURSE IN SERUM STUDY
LESSON I
STUDENTS in this course will prepare their own materials for injec-
tion, will immunize and bleed animals themselves, in fact will do all the
manipulations necessary in the preparation of materials for the experi-
ments that are done in the class. In some cases students will work in
groups of two or more in order to save time and animals.
It is suggested that each student be required to enter his results on a
joint tabulation sheet prepared for each day's exercise and that these results
be discussed by the instructor at the beginning of the following exercise.
PREPARATION OF MATERIALS FOR INJECTION
1. SHEEP ERYTHROCYTES
The students who are to immunize their rabbit to blood cells
should obtain the blood from the sheep, and at subsequent lessons
other students will be given experience in this procedure.
Bleeding of Sheep. — The animal is held by two assistants
with the head thrown back and one side of the neck is clipped
and carefully shaved.
A rubber tourniquet is then placed around the neck as near
as possible to the shoulders and tied tightly so as to compress the
external jugular vein. The vein distends and will be seen to stand
out as a ridge the size of a finger. A small part of the shaved area
of the skin is painted with tincture of iodine, and a large sterile
needle with four inches of rubber tubing attached is plunged
through the skin into the vein. The blood is allowed to flow
into small Erlenmeyer flasks containing about 20 large beads,
and when the desired amount of blood is obtained the flask is
shaken thoroughly until coagulation is completed, this procedure
defibrinating the blood. If preferred, the blood may be received
into citrate solution, containing 1 per cent sodium citrate and 0.5
per cent sodium chloride (not more than 1 part of blood to 1 part
of solution) or into ammonium oxalate solution (1 per cent).
To obtain washed erythrocytes and serum for injection
the defibrinated blood is poured into sterile centrifuge tubes.
The cotton plugs of these tubes should be fastened by turning
the free edges back over the neck of the tube and securing
them there by means of rubber bands; this will prevent the
4 LABORATORY COURSE IN SERUM STUDY
plug being thrown to the bottom of the tube during centrifuga-
tion. The cotton plug may also be replaced by a cap of sterile
tin foil. After careful balancing, the tubes are centrifugalized
at high speed until the sediment is thrown down. The super-
natant fluid is removed with a sterile pipette and in this case
should be preserved for the injection of rabbits for the production
of precipitins. The tubes are then filled with sterile salt solution,
the blood cells resuspended by drawing them in and out of a
pipette and again thrown down in the centrifuge. This process
is repeated three times. The washed sediment is then trans-
ferred to another tube with a graduated pipette and an equal
amount of salt solution added to make the 50 % suspension which
is used for injection.
»
2. PREPARATION OF BACTERIAL EMULSIONS
Ten c.c. of sterile salt solution are added by means of a pipette
to a 24-hour culture on slant agar of the particular organism to
be used. Surface growth is scraped from the agar by means of
a platinum loop and the bacteria suspended in salt solution by
gentle shaking. The suspension is then poured off into a sterile
test tube, carefully flaming the mouths both of the culture tube
and the sterile test tube before pouring. The second tube may
then be drawn out and sealed in a blowpipe flame, and the tube
entirely immersed in a water bath for sterilization ; or the upper
portion of the tube may be carefully heated in a Bunsen flame to
kill any bacteria which may be adherent to the side of the upper
portion, and when the tube has cooled the lower portion is then
immersed in a water bath, taking care that the level of the water
comes well above the portion which has not been sterilized by
flaming. The bacteria should be heated at 60° C. for one half
hour and are then ready for injection.
3. PREPARATION OF SERUM FOR INJECTION
The serum or ascitic fluid used for the production of precipitins
should be clear and sterile and is less toxic if heated for 30 minutes
at 56° C. before injection. It requires no other preparation.
6 LABORATORY COURSE IN SERUM STUDY
CARE OF ANIMALS
The animals used for the routine work of the course are mainly
guinea pigs and rabbits ; in some cases dogs or cats are used, and
for demonstration a sheep is used on one or two occasions.
Animals used should be selected from among the healthy stock ;
animals having suppurations, diseased eyes, or skin diseases
should be discarded. They should be well nourished and before
immunization is begun should be weighed and the weight recorded.
During the course of treatment students should, if possible,
supervise the feeding and caging of their animals. Care should
be taken that the cages are kept reasonably dry, that the animals
have plenty of water to drink and the food is abundant, and
that not too much green feed is given. If any of the rabbits
under treatment show a catarrhal discharge from the nostrils,
a condition spoken of by animal dealers as "wet mouth", these
animals should be segregated and their nostrils and forepaws
washed daily with weak bichloride solution. Great care should
be taken that the food soiled by these animals is not put into
cages of the healthy ones. Rabbits and guinea pigs are best
preserved in a warm place, and wood bottoms on the floors of
bins and cages are much better than either cement or tin unless
these are covered. They should not be allowed to live for days
on floors wet and soiled with their own discharges. During the
immunization the animals should be weighed periodically and
their weight recorded, and if great emaciation and loss of weight
ensues in an individual case, the animal should be given a rest
and carefully fed. Unless this is done many of the animals will
die in the course of immunization.
There is certain to be a considerable mortality among the
animals even if the greatest care is used, and it is wise to start im-
munizing two individuals for each sort of substance used as antigen.
METHODS OF INJECTION
1. INTRAVENOUS INJECTION
When a rabbit is to be injected the animal is held by an assist-
ant. The outer border of the hairy side of the ear is shaved and
8 LABORATORY COURSE IN SERUM STUDY
rubbed vigorously with alcohol until the marginal vein distends.
The needle of the syringe is then introduced into the vein, point-
ing toward the base of the ear, and the material injected, care
being taken to avoid the introduction of bubbles. When the
needle is withdrawn a small piece of absorbent cotton is pressed
over the puncture to stop bleeding. It is well to leave this cotton
adherent to the puncture when the animal is returned to the cage.
In injecting guinea pigs intravenously it is necessary to incise
the skin of the neck and expose the external jugular vein, and unless
a very small needle is used the vein must be tied off after injection
has been made. Etherization is needed for this procedure.
The injection of mice and rats intravenously is difficult, but
can be accomplished by rubbing the tail with xylol and holding
the mouse so that the tail bends sharply over the edge of the jar.
The four parallel veins will then stand out and with care a very
fine needle can be introduced into one of them.
2. INTRAPERITONEAL INJECTIONS
A rabbit or guinea pig is held back down by an assistant and
a small area of the abdominal wall median line clipped and the
skin disinfected with alcohol or iodine. A fold of skin and muscle
is pinched up with the left hand, and the needle, which should not
be too sharp, is cautiously introduced directly into the peritoneal
cavity and the injection made. The safest point is the median
line below the umbilicus, as in this area there is little danger of
puncturing the stomach or large intestine.
3. SUBCUTANEOUS INJECTIONS
Subcutaneous injections are made in a similar way, after
clipping the hair of the area selected and pinching up a fold of
skin, taking care that the needle does not pass into the muscles.
Rabbits and guinea pigs are usually injected into the surface of
the abdomen, rats and mice at the root of the tail. If there is
danger of the substance injected causing ulceration, as in the case
of red cells, it is best to inject rabbits under the skin of the back,
as this area is less likely to become infected.
10 LABORATORY COURSE IN SERUM STUDY
For injection a syringe is used which will withstand boiling.
For general work all glass syringes of the Luer type are the most
satisfactory. A tightly fitting needle of gauge 20 to 22 should be
used for "Inrra venous injections; for intraperitoneal injections
larger needles may be used if desired. If glass syringes are used
the needle may be attached to the barrel of the syringe, the
plunger withdrawn, and the two parts boiled separately for 5
minutes in water containing about 1 per cent of sodium carbonate.
A pair of forceps should be placed in the sterilizer with the points
under the water. The barrel is first removed with the forceps,
the plunger then removed and inserted. The fluid to be injected
is drawn into the syringe by tilting the test tube or container
and placing the beveled tip of the needle closely against the
lower side of the wall. If air is drawn into the syringe at the
same time, it should be expelled by holding the syringe vertically,
needle up, covering the tip of the needle with a small piece of
cotton wet with alcohol or some other disinfectant and expelling
the air. After use the syringe should be resterilized if any
infected material has been used, washed with water, and the
needle washed with water, then with alcohol and then with ether
to dry it thoroughly. If care is not taken to clean the needles
thoroughly after use, they are certain to become clogged. If blood
or serum have been injected, the needle and syringe must be
cleaned with cold water at once. Otherwise if alcohol or boiling
water is used, the protein will be coagulated and the needle clogged.
Animals should be labeled by attaching a number tag by means
of a metal staple to the ear, should be placed in a cage with a tag
recording the number of the animal, the name of the student in
charge and the nature of the material injected, and the date of
each injection.
SCHEME FOR INJECTIONS (APPROXIMATE DOSES AND INTERVALS)
1. Rabbit immunized to B. typhosus by intravenous injection.
1st day 1st dose 0.05 agar slant
6th day 2d dose 0.1 agar slant heated as
llth day 3d dose 0.2 agar slant indicated
16th day 4th dose 0.2 agar slant below
24th day 5th dose 0.2 agar slant
12 LABORATORY COURSE IN SERUM STUDY
2. Rabbit immunized to B. typhosus by subcutaneous injections.
Injections at five or six day intervals, followed by intravenous injections.
1st dose 0.1 agar slant
2d dose 0.2 agar slant
3d dose 0.3 agar slant
4th dose 0.5 agar slant
5th dose 0.5 agar slant
3. Rabbit immunized to B. typhosus and to B. coli communior by
intravenous injection. Intervals as above.
1st dose 0.05 agar slant of B. typhosus
2d dose 0.1 agar slant of B. typhosus
3d dose 0.2 agar slant of B. typhosus
4th dose 0.2 agar slant of B. typhosus, plus 1
0.05 agar slant of B. coli communior J
5th dose 0.2 agar slant of B. typhosus, plus
0.05 agar slant of B. coli communior
6th dose 0.2 agar slant of B. typhosus, plus j
0.1 agar slant of B. coli communior J
7th dose 0.2 agar slant of B. typhosus, plus 1
0.2 agar slant of B. coli communior J
4. Guinea pig immunized to Sp. cholerse by intraperitoneal injec-
tion. An old laboratory strain of the cholera vibrio should be used
for immunization. Intervals as above.
1st dose 0.05 agar slant
2d dose 0.1 agar slant
3d dose 0.2 agar slant
4th dose 0.2 agar slant
5th dose 0.2 agar slant
5. Rabbit immunized to micrococcus aureus by intravenous injec-
tion. Intervals as above. In the case of this microorganism the
intervals should be lengthened if there is any indication of illness.
Great care is necessary in avoiding overdosage.
1st dose 0.05 agar slant
2d dose 0.1 agar slant
3d dose 0.2 agar slant
4th dose 0.2 agar slant
5th dose 0.2 agar slant
14 LABORATORY COURSE IN SERUM STUDY
6. Rabbit immunized to sheep serum by intravenous injection.
Five-day intervals.
1st dose 2.0 c.c.
2d dose 2.0 c.c.
3d dose 2.0 c.c.
7. Rabbit immunized to human serum (ascitic fluid) by intravenous
injection. Five-day intervals.
1st dose 4.0 c.c.
2d dose 4.0 c.c.
3d dose 4.0 c.c.
8. Rabbit immunized to horse serum. (Immunize noteless than
three in order to obtain sufficient serum for later exercises.)
1st dose 2.0 c.c.
2d dose 2.0 c.c.
3d dose 2.0 c.c.
9. Rabbit immunized to sheep erythrocytes by intravenous injec-
tion. Four or five day intervals.
1st dose 4.0 c.c. of 50 % suspension
2d dose 4.0 c.c. of 50 % suspension
3d dose 4.0 c.c. of 50 % suspension
A fourth injection may be given in some cases, but is not often
necessary.
10. Rabbit immunized to sheep erythrocytes by intraperitoneal
injections. Intervals as above.
1st dose 4.0 c.c. of 50 % suspension
2d dose 8.0 c.c. of 50 % suspension
3d dose 8.0 c.c. of 50 % suspension
4th dose 8.0 c.c. of 50 % suspension
11. Rabbit immunized to cat erythrocytes by intravenous injec-
tions of 2 c.c. at 4-day intervals. — See Lesson XII.
A preliminary titration of the serum done with a specimen
obtained from an ear vein should be done about the eighth or
ninth day after the last injection. If this is satisfactory, the
bleeding of the animal is usually done the ninth or tenth day. No
absolute rule can be established as to exact period at which the
serum has the highest antibody concentration, this depending to
16 LABORATORY COURSE IN SERUM STUDY
some extent on the health of the animal, the size of the injec-
tions, and the intervals. It is a safe rule to assume that the
high point in the curve is apt to lie between the eighth and the
twelfth day after the last injection and to be guided by this in
preliminary titrations.
THE BLEEDING OF ANIMALS
Before the immunization of the animal is begun a sample of
blood should be taken from the ear vein of the animal and the
serum stored in the refrigerator for use as a control at a later exer-
cise when the serum of this animal is tested for immune bodies.
The blood can be obtained from the marginal vein in the ear in
the following manner.
The animal is firmly held with the head down by an assistant.
The lateral portion of the hairy surface of the ear is carefully
shaved and the skin rubbed vigorously to cause the veins to dis-
tend. If necessary the ear may be rubbed with xylol. The skin
over the vein is washed over with alcohol and a cut made in the
vein with a razor blade or other sharp instrument. The blood is
allowed to drip into a clean centrifuge tube until about 4 c.c.
(60 drops) are obtained. It is then allowed to coagulate and the
edges of the clot are freed from the side of the tube by means of a
sterile platinum wire. The tube is centrifugalized and the clear
serum thus expressed is transferred to a sterile test tube by
means of a capillary pipette with a rubber nipple attached. It is
then labelled carefully and stored in the icebox for future use.
Later bleeding from the ear veins of a rabbit can be facilitated
by strapping the animal down on a flat, hot water bag. A techni-
cal point of great simplicity but which helps to make the sort of
cut in the vein which favors free bleeding, consists in taking a
pointed steel writing pen, breaking off one of the prongs and
plunging the stilette thus made straight into the vein in a direc-
tion toward the heart. The entry thus made through the wall
of the vein does not easily collapse and very free bleeding results.
To bleed a rabbit from the carotid the rabbit is strapped down
on its back on a dissecting board and ether administered. A
18 LABORATORY COURSE IN SERUM STUDY
central incision along the neck above the trachea is made, and the
carotid will be found lying very close to the trachea on either side.
A ligature is applied distally, the artery raised on the handle of a
forceps and a bulldog clamp applied toward the ridge of the neck.
A sterile glass canula can be inserted, but we have found that
with a little skill it is quite easy to lift up the artery and hold it
by its wall with a forceps in such a way that the blood stream
is not blocked. The vessel is then cut, the clamp removed,
and by aiming the artery with the forceps in which it is held the
stream of blood can be directed into a sterile test tube. In this
way rabbits are usually bled until distinct respiratory distress
ensues and the stream of blood ceases, the blood coming in
drops. The clamp is then applied, the vessel tied and the rabbit
sewed up aseptically. Such a rabbit can be kept alive and
further used after recovery for continuation of the immunization.1
The bleeding of guinea pigs from the carotid is in every way
similar, though a little more difficult than the bleeding of rabbits.
When guinea pigs are bled, as they will be for the purpose of ob-
taining complement or alexin, it is necessary to do this carefully
in order to obtain the best results. The blood is best taken into
large sterile test tubes with as little violence as possible in order
that there may be no breaking up of blood cells with consequent
hemolysis. It is best to take the blood the evening before it is
to be used, to allow it to clot in the test tube and then to separate
the clot very gently from the sides of the tube. The tube should
then be set aside in the refrigerator at a low temperature (1-4° C.).
The rapidity with which complement degenerates at higher tem-
peratures is generally underestimated. The following day the
serum is pipetted away from the clot.
Some observers believe that complement increases in potency
on standing for some hours on the clot. This was the con-
tention of Henderson-Smith, though it has recently been con-
tradicted by Addis and others. It is a good practice to obtain it
in this way, however, because blood so taken is apt to be clearer
and freer from haemolysis than blood centrifugalized immediately
after clotting. Although a slight tinge of haemolysis does not
1 Intravenous injections of 10 to 20 c.c. of warm salt solution will often help to
keep such animals alive.
\
20 LABORATORY COURSE IN SERUM STUDY
f-'-1- v/
^bes
^ • u~
render blood useless for haemolytic purposes, nevertheless the
products of haemolysis do affect the haemolytic reaction and it is
t to have an entirely clear alexin for use.
The technique of bleeding of sheep or other large animals has
been described on page 2.
LESSON II
BACTERICIDAL POWER OF NORMAL SERUM
NORMAL blood plasma or serum possesses the power of killing
bacteria. Not all bacteria are equally susceptible to this effect.
Some, like the Gram-positive cocci, the anthrax bacillus, and
others, are probably killed in the circulation only by the co-
operation of serum and leucocytes in phagocytosis. When the
serum is heated to 56° C., its bactericidal power is suspended.
Reagents :
1. Fresh normal rabbit serum, unheated.
2. Bacillus typhosus — 24-hour broth culture diluted 1-5000 in
sterile salt solution.
3. Sterile salt solution.
The materials in this experiment must be measured with sterile
pipettes and placed in sterile test tubes, with care to avoid con-
tamination during the process of the experiment.
(a) Place 0.5 c.c. of normal rabbit serum in each of two sterile
test tubes and heat one tube for half an hour at 56° C. Then
set up the following preparations in sterile plugged test tubes :
1. Normal rabbit serum 0.5 c.c. + Typhoid broth (1-5000) 0.5 c.c.1
(fresh)
2. Normal rabbit serum 0.5 c.c. + Typhoid broth (1-5000) 0.5 c.c.
(heated)
3. Salt solution 0.5 c.c. + Typhoid broth (1-5000) 0.5 c.c.
1 To make dilutions for this and other purposes proceed as follows :
1. 1 c.c. of original substance to 9 c.c. of salt solution = 1-10
2. 1 c.c. of dilution (1.) to 9 c.c. of salt solution = 1-100
3. 1 c.c. of dilution (2.) to 4 c.c. of salt solution = 1-500
4. 1 c.c. of dilution (3.) to 1 c.c. of salt solution = 1-1000
5. 1 c.c. of dilution (4.) to 4 c.c. of salt solution = 1-5000
This illustrates the general method, which can be shortened or lengthened
by longer or smaller intervals as the particular occasion requires.
22 LABORATORY COURSE IN SERUM STUDY
(6) Place the tubes in the incubator for two hours, and then
pour into each tube a tube of agar which has been melted in
boiling water and cooled to 42° C. Pour the entire contents
of each tube into a sterile Petri dish. When the plates are
hardened, place them in the incubator in an inverted position,
to be observed at the following lesson. The plates made from
tubes 2 and 3 will show an enormous number of minute
typhoid colonies after incubation. The plate from tube 1 will
be sterile or may show a few large colonies.
ILEMOLYTIC AND ILEMAGGLUTINATIVE POWER OF
NORMAL SERUM
The normal blood serum of many animals has haemolytic
and haemagglutinative action upon the red blood cells of animals
of some other species. This hsemolytic action, like the bacteri-
cidal action, is lost when the serum is heated to 56° C. The
haemagglutinating property, on the other hand, is relatively
resistant to heat and is not destroyed until the serum is heated to
70° C. or above. Such a hsemolytic normal serum acts only on
the erythrocytes of certain other species, sometimes upon 2 or 3
varieties with differing intensity. Thus goat serum is strongly
haemolytic for rabbit cells — less so for those of guinea pigs.
Dog serum contains a relatively large amount of normal haemo-
lysin for most cells used in laboratory work. Guinea pig cells
are highly susceptible, and human cells relatively resistant to
haemolysis by dog serum.
Reagents :
1. Fresh dog serum.
2. 2 % suspension of washed guinea pig cells.
3. 2 % suspension of washed human cells.
4. Salt solution.
Place 0.25 c.c. of dog serum in a test tube and heat for one half
hour in water bath at 56° C. Bring up volume to 2.5 c.c. with normal
salt solution. Prepare a similar 1-10 dilution of unheated dog serum.
Set up the following experiment in half-inch tubes :
24 LABOKATORY COURSE IN SERUM STUDY
1. Dog serum unheated (1-10) 0.5 c.c. + Guinea pig cells 2 % 0.5 c.c.
2. Dog serum unheated (1-10) 0.5 c.c. + Human cells 2 % 0.5 c.c.
3. Dog serum heated (1-10) 0.5 c.c. + Guinea pig cells 2 % 0.5 c.c.
4. Dog serum heated (1-10) 0.5 c.c. + Human cells 2 % 0.5 c.c.
5. Salt solution 0.5 c.c. + Guinea pig cells 2 % 0.5 c.c.
6. Salt solution 0.5 c.c. -f Human cells 2 % 0.5 c.c.
Incubate the tubes one hour at 37° C. in water bath, observing
changes in cells at intervals of 5, 10, 15, 30, and 60 minutes. Tabulate
the results. Tube 1 should show prompt haemolysis; tube 3 should
show agglutination of the cells. In tubes 2 and 4 there should be no
observable change in the cells in most samples of dog serum.
Each student needs: 10 J^-inch test tubes, test tube rack, 5 one-c.c.
pipettes, 1 five-c.c. pipette.
LESSON III
HAEMOLYSIS. (EHRLICH AND MORGENROTH EXPERI-
MENTS)
1. To a group of students is assigned the task of bleeding a sheep
from the jugular vein into a flask containing glass beads. After defibrina-
tion the cells are washed three times in salt solution and a 5 per cent
suspension of the well-packed sediment is made (1.0 c.c. of sediment and
19.0 c.c. of salt solution).
2. To another group of students is assigned the bleeding of guinea
pigs from the carotid artery into centrifuge tubes. The blood is allowed
to clot, this clot is "rimmed" and the serum separated. The process
may be hurried by centrifugation. The serum is then diluted, 1 part of
serum to 9 parts of salt solution.
3. Immune rabbit serum (lytic for sheep cells) will be given out
diluted.1 This serum has been heated for one half hour at 56° C.
before dilution, in order to destroy its alexin or complement. This is
done so that no unknown amount of complement may be present in the
final tests.
All these materials must be prepared before the regular class period,
as the experiments themselves consume several hours. Provided the
guinea pig serum is kept very cold (0°-^i0 C.), all the materials can be
prepared the preceding day.
1 In order to save time in the course an immune serum previously prepared by
the instructor is used here. The immune haemolytic serum whose preparation
was begun by the students in Lesson I is to be titrated later and used for the
lessons on complement fixation .
26 LABORATORY COURSE IN SERUM STUDY
Experiment 1
To SHOW THAT RED BLOOD CELLS WILL ABSORB AMBOCEPTOR BUT
NOT COMPLEMENT
Set up two mixtures in centrifuge tubes as indicated below:
A. Sheep cells and heated immune rabbit serum
B. Sheep cells and fresh normal guinea pig serum.
Incubate for 30 minutes at 37 C., then centrifugalize and remove
the supernatant fluid, using a capillary pipette with rubber nipple.
Wash the sediment once in salt solution.
Resuspend the washed sediment in each tube in 3 c.c. salt solution.
Add the reagents as indicated and incubate for one hour at 37 C.,
observing at short intervals. Tabulate the results.
Tube A
Sheep cells 5% 1.0 c.c.
Inactivated immune rabbit serum l .... 1.0 c.c.
Incubate 30 minutes at 37° C., centrifugalize, remove the superna-
tant fluid to another tube and wash the sediment.
We now have 2 tubes — one (Tube 1) containing the supernatant
fluid, the other (Tube 2) the sediment from the original Tube A. To
these tubes add reagents as follows :
*
Tube 1 Tube 2
(Supernatant fluid from (Sediment from Tube A
Tube A) 2.0 c.c. resuspended in salt
Guinea pig serum (1-10) 1.0 c.c. solution) 3.0 c.c.
Sheep cells 5% . . . 1.0 c.c. Guinea pig serum (1-10) 1.0 c.c.
Incubate and observe.
In which of the two does haemolysis take place? What conclu-
sion as to the reaction that has taken place in the original mixture in
Tube A can you draw from this?
TubeB
Sheep cells 5% 1.0 c.c.
Guinea pig serum (1-10) 1.0 c.c.
Incubate 30 minutes at 37° C., centrifugalize, remove supernatant
fluid to another tube and wash sediment.
1 Diluted by the instructor so as to contain two hemolytic units in 1 c.o.
28 LABORATORY COURSE IN SERUM STUDY
Then, as in the preceding case, we have two tubes to which the
following additions are made :
Tube 1 Tube 2
(Supernatant fluid from (Sediment from Tube B
Tube B) about . . . 2.0 c.c. resuspended in salt
Immune rabbit serum 1.0 c.c. solution) 3.0 c.c.
Sheep cells 5% . . 1.0 c.c. Immune rabbit serum . 1.0 c.c.
Incubate and observe.
In which tube does haemolysis take place? Contrast this with
results in Tube A and draw conclusions.
Experiment 2
TO SHOW THAT AT 0° C. CELLS WILL ABSORB AMBOCEPTOR ALONE
FROM A MIXTURE OF AMBOCEPTOR AND COMPLEMENT
The same reagents will be used in this as in the preceding experi-
ment. The three reagents will first be cooled in ice water and then be
mixed in a cold centrifuge tube, and allowed to stand at 0° C. for an
hour. With proper care this can easily be done in a battery jar filled
with cracked ice and brine.
The success of the experiment depends on having the reagents and
the centrifuge tube and metal holder thoroughly cooled in ice water
before the mixture is made and in centrifugalizing and removing the
supernatant fluid before they have time to regain the temperature of
the room. The protocol for this experiment is given below :
Place centrifuge tube in holder in a cup of cracked ice and water and
add:
5% sheep cells 1.0 c.c.
Inactive immune rabbit serum (diluted) . . 1.0 c.c.
Guinea pig serum (1-10) 1.0 c.c.
After twenty minutes the tube is centrifugalized, the supernatant
fluid removed and half of it placed in each of two small test tubes.
The sediment is to be washed once in cold salt solution, resuspended
in 4 c.c. of salt solution and half of this suspension placed in each of
two small test tubes.
We now have four tubes — two of which contain washed sediment
and two of which contain supernatant fluid of the original mixture.
To these the following additions are now made :
30 LABORATORY COURSE IN SERUM STUDY
Tube 1
Supernatant fluid 1.5 c.c.
Fresh guinea pig serum 1-10 (complement) 0.5 c.c.
Washed sheep cells 5 % 0.5 c.c.
Tube 2
Supernatant fluid 1.5 c.c.
Immune rabbit serum 0.5 c.c.
Washed sheep cells 5 % 0.5 c.c.
TubeS
Sediment 2.0 c.c.
Guinea pig serum 1-10 0.5 c.c.
Tube 4
Sediment 2.0 c.c.
Immune rabbit serum 0.5 c.c.
Incubate one hour at 37° C., observing at frequent intervals.
The results will show that the haemolytic amboceptor or sensitizer
present in the original heated immune serum was bound by the cells at
0° C. — but at this low temperature the complement or alexin remained
not only inactive but unbound and free.
APPARATUS
Each student will require :
3 graduated centrifuge tubes
8 half-inch test tubes
9 one c.c. pipettes
1 test tube rack
1 bottle of salt solution
1 jar of cracked ice (pint measure)
1 rubber nipple and capillary pipette
The class will require :
1 outfit for bleeding sheep :
needle,
tourniquet,
razor and scissors,
bottle of tincture of iodine,
sterile flask containing beads.
32 LABORATORY COURSE IN SERUM STUDY
1 or more outfits for bleeding guinea pigs :
scissors,
forceps,
ether cone,
centrifuge tubes.
MATERIALS
Each student will require :
Washed sheep cells 5 % 6.0 c.c.
Guinea pig serum 1-10 6.0 c.c.
Inactive serum of rabbit immunized against
sheep cells, diluted so that each c.c. con-
tains 2-4 hsemolytic doses 6.0 c.c.
LESSON IV
QUANTITATIVE RELATIONSHIPS OF AMBOCEPTOR AND
COMPLEMENT
METHODS OF TITRATION
THE haemolysis of red cells by specific sensitizer [or ambocep-
tor] and alexin [or complement] is a delicately quantitative
reaction in which very definite amounts of the three substances
are necessary to produce a complete reaction. As a unit of red
cells, one cubic centimeter of a 5 per cent suspension of red
cells is conveniently taken. This means that the cells after
washing are sedimented in the centrifuge and 1.0 c.c. of the
sediment is added to 19.0 c.c. of salt solution. (Some investi-
gators have worked with suspensions representing 5 per cent of
the original blood. Assuming the cells to constitute 50 per
cent of the blood volume, suspensions so made up are just half
as concentrated as the ones here used. In all the work here de-
scribed the term 5 per cent suspension is taken to represent 5 per
cent by actual volume of erythrocyte sediment in salt solution.)
Definite amounts of sensitizer and of alexin are necessary to
lake completely one cubic centimeter of such a suspension, and it
is necessary for accurate experimentation to determine for each
set of reagents the minimal amounts necessary to accomplish
34 LABORATORY COURSE IN SERUM STUDY
this purpose. It must be remembered that no two suspensions
of red cells are exactly alike — since it is impossible to measure
them with absolute accuracy, and since the resistance of erythro-
cytes to haemolysis, even when taken from the same animal on
different days, may vary. No two fresh sera, moreover, are
entirely alike in alexic activity. The experiments given below
will demonstrate the method of determining by titration the
minimal haemolytic dose of complement and amboceptor ; they
will also demonstrate that this dose varies reciprocally, that is,
that with a small amount of complement a relatively larger
amount of amboceptor will be necessary to cause complete
haemolysis, whereas with a large amount of complement only a
small amount of amboceptor is necessary. There is, however, no
simple proportionality.
Experiment 1
TITRATION OF H^MOLYTIC SENSITIZER OR AMBOCEPTOR. DETERMINA-
TION OF UNIT
The unit of sensitizer, or amboceptor, is the smallest amount
which will cause complete laking of a unit of cells (1.0 c.c. of a
5 per cent emulsion) 1 in one hour, in the presence of an excess of
complement.
As the degree of dilution is an important factor it is necessary that
the volume of all the tubes be brought to a uniform standard before
the reaction actually begins. For this reason the reagents are added in
the following order :
1. Sensitizer [amboceptor]
2. Alexin [complement]
3. Saline to make all volumes equal
4. Cells
A control tube must be set up to prove that the amount of alexin or
complement used will not of itself lake the cells.
1 For convenience or economy 0.5, 0.25 or 0.1 c.c. of 5 per cent red cells are
sometimes taken as the unit, and in such cases the unit of amboceptor and com-
plement must be proportionately reduced. For the sake of uniformity results
so obtained can easily be transposed to terms of the 1 c.c. unit, as is often done
in reporting Wassermann tests.
C6
LABORATORY COURSE IN SERUM STUDY
Add saline
to 2 c.c.
and then
0.5 c.c. of
the red cell
emulsion. 1
Set up the following tests :
Series 1
Immune Serum (Sensitizer) Complement
Tube 1. (diluted 1-100) 0.5 c.c. 0.05 c.c.
2. (diluted 1-100) 0.2 c.c. 0.05 c.c.
3. (diluted 1-1000) 1.0 c.c. 0.05 c.c.
4. (diluted 1-1000) 0.5 c.c. 0.05 c.c.
5. (diluted 1-1000) 0.4 c.c. 0.05 c.c.
6. (diluted 1-1000) 0.3 c.c. 0.05 c.c.
7. (diluted 1-1000) 0.2 c.c. 0.05 c.c.
8. (diluted 1-1000) 0.1 c.c. 0.05 c.c.
9. (diluted 1-1000) 0.0 c.c. 0.05 c.c.
(Complement Control)
Series 2
Immune Serum (Sensitizer) Complement
Tube 1. (diluted 1-100) 0.5 c.c. 0.02 c.c.
2. (diluted 1-100) 0.2 c.c. 0.02 c.c.
3. (diluted 1-1000) 1.0 c.c. 0.02 c.c. Add saline
4. (diluted 1-1000) 0.5 c.c. 0.02 c.c. to 2 c.c.
5. (diluted 1-1000) 0.4 c.c. 0.02 c.c. and then
6. (diluted 1-1000) 0.3 c.c. 0.02 c.c. 0.5 c.c. of
7. (diluted 1-1000) 0.2 c.c. 0.02 c.c. the red cell
8. (diluted 1-1000) 0.1 c.c. 0.02 c.c. emulsion.
9. (diluted 1-100) 0.5 c.c. 0.00 c.c. (
(Sensitizer Control)
Series 3
Immune Serum (Sensitizer) Complement
Tubel. (diluted -100) 0.5 c.c. 0.01 c.c.
2. (diluted -100) 0.2 c.c. 0.01 c.c.
3. (diluted -1000) 1.0 c.c. 0.01 c.c. Add saline
4. (diluted -1000) 0.5 c.c. 0.01 c.c. to 2 c.c.
5. (diluted -1000) 0.4 c.c. 0.01 c.c. and then
6. (diluted -1000) 0.3 c.c. 0.01 c.c. 0.5 c.c. of
7. (diluted 1-1000) 0.2 c.c. 0.01 c.c. the red cell
8. (diluted 1-1000) 0.1 c.c. 0.01 c.c. emulsion.
9. (diluted 1-1000) 0.0 c.c. 0.00 c.c. J
(Salt Solution Control)
1 A half c.c. instead of one c.c. is used in order to save materials. The amounts
of complement and amboceptor needed are of course half those that would be needed
for 1 c.c. of cells.
38
LABORATORY COURSE IN SERUM STUDY
In measuring 0.05, 0.02 and 0.01 c.c. of complement it is best to use
a half c.c., .2 c.c. and .1 c.c. of a 1-10 dilution. Shake each tube
thoroughly when all the reagents have been added. Incubate one hour
in water bath at 37° C. and record by -f- or — signs haemolysis at 15,
30 and 60 minutes in the following protocol:
IMMUNE SERUM (SENSITIZER OR AMBOCEPTOR)
Diluted 1-100
Diluted 1-1000-
0.5
0.2
1.0
0.5
0.4
0.3
0.2
0.1
0.0
Complement 0.05 .
Complement 0.1
Complement 0.2
Indicate what the unit is in this case.
The tabulation given for final record is the manner in which an
experiment of this kind is best recorded by experienced workers. We
have inserted the simpler protocols of the three series before this in
order to make the purpose of this experiment and its execution a little
more easy.
(On account of the costliness of guinea-pig serum students should
divide themselves into groups of 3, of whom each student should set
up one series of amboceptor dilutions with one of the complement quan-
tities.)
Individual guinea pigs vary in the complementary activity of
their serum. For accurate hsemolytic experiments, however, it
is necessary to use a constant amount of complement activity.
For this reason the unit of amboceptor (which if carefully pre-
served remains constant over long periods) is taken as a standard
to which the strength of each fresh complement serum is adjusted.
Strictly speaking, the unit of complement should be that
amount which gives complete laking with one unit of cells and
one unit of amboceptor. In practice, however, this turns out to
be too large an amount, and for this reason complement is usually
titrated with two units of amboceptor. The unit of complement
then may be defined as the least amount that completely hemo-
lyses 1.0 c.c. of a five per cent emulsion of red cells in the presence
of two units of amboceptor in thirty minutes in the water bath.
If the test is done with smaller quantities of red cells the unit is
correspondingly smaller. Set up titrations with both two and
one units as follows:
40
LABORATORY COURSE IN SERUM STUDY
TUBE
COMPLEMENT 1-10
AMBOCEPTOR
1
0.5 c.c.
2 units '
2
0.4
2 units
Add
3
0.3
2 units
Saline to 2 c.c.
4
0.2
2 units
5% cells, 0.5 c.c.
5
0.1
2 units t
6
0.5
1 unit
7
0.4
1 unit
Add
8
0.3
1 unit
Saline to 2 c.c.
9
0.2
1 unit
5% cells, 0.5 c.c.
10
0.1
1 1 unit
Incubate. Read results at 15, 30 and 60 minutes.
(Indicate unit found.)
It should be remembered that even with these two reagents, com-
plement and amboceptor, titrated, the amounts determined might vary
somewhat if cells from another sheep or taken from this sheep on another
day were used — since the resistance of the cells is also a variable
factor.
In many experiments it is expedient to use red cells previously
sensitized with a definite number of amboceptor units. In order to do
this the following method is employed. The red cells after washing are
taken up in salt solution in a definite concentration which for ordinary
work is 5 per cent. Inactivated immune serum (amboceptor or sensi-
tizer) diluted in salt solution is then added so that the required number
of previously determined haemolytic units shall be present for every unit
(1.0 c.c. of 5 per cent) of red cells. Therefore, supposing that 0.001 c.c.
of the immune serum was found to be one haemolytic amboceptor unit,
1.0 c.c. of the 1-1000 dilution of this serum added to 1.0 c.c. of a 5 per
cent emulsion of red cells would sensitize them with one unit. Having
made such a mixture and having given it 15 minutes to allow the union
to take place, every 2.0 c.c. would represent 1.0 c.c. of 5 per cent red
cells and 1 unit of amboceptor united. Or if desired the cells could be
centrifugalized, washed, and made up to the original concentration of
5 per cent. On this principle any number of units up to the maximum
absorption power can be added to the red cells. As we approach the
maximum absorption power of amboceptor by red cells it is always
well to centrifugalize and reemulsify the red cells so that our experi-
ment may not be confused by the presence of excessive unabsorbed
amboceptor.
42
LABORATORY COURSE IN SERUM STUDY
Each student needs:
10 c.c. of 5% sheep cells
2 c.c. amboceptor (diluted 1-100)
2 c.c. complement undiluted, for each three students
1 test tube rack
20 half-inch test tubes
4 1-c.c. pipettes, and flask salt solution
4 f -inch test tubes
LESSON V
QUANTITATIVE RELATIONS OF AMBOCEPTOR AND COMPLE-
MENT (Continued)
Experiment 1
THE ACTIVITY OF COMPLEMENT DEPENDS TO SOME EXTENT ON ITS
CONCENTRATION AND NOT ONLY ON THE TOTAL AMOUNT PRESENT
A 25 per cent suspension of washed sheep cells is made.
A series of tubes is set up (if necessary by instructor) in which the
minimal amount of amboceptor necessary to lake 0.1 c.c. of these cells
in the presence of 0.05 c.c. of undiluted complement and in a total volume
of 1.0 c.c. is determined. The hsemolytic serum (amboceptor) is then
diluted so that this minimal amount is contained in 0.1 c.c.
The student then mixes 2.0 c.c. of this amboceptor dilution with
2.0 c.c. of the 25 per cent red cell suspension. He then determines, in
the following preliminary titration, the minimal amount (unit) of com-
plement which will lake 0.1 c.c. of these cells in one hour, in a total
volume of 1 c.c.
GUINEA-PIG SERUM 1-10
SENSITIZED CELLS
(.05 C.C. CELLS + .05 C.C.
OF AMBOCEPTOR)
SALINE UP TO 1.0 C.C.
Tube 1
Tube 2
TubeS
Tube 4
TubeS
Tube 6
Tube?
TubeS
0.15 C.C.
0.2
0.25
0.3
0.4
0.5
0.0
.5
0.1 c.c.
0.1 c.c.
0.1 c.c.
0.1 c.c.
0.1 c.c.
0.1 c.c.
0.1
.05 unsensi-
tized cells
.75 C.C.
.7
.65
.6
.5
.4
.9
.45
Then with 1, 5, and 10 times this minimal amount in the tubes as indi-
cated in the protocol the experiment is set up. The protocol given
below is constructed on the supposition that, in the above titration, 0.2
c.c. of a 1 to 10 dilution of complement was the smallest amount of the
complement which gave complete laking.
44
LABORATORY COURSE IN SERUM STUDY
GUINEA Pio SERUM
1 IN 10
SALT SOLUTION UP TO
RESULTING DILUTION
OF GUINEA PIG SERUM
Tube 1
0.2 C.C.
1.0 c.c.
in 50
Tube 2
0.2
2.0
in 100
Tube 3
0.2
5.0
in 250
Tube 4
0.2
10.0
in 500
Tube 5
1.0
1.0
in 10
Tube 6
1.0
5.0
in 50
Tube 7
1.0
10.0
in 100
Tube 8
1.0
20.0
in 200
Tube 9
2.0
5.0
in 25
Tube 10
2.0
10.0
lin 50
Tube 11
2.0
20.0
1 in 100
To each tube add 1 unit (0.1 c.c.) of cells previously sensitized with
1 unit of amboceptor.
Calculate the concentration of complement in the tubes used in the
preliminary complement titration, and compare the results. If the
activity of complement does depend upon the concentration, complete
laking should occur in one hour in those tubes of the second series
which contain a dilution of guinea pig serum equal to that in the first
tube of the first series which showed complete haemolysis.
Experiment 2
THE ACTIVITY OF SENSITIZER OR AMBOCEPTOR DEPENDS ON ITS
TOTAL AMOUNT AND NOT ON ITS CONCENTRATION
Suspend 1 unit (0.05 of 25%) of cells in varying amounts (1, 5, 10,
20 c.c.) of salt solution.
To each tube add 1 unit of amboceptor.
Mix and keep at 37° C. for one half hour.
Centrifugalize.
To the sediment in each tube add 1 unit of complement, as previously
determined, and sufficient salt solution to bring the volume to 1 c.c.
Incubate.
All tubes should show complete haemolysis in 1 hour if the cells
have absorbed the entire unit of amboceptor.
Each student needs:
2 c.c. of 25% sheep cells 1 ten-c.c. pipette
(about) 2 c.c. of undiluted com- 2 test tube racks for 12 J-inch
plement test tubes
25 units of amboceptor 20 J-inch test tubes
Flask of saline 1 fifty-c.c. centrifuge tube
4 one-c.c. pipettes 3 fifteen-c.c. centrifuge tubes
6 £-inch test tubes
46 LABORATORY COURSE IN SERUM STUDY
LESSON VI
THE UNION OF IMMUNE-BODY (AMBOCEPTOR OR SENSI-
TIZER) AND CELLS
\
1. DISSOCIATION OF AMBOCEPTOR (Mum)
RED cells have a very great affinity for their specific immune-
body. They can absorb a great number of units. Nevertheless at
37° C. corpuscles containing multiple doses of amboceptor give off
a certain amount to the surrounding fluid when it is free of it.
There appears to be an equilibrium between combined and free im-
mune-body. This can be illustrated by the following experiment :
To 1.5 c.c. of a" 5 per cent emulsion of sheep corpuscles is added
inactivated haemolytic serum so that there shall be 20 units of ambo-
ceptor x to every 0.5 c.c. of cells. (The student should calculate and
make dilutions for this purpose after being told the unit of the hsemolytic
serum given him.)
This mixture is allowed to stand at room temperature for 30 minutes.
Centrifugalize and set aside the supernatant fluid. This is Tube (a).
Wash the cells obtained in the sediment of the preceding centrif uga-
tion three times and set aside the salt solution remaining as supernatant
fluid of the last washing. This is Tube (6).
Make the suspension of red cells up to the original volume by the
addition of saline and incubate this at 42° C. for one hour. Then centrif-
ugalize and set aside the supernatant fluid of this, which constitutes
Tube (c).
Make the sediment of the cells obtained in the preceding up to 1.5
c.c. Shake. Remove 0.5 c.c. and add to it 1.0 c.c. of 10 per cent fresh
guinea pig complement. This is Tube (d).
To (a), (6) and (c), each, add 0.5 c.c. of 5 per cent sheep corpuscles
and .5 c.c. of complement (10 per cent fresh guinea pig serum in salt
solution).
All four tubes are incubated at 37° C. for one hour.
Record the degree of laking in each tube.
Tube (a) will show how much of the 20 units of amboceptor failed to
be absorbed. The exact amount could be determined by titration, which,
however, would needlessly prolong the experiment and add nothing
to the illustration of the principle.
1 The unit here is the minimal amount which lakes 0.5 c.c., not 1 c.c., of cells.
If Experiments 1 and 3 of this lesson are to be done on the same afternoon it is
well to sensitize a considerable amount of cells at one time.
48
LABORATORY COURSE IN SERUM STUDY
Tube (6) will show whether the three washings were sufficient to
remove all amboceptor from the fluid bathing the cells. Any excess of
amboceptor remaining unattached to the cells after the washing would
show in the supernatant fluid by laking.
Tube (c) will show how much, if any, amboceptor was dissociated
after one hour at 37° C.
2. VELOCITY OF AMBOCEPTOR ABSORPTION
Red cells absorb their homologous immune-body very rapidly.
A knowledge of this is of great importance as the following ex-
periments show.
A
Take 3 c.c. of 5 per cent sheep corpuscles in a wide test tube and add
3 c.c. of diluted amboceptor drop by drop, shaking constantly (the
dilution of the amboceptor is such that 1 unit is contained in 0.5 c.c. ;
the unit here is taken as the minimal amount which lakes 0.5 c.c. of
ceUs).
B
Take 3 c.c. of diluted amboceptor and add 3 c.c. of 5 per cent sheep
cells drop by drop, shaking constantly.
Set up two parallel series of five tubes each containing varying
amounts of guinea pig serum (1-10).
Series 1
GUINEA Pro SERUM
SALT SOLUTION (TO
TUBE
DILUTED 1-10
MAKE ALL VOLUMES
MIXTURE A
(COMPLEMENT)
EQUAL)
1
0.75 c.c.
0.0 C.C.
1.0 c.c.
2
0.5 c.c.
0.25 c.c.
. 1.0 c.c.
3
0.4 c.c.
0.35 c.c.
1.0 c.c.
4
0.3 c.c.
0.45 c.c.
1.0 c.c.
5
0.2 c.c.
0.55 c.c.
1.0 c.c.
Series 2
Duplicate above with mixture B.
Incubate one hour and compare results.
The observed differences are probably explained by the fact that
the first cells which are added to B absorb nearly all the amboceptor,
leaving insufficient to sensitize the last cells added.
50 LABORATORY COURSE IN SERUM STUDY
3. LIBERATION OF AMBOCEPTOR IN HAEMOLYSIS
When red cells containing several hsemolytic doses of ambo-
ceptor are laked by a not excessive amount of complement, some
of the amboceptor is liberated in the solution.
To 2.0 c.c. of 5 per cent sheep cells add sufficient amboceptor so
that there will be 20 units for every unit (0.5 c.c.) of 5 per cent sheep
cells. Allow to stand for one half hour. Centrifuge and wash with
saline three times and after the third washing make the volume again
up to 2.0 c.c.
To 1.0 c.c. of cells so sensitized add 1.0 c.c. of 10 per cent guinea-pig
complement. Incubate one half hour. Then divide into two equal
portions of 1.0 c.c. each. To the first portion add 0.5 c.c. of the sen-
sitized cells and incubate again for one hour. Note whether haemolysis
of the added cells occurs. To the second portion add 0.5 c.c. of un-
treated corpuscle suspension and 1.0 c.c. of 10 per cent complement.
Haemolysis, after incubation at 37J degrees centigrade, will indicate
that amboceptor was liberated by the laking of the sensitized cells dur-
ing the first incubation and was taken up by the added cells in the second
incubation.
How does the amount of amboceptor liberated compare with the
amount dissociated by simple incubation without haemolysis in Experi-
ment 1 ?
Each student needs :
12 c.c. of 5% sheep cells
160 units of amboceptor
10 c.c. of complement (1-10)
Flask saline
4 one-c.c. pipettes
1 test tube rack for half-inch test tubes
1 graduated centrifuge tube
20 half-inch test tubes
3 three-quarter-inch test tubes
1 five-c.c. pipette
1 nipple pipette1
1 If experiments 1 and 3 of this lesson are to be done on the same afternoon, it
is well to sensitize a considerable amount of cells at one time.
52 LABORATORY COURSE IN SERUM STUDY
LESSON VII
PFEIFFER PHENOMENON
VIRULENT cholera spirilla injected into the peritoneal cavity
of a normal guinea pig proceed to multiply rapidly after a pe-
riod of about half an hour, and the animal dies usually within 24
hours with symptoms of profound intoxication. The same spirilla
injected into an animal which has been immunized by previous
injections of killed spirilla or of sub-lethal doses of the living
microorganisms become granular and in some cases swell, take
on globular and vacuolated forms and gradually undergo lysis.
The guinea pig in such cases recovers completely from the in-
jection. This destruction of invading bacteria by the immune
animal was termed by Pfeiffer " bacteriolysis." It has been ob-
served with certain of the Gram-negative bacilli such as the
typhoid and the dysentery bacillus, but is best observed in
cholera. Similar lysis is observed if the spirilla are injected into
a normal pig together with serum of an immunized guinea pig or
rabbit.
For this experiment it is necessary to employ a virulent 1 strain
of cholera spirilla, since old laboratory cultures do not show the
phenomenon sharply and rarely kill the guinea pigs in the con-
trols.
The lethal dose of the particular strain of cholera used for
the guinea pigs should be determined roughly before carrying out
the experiment.
It is inadvisable to allow students, except in very small groups,
to handle virulent cholera cultures, so that in carrying out this
experiment, the preparation of the cholera suspension, the in-
jection of the pig and the removal of specimens from the peri-
toneum, as described below, should be carried out by the instruc-
tor. The smears should be fixed immediately in 10 per cent
foimalin, after which they may be distributed to the members of
the class for study.
Two pigs — one normal, the other previously immunized with cholera
spirilla — are intraperitoneally injected with the determined dose, say a
1 The strain must be recently isolated or one that has been passed through guinea
Digs until it will kill in quantities of one loopful of an agar slant.
54 LABORATORY COURSE IN SERUM STUDY
twentieth of the emulsion obtained from an agar slant, and after ten
minutes a specimen of the peritoneal fluid is withdrawn for observation.
This is done by shaving the left side of the abdomen of the pig, holding
the pig, back down, on a tray. This can be done satisfactorily with the
operator's left hand. The shaved surface of the skin is sterilized by
wiping with 5 per cent carbolic, a small cut made through the skin,
but not through the muscles, with a sharp pair of scissors. Then the
sharp tip of a capillary pipette is inserted through the muscles and the
peritoneum. If the pipette is held horizontally or with the open end
depressed, a small amount of peritoneal fluid will run up into the capillary
portion even if no suction is used. When sufficient amount of fluid
[0.1 or 0.2 c.c.] is obtained, the pipette is withdrawn and the puncture
covered with cotton soaked in carbolic solution.
A hang-drop preparation is made with a drop of this fluid and the
remainder blown out into a watch glass. With a platinum loop a series
of thin smears are made of this fluid on glass slides, allowing one slide
for each member of the class, and as soon as the films are dried they are
placed in Coplin jars containing a 10 per cent solution of formalin
(4 per cent formaldehyde) for five minutes. They are then withdrawn,
allowed to dry, and are stained by the students with methylene blue and
studied under the oil immersion lens. The hang-drop preparation
should be observed under a high-power dry lens and one preparation
may be observed by the whole class.
Preparations are made in this way from the normal and immune pig
ten minutes, thirty minutes, one hour and two hours respectively after
injection. By the end of this period if a proper dose of the organisms
has been injected, the spirilla will be found to have multiplied enor-
mously in the peritoneal fluid of the normal animal, whereas in the
smears made from the immune animal after thirty minutes to an hour
the spirilla will appear granular or swollen, and after two hours, or, if
the observation is followed that long, after three or four hours, will be
found to have practically disappeared.
The two animals should be kept for observation after the experiment,
and the unprotected animal will probably be found dead the next day.
The passive transference of bacteriolytic immunity to another
animal by simultaneous injection of cholera spirilla and anti-cholera
serum obtained from the rabbit immunized to cholera may be demon-
strated in a similar way, and by this technic the strength of a bacteriolytic
serum may be titrated by injecting series of guinea pigs with varying
amounts of the serum.
56 LABORATORY COURSE IN SERUM STUDY
A type protocol of such an experiment is given below:
WEIGHT or
GUINEA Pio
DOSE OF
BACTERIA1
CHOLEBA
SPIRILLA
AMOUNT OP
INACTIVATED
IMMUNE SERUM
RESULT
(1) 215 gm.
2 mg.
0.1 c.c. in 1 c.c.
Complete dissolution in less
salt solution
than one hour. Lives.
(2) 230 gm.
2 ing.
0.05 c.c.
About the same as first.
(3) 200 gm.
2 ing.
0.01 c.c.
Somewhat slower than in other
two ; a few unchanged spirilla
after 1 hour. Final dissolution.
Pig lives.
(4) 245 gm.
2 mg.
0.005 c.c.
Similar to (3) but complete
dissolution in 2 hours. Pig lives.
(5) 220 gm.
2 mg.
0.001 c.c.
After 30 minutes the spirilla
seem to have begun to multiply.
Dies with innumerable active
spirilla in peritoneum.
Normal Con-
trol
(6) 210 gm.
2 mg.
0.1 c.c. normal
Very slight lysis at the begin-
inactive rab-
ning. Soon rapid multiplication.
bit serum
Dies.
LESSON VIII
BACTERICIDAL TEST IN VITRO — STERN KORTE
THE destruction of bacteria which takes place in the peri-
toneum of immune animals may also be demonstrated for certain
species of bacteria in the test tube. Small quantities of typhoid
bacilli, as shown in a previous experiment, are killed by exposure to
certain normal sera such as those of the rabbit and the guinea pig.
A more powerful action is observed in sera of animals immunized
to the typhoid bacillus, and, as in the case of red cells, the immune
body or sensitizer can be shown to be relatively thermostable in
that it will act after exposure to 56° C. for half an hour provided it
is reactivated by the addition of a small amount of normal serum.
The bactericidal effect of immune serum shows more clearly
than most other antibody phenomena the presence of a prezone,
1 The bacteria may be measured for such an experiment by standard loop-
fuls (one loop being equal to 2 milligrams), or by volume in emulsion with salt
solution.
58
LABORATORY COURSE IN SERUM STUDY
that is, the failure of the antibodies to act if present in excessive
concentration. In the sera of artificially immunized animals or of
typhoid patients amounts greater than 0.01 c.c. of a strongly im-
mune serum are usually ineffective, the best results being obtained
with amounts varying from 0.001 to 0.0001 c.c. in a volume of 1 c.c.
Since the inactivated immune serum must, of course, be re-
activated by fresh normal serum used as complement or alexin,
it is important to use an amount of the normal serum too small to
be of itself bactericidal ; and the first step to determine is the
bactericidal power of the normal serum against the strain of
bacteria to be used. On the other hand, to secure striking results
it is necessary to use as large an amount of fresh normal serum
as possible without entering the zone of normal bactericidal
power. Normal rabbit serum is usually definitely bactericidal
for typhoid bacilli in amounts ranging from 0.1 to 0.02 c.c. (in a
total volume of 1 c.c.).
Reagents :
1. Anti-typhoid serum — serum of a rabbit immunized against
typhoid bacilli obtained in sterile condition and heated at 56° C. for half
an hour.
2. Normal rabbit serum, which must also be sterile.
3. 24-hour broth culture of typhoid bacilli.
The glassware and salt solution used in the experiment must, of
course, be sterile, and the reagents handled with care to avoid contam-
ination during the setting up of the experiment.
TENTATIVE SCHEDULE l
Set up the following tubes:
IMMUNE SERUM
TUBE
Actual Quantity of the
SOLUTION
SERUM 1-50
Immune Serum
1
0.01 C.C.
0.5 c.c. (1-50)
0.0 c.c.
0.5 c.c.
2
0.002 c.c.
0.1 c.c. (1-50)
0.4 c.c.
0.5 c.c.
a
0.001 c.c.
0.5 c.c. (1-500)
0.0 c.c.
0.5 c.c.
4
0.0001 c.c.
0.05 c.c. (1-500)
0.45 c.c.
0.5 c.c.
5
* 0.01 c.c.
0.5 c.c. (1-50)
0.5 c.c.
—
6
None
— —
0.5 c.c.
0.5 c.c.
7
None
— —
1.0 c.c.
—
8
None
— —
1.0 c.c.
—
1 By a previous test of the serum the instructor should adjust the schedule so
that one or two tubes lie within the "killing " zone and others above and below it.
2 The serum in this control tube should be that amount which shows maximum
bactericidal power when used with active serum.
60 LABORATORY COURSE IN SERUM STUDY
Add to each tube about 0.1 c.c. of a 24 hour broth culture of B.
typhosus, diluted 1-500. This should be measured by making a mark
on a sterile capillary pipette, filling accurately to this mark and dis-
charging the contents against the side of the tube, close to the level of
the fluid, being careful not to touch the upper portion. The actual
amount used is unimportant, but precisely equal amounts must be added
to each tube. Shake. Incubate the tubes two hours at 37° C. At the
end of this time convey two loopsful from each tube into melted and cooled
agar tubes and pour plates in the usual way. Transfer of two loops from
tube eight should also be made into agar in this way immediately after
mixing without incubating.
In careful experiments, two additional controls, the one containing
0.5 c.c. of 1-50 normal serum, the other containing 0.5 c.c. of 1-50 im-
mune serum without the addition of typhoid bacilli, should also be set
up and transfers should be made from them into agar to prove the sterility
of the reagents used. A successful experiment should show an enormous
number of colonies, over 10,000 to the plate, in plates Nos. 5, 6 and 7.
Plate 8 should also show a large number of colonies, though perhaps less
than the other controls. In plates 2 and 3 there should be very few col-
onies or none at all; in plates 1 and 4 some reduction in the number,
though in some cases there may be no observable difference between
these plates and the controls.
Each student needs: Five f-inch test tubes, twelve petri dishes,
twelve tubes of agar, four one-c.c. pipettes.
LESSON IX
I. TITRATION OF AGGLUTININS BY MACROSCOPIC METHOD
THE clumping of bacteria by immune serum causes a homo-
geneous suspension to form flocculi easily visible to the naked eye,
which, on standing, settle to the bottom of the tube, making
the supernatant fluid clear. The macroscopic test is considered
more accurate than the microscopic since one is less often de-
ceived by small clumps of bacteria which may form spontaneously
in salt solution or broth for various reasons and are readily
visible under the microscope but do not come together in large
enough masses to cause a precipitate visible to the naked eye. It
is much simpler to carry out a quantitative titration of the aggluti-
native power of the serum by setting it up in test tubes than
by preparing hang-drops of each dilution. Furthermore, since
the quantities used in these tests are relatively large, slight inac-
curacies in measurement do not lead to such gross errors as would
62 LABORATORY COURSE IN SERUM STUDY
similar inaccuracies in the microscopic method. For these
reasons the macroscopic method is the one usually employed in
experimental investigations. On the other hand, in testing
patient's sera against the typhoid bacillus (the Widal reaction)
considerable information is given by the cessation of motility of
the bacilli in the presence of immune serum. It is desirable to
use small amounts of serum and unnecessary to set up more than
three or four dilutions in one test.
The macroscopic test may be carried out with living bacteria,
with bacteria killed either by exposure to heat (56° C.) or by ex-
posure to weak antiseptics such as phenol, 1 per cent solution, or
formaldehyde 0.8 per cent. Killed suspensions of bacteria may
be kept a long time in the ice box and still remain serviceable.
The flocculation is most easily observed in narrow test tubes,
f of an inch in diameter, but larger tubes can also be used with-
out difficulty. Some workers prefer to use tubes pointed at the
bottom, similar to the centrifuge tubes, since the sediments
which settle out can be readily observed in the tips of these tubes.
The period of incubation varies. A marked reaction is visible
after an hour at 37°, but with many organisms it is necessary to
incubate for two hours to obtain clear-cut results, and the floc-
culation often becomes more distinct when the tubes are removed
from the water bath and placed in the ice box for from half an
hour to 12 hours. Such organisms as the streptococcus1 and the
staphylococcus usually require the longer periods of incuba-
tion, and agglutination is favored by a temperature of 56°. On
the other hand, for organisms of the typhoid-colon group long in-
cubation is unnecessary.
Reagents :
1. Serum of a rabbit immunized against typhoid. For this test the
serum of animals immunized by the class should be used, the members
in charge of the rabbits bleeding them from the carotid at the previous
exercises and distributing to each other member of the class 2 c.c. of a
1-10 dilution of the serum.
2. B. typhosus — 24 hour culture on agar slant.
3. Normal salt solution.
1 Streptococcus agglutination requires a special and complic.it c<l technique to
be satisfactory and then isn't.
64 LABORATORY COURSE IN SERUM STUDY
Prepare a suspension of typhoid bacilli by adding 10 c.c. of salt
solution to an agar slant culture and gently rubbing the growth from
the surface of the agar by means of a platinum loop or a capillary glass
pipette, being careful not to cut the surface of the agar. Shake the tube
gently without wetting the stopper and allow to stand for 10 minutes
so that the larger clumps may settle to the bottom. Take a capillary
glass pipette, plug it at the suction end with non-absorbent cotton and
provide it with a rubber nipple. Draw off 8 or 9 c.c. of the upper por-
tion of the suspension, being careful not to disturb the sediment and
place in a separate test tube, taking care that the suspension does not
flow against the upper portions of the tube. Add 0.2 c.c. of 40 per cent
formaldehyde and place in water bath at 56° for thirty minutes. If
clumps are still visible the suspension may be filtered through a moist
paper.
B
Prepare the following dilutions of the immune serum in test tubes: 1
1. 1-10 = dilution as distributed
2. 1-50 = 0.5 c.c. of 1-10 dilution + 2 c.c. salt solution
3. 1-250 = 0.5 c.c. of 1-50 dilution + 2 c.c. salt solution
4. 1-500 =1.0 c.c. of 1-250 dilution + 1 c.c. salt solution
5. 1-1000 =1.0 c.c. of 1-500 dilution + 1 c.c. salt solution
6. 1-2000 = 1.0 c.c. of 1-1000 dilution + 1 c.c. salt solution
Place 0.5 c.c. of each dilution of serum (1-10 to 1-2000) in a series of
agglutination tubes and in an additional tube 0.5 c.c. of salt solution for
control.
To each tube add 0.5 c.c. of the killed typhoid suspension. This,
of course, doubles the dilution in each case and the tubes should be
labeled 1-20, 1-100, 1-500, etc., up to 1^000. Shake gently till the
two fluids mix. The typhoid suspension should be distributed by means
of a pipette carefully plugged at the upper end with non-absorbent
cotton. This should be used wherever bacteria are handled with a
pipette, even when they are thought to have been killed.
Place the tubes in the water bath, at 37° C., for one hour, and
observe carefully by transmitted light. Record the results and place
the tubes in the ice box for one half hour and again make a reading.
Read again on the following day.
1 Instructor should adjust this schedule by a previous titration.
66
LABORATORY COURSE IN SERUM STUDY
TUBE
SERUM
TYPHOID EMULSION
RESULT
1
1-10 0.5 c.c.
0.5 c.c.
2
1-50 0.5 c.c.
0.5 c.c.
3
1-100 0.5 c.c.
0.5 c.c.
4
1-250 0.5 c.c.
0.5 c.c.
5
1-500 0.5 c.c.
0.5 c.c.
6
1-1000 0.5 c.c.
0.5 c.c.
7
Salt sol. 0.5 c.c.
0.5 c.c.
8
Normal serum 1-20 0.5 c.c.
0.5 c.c.
D
The students in charge of the immune rabbits should test at the same
time the samples of serum which were obtained from the rabbits before
they received their first injection and which have been stored in the
ice box meanwhile. In this case one tube of undiluted serum should be
tested, one of a 1-10 dilution, one of 1-50. It is not necessary to test
higher dilutions.
H. MACROSCOPIC METHOD OF PROSCHER
Take four glass saltcellars. In the first place 0.5 c.c. of 1-10 dilu-
tion of serum used in the first experiment ; in the second 0.5 c.c. of 1-500
serum ; and in the third 0.5 c.c. of 1-2000 dilution ; in the fourth 0.5 c.c.
of salt solution. To each add 0.5 c.c. of typhoid suspension. The salt-
cellars are stacked one on top of the other and placed in the incubator
for one hour. At the end of this time they are taken out and may be
studied under low power of the microscope. A record is made of the
amount of clumping in each preparation.
m. THERMOSTABILITY OF AGGLUTININS
The agglutinins like the bactericidal sensitizing substance are
resistant to moderate heating but they do not require the reacti-
vation by the heat-sensitive alexin which is present in normal
serum. They are, however, destroyed by exposure to tem-
peratures of from 70° to 80°, in which respect they also resemble
the sensitizing substance.
In each of three agglutination tubes place 0.5 c.c. of a 1-10 dilution
of the immune serum. Place one tube in a water bath at 56° C. for one
half hour. Place a second tube in a water bath at 73° C. for one half
hour. Reserve the third tube as a control. After heating add 0.5 c.c. of
68 LABORATORY COURSE IN SERUM STUDY
the killed typhoid suspension to each tube and incubate as in the first
experiment. It should be noted that the sensitiveness of the immune
bodies to heat varies somewhat according to the dilution in which they
are exposed, and if desired, exposure to 56° C. may be repeated with un-
diluted serum.
The student is referred to the literature given in the text-book on
the work of Joos and others on the variation in heat stability of agglu-
tinins according to whether they are produced by the injection of heated
or unheated bacteria.
Each student needs: 2 c.c. typhoid immune serum, 1 typhoid
culture on agar slant.
Normal saline, 8 f-inch test tubes, 2 1-c.c. pipettes, 12 f-inch test
tubes, 4 salt cellars.
LESSON X
AGGLUININS (Continued)
I. AGGLUTINATION BY MICROSCOPIC METHOD
(WIDAL TEST)
DILUTIONS for the microscopic test may be made in the same
manner as those for the macroscopic, but it is often desirable in
clinical tests to work with smaller amounts of serum than can be
measured accurately in the ordinary graduated pipettes. In this
case capillary pipettes are used and the serum is measured either
by counting drops of serum and of salt solution, the same pipette
held at the same angle being used for both to insure accuracy,
or by using as an arbitrary unit the amount of fluid contained in
a capillary pipette from the tip to a wax pencil mark placed about
an inch from the tip. Students should practice making dilu-
tions by both of these methods. The former is the usual method
employed in diagnostic work ; the latter, however, is more ac-
curate.
Reagents :
1. Serum of rabbit immunized to B. typhosus.
2. B. typhosus — 24 hour culture in broth.
3. Salt solution.
A
Take four watch glasses or saltcellars and by means of a capillary
pipette provided with a rubber nipple place 9 drops of salt solution in the
first watch glass and 5 drops in each of the other three. With the same
pipette place one drop of serum in the first saltcellar; (if a highly
70
LABORATORY COURSE IN SERUM STUDY
immune animal serum is used this should previously be diluted about
1-100). Mix this dilution of serum thoroughly by drawing it in and out
of the pipette several times. Then place 5 drops of this first dilution
in the second watch glass. Mix the contents of the second glass as
before. Place 5 drops from the second glass in the third glass ; return
the remainder of the fluid to the second glass, mix the contents of the
third watch glass and remove 5 drops of this last to make its volume
equal to that of the others. We now have four watch glasses, each con-
taining 5 drops of fluid, representing 1-10, 1-20, 1^0 dilutions of the
serum in the first three, and salt solution in the fourth. The pipette is
now thoroughly rinsed with salt solution and filled with the typhoid
broth. Five drops of the broth are allowed to fall into each of the
watch glasses, reducing the dilutions to 1-20, 1^0, and 1-80. These are
the usual dilutions used in the Widal test for diagnosis. By means
of a platinum loop a series of hang-drop preparations is then made,
first with the control, then of the highest dilution, etc. The drops are
allowed to stand at room temperature for an hour and are then studied
under a high, dry lens of a microscope and the completeness of the clump-
ing and the loss of motility in the different preparations recorded.
WATCH GLASS No. 1
WATCH GLASS No. 2
WATCH GLASS No. 3
WATCH GLASS No. 4
Salt solution
9 drops
Salt solution
5 drops
Salt solution
5 drops
Salt solution
5 drops
Immune serum
1 drop
Mixture No. 1
5 drops
Mixture No. 2
5 drops
0
Discard 5 drops of Mixture No. 3 and add 5 drops of a broth culture
of B. typhosus to each glass.
B
A similar test, using the same reagents, measured by the Wright
technique, should also be set up :
Take four watch glasses as before and a capillary pipette, broken off,
squarely tipped, stoppered with cotton, and provided with a rubber
nipple. Place a mark with a wax pencil about one inch from the tip of
the pipette. Pour out salt solution into a watch glass or cover of a
Petri dish so that it is easily reached with a pipette. Draw up salt solu-
tion to the mark, admit a bubble of air, draw up salt solution again to the
mark, admit a bubble of air, and proceed until 9 units of salt solution have
been taken up. Then take up serum to the mark and discharge the
72 LABORATORY COURSE IN SERUM STUDY
entire contents of the pipette into the first watch glass ; mix by drawing
the solution in and out of the pipette several times. Take up five units
of salt solution into the pipette in similar manner, then five units of the
first dilution, and place this mixture in the second watch glass. Place
five units of salt solution and five units of the second dilution in the third
watch glass. Remove five units from the third watch glass and discard.
Place five units of salt solution alone in the fourth glass. Then with this
same pipette measure five units of typhoid broth into each of the watch
glasses. Prepare hang-drops and incubate and observe as above.
Another method commonly used is to prepare the dilutions of serum
in one of the two ways given above, and then place on a series of cover
glasses with a platinum loop a drop of typhoid broth, then on each glass
with the same loop a drop of diluted serum, after which the two drops
are stirred together by means of a capillary pipette sealed at the end.
This method offers no advantages over the preceding.
The method sometimes used for clinical diagnosis, of diluting with
counted loopfuls of saline drops of dried blood sent to the laboratory
on glass slides, is exceeding inaccurate.
•
II. EFFECT OF SALT ON AGGLUTINATION
The agglutination of bacteria which occurs in the presence of
immune serum has many analogies to the flocculation of colloidal
suspensions by electrolytes and is dependent on the presence of
electrolytes in the solution. Bacteria sensitized with immunized
serum agglutinate in the presence of traces of sodium chloride
and other inorganic salts, but not if suspended in distilled water.
These salts may also cause unsensitized bacteria to agglutinate,
and false clumping due to their action is sometimes observed in
the control tubes of agglutination tests, especially if the bacteria
are grown in media which have become concentrated by drying.
This effect is more easily demonstrated with certain salts of the
heavy metals than with sodium chloride. The following experi-
ment will serve to illustrate the effect of electrolytes on the agglu-
tination of sensitized and unsensitized bacteria :
Reagents :
1. Serum of animal immunized to B. typhosus.
2. B. typhosus — 24 hour agar slant.
3. Distilled water.
74 LABORATORY COURSE IN SERUM STUDY
4. 10 per cent salt solution.
5. Copper sulphate solution 0.8 per cent.
Prepare typhoid suspension (killed with formalin as in previous
lesson for the sake of safety) . Place in each of two centrifuge tubes, with
pointed tip, 2 c.c. of the suspension. To tube A add 2 c.c. of agglutinat-
ing serum diluted 1-50. To tube B add 2 c.c. of distilled water. Allow
the tubes to stand at 37° C. for 30 minutes. Centrifugalize the tubes at
high speed until the supernatant fluid is clear. Resuspend the sedi-
ments in 5 c.c. of distilled water and again centrifugalize. To the washed
sediments add 2 c.c. of distilled water and draw the mixture repeatedly in
and out of the capillary pipette in order to break up the clumps and
obtain an even suspension. Set up the following tests in agglutination
tubes :
1. Sediment A 0.5 c.c. Distilled water 0.5 c.c.
2. Sediment A 0.5 c.c. 10% salt sol. 0.09 c.c. Dist. water 0.4 c.c.
Copper sulphate sol. 0.8 % Distilled water
3. Sediment A 0.5 c.c. 0.02 c.c. 0.5 c.c.
4. Sediment B 0.5 c.c. 0.02 c.c. 0.5 c.c.
5. Sediment B 0.5 c.c. 0.1 c.c. 0.5 c.c.
6. Sediment B 0.5 c.c. 0.5 c.c.
The tubes are placed in the water bath at 37° for one hour and then
observed. Tubes 2, 3 and 5 should show agglutination.
This experiment shows that electrolytes are necessary for the
appearance of agglutination and that bacteria even though they
may have united with the specific serum agglutinins do not clump
in the absence of such salts. It is the mainstay of Bordet's view
of agglutination as a "two-phase" reaction in which one step is
the union with agglutinins, the other the actual clumping brought
about by the salts.
Each student needs: 8 watch glasses, 2 centrifuge tubes, 6 f-inch test
tubes, glass tubing, test tube rack.
Bacillus typhosus, 24-hr broth culture, 24-hr, agar slant.
LESSON XI
ABSORPTION OF BACTERIAL AGGLUTININS
THE serum of an animal immunized simultaneously with two
species of bacteria develops agglutinative power for each of these
species. The agglutinins for the two species may be shown to
be distinct by treating the serum with a heavy emulsion of one of
76 LABORATORY COURSE IN SERUM STUDY
them, and subsequently removing the agglutinated bacteria by
centrifugation. The agglutinins for this species having been ab-
sorbed by the bacteria will be removed by this procedure, leaving
the agglutinins for the species not used in the absorption unin-
fluenced. For the complete removal of agglutinins it is necessary
to leave the serum in contact with the bacteria for a long period
and often to repeat the treatment of the serum with bacterial
suspension several times. These facts are illustrated in Experi-
ment 1.
The experiment illustrates the general principle that an animal
may produce specific and distinct antibodies simultaneously
against a number of different antigens.
Experiment 1
ABSORPTION OF AGGLUTININS FROM THE SERUM OF AN ANIMAL IM-
MUNIZED AGAINST Two SPECIES OF BACTERIA
Reagents :
1. Serum of an animal simultaneously immunized against Bacillus
typhosus and Bacillus coli. The students who have immunized this
animal should bleed it from the carotid at a preceding lesson and
should distribute the serum to other members of the class, diluted 1-10
with salt solution. Each student requires 5 c.c. of this dilution.
2. Thick suspension of typhoid bacilli killed by heating one hour at
55° C.
3. Thick suspension of colon bacilli killed by heating one hour at
60° C.
Both these suspensions should be used undiluted for the absorption,
and in a dilution of 1-10 for titrating the agglutination.
4. Salt solution.
A. PRELIMINARY TITRATION OF AGGLUTINATIVE POWER OF IMMUNE
SERUM
The dilutions of serum required for this test should be prepared
in f-inch test tubes according to a protocol to be made out by the indi-
vidual student. Before making any such series of dilutions it is highly
important to write out plainly the amount of serum and salt solution
to be placed in each tube in order to avoid errors.
78 LABORATORY COURSE IN SERUM STUDY
Set up the following experiment in agglutination tubes :
Tube No. 1 Serum (1-20) 0.5 c.c. + Typhoid suspension 0.5 c.c.
2 Serum (1-100) 0.5 c.c. + Typhoid suspension 0.5 c.c.
3 Serum (1-500) 0.5 c.c. + Typhoid suspension 0.5 c.c.
4 Serum (1-2000) 0.5 c.c. + Typhoid suspension 0.5 c.c.
5 Salt solution 0.5 c.c. -f- Typhoid suspension 0.5 c.c.
6 Serum (1-20) 0.5 c.c. + Colon suspension 0.5 c.c.
7 Serum (1-100) 0.5 c.c. + Colon suspension 0.5 c.c.
8 Serum (1-500) 0.5 c.c. + Colon suspension 0.5 c.c.
9 Serum (1-2000) 0.5 c.c. + Colon suspension 0.5 c.c.
10 Salt solution 0.5 c.c. -f Colon suspension 0.5 c.c.
Incubate one hour at 37° C. and record the results.
B. ABSORPTION
Place in small centrifuge tubes :
A. Serum (1-10) 1.5 c.c. + Thick typhoid suspension 1.5 c.c.
B. Serum (1-10) 1.5 c.c. + Thick colon suspension 1.5 c.c.
Incubate one hour. (For complete absorption, incubation in the
thermostat should be followed by 12 or more hours in the refrigerator.)
Centrifugalize at high speed and remove the clear supernatant fluid.1
C. TITRATION OF THE SERUM AFTER ABSORPTION
The supernatant fluid obtained by centrifugation should be tested
undiluted (it corresponds to a 1-20 dilution of the original serum) against
the same organism used for absorption, and as the absorption is often
incomplete after this short incubation a similar test should be set up
of the supernatant fluid diluted 1-5 (corresponding to a 1-100 dilution
of the original serum). The supernatant fluid should also be tested
against the organism not used in absorption in the highest dilution in
which agglutination was found in the preliminary tit ration.
1. Supernatant fluid A 0.5 c.c. + Typhoid suspension 0.5 c.c.
2. Supernatant fluid A (1-5) 0.5 c.c. + Typhoid suspension 0.5 c.c.
3. Supernatant fluid A 0.5 c.c. + Colon suspension 0.5 c.c.
4. Supernatant fluid in high-
est dilution in which it
agglutinated colon in pre-
liminary titration 0.5 c.c. + Colon suspension 0.5 c.c.
1 It should be noted that absolutely complete removal of agglutinins is not
possible except by many times repeated absorption.
80 LABORATORY COURSE IN SERUM STUDY
5. Supernatant fluid B 0.5 c.c. + Colon suspension 0.5 c.c.
6. Supernatant fluid B (1-5) 0.5 c.c. -f Colon suspension 0.5 c.c.
7. Supernatant fluid B 0.5 c.c. + Typhoid suspension 0.5 c.c.
8. Supernatant fluid in high-
est dilution in which it
agglutinated typhoid in
original titration 0.5 c.c. + Typhoid suspension 0.5 c.c.
9. Salt solution . 0.5 c.c. + Typhoid suspension 0.5 c.c.
10. Salt solution 0.5 c.c. + Colon suspension 0.5 c.c.
Incubate one hour at 37° C. and record results.
Tubes 1, 2, 5 and 6 should show no agglutination if absorption has
been complete. If all the agglutinin has not been absorbed, however,
there may be clumping in tubes 1 and 5. The bacteria in tubes 3, 4, 7
and 8 should be agglutinated.
GROUP AGGLUTININS
The serum of animals highly immunized to one species of
bacteria will often agglutinate closely related bacteria, though
this action is invariably weaker than the agglutinative power for
the bacteria used in immunizing the animal.
Thus the serum of an animal which before treatment agglu-
tinated Bacillus coli in dilutions of 1-20 and Bacillus typhosus in
dilutions of 1-5, after immunization with Bacillus typhosus may
agglutinate Bacillus coli as highly as 1-100, and Bacillus typhosus
up to 1-10,000 or more. This indicates a close similarity in the
chemical constituents of the two antigens — the Bacillus typhosus
antigen containing small amounts of the protein present in Bacillus
coli. Absorption with Bacillus typhosus will remove both the
Bacillus typhosus and the colon agglutinins. Absorption with
Bacillus coli will take out only the colon agglutinin, the serum still
agglutinating Bacillus typhosus 1-10,000.
The serum here contains at least two types of agglutinins, the
so-called major or specific agglutinin which reacts only with the
homologous bacteria, and the minor or group agglutinins which
react both with the homologous bacteria and with the allied
species. If such a serum is added to a suspension of the homolo-
gous bacteria both major and minor agglutinins are absorbed
82 LABORATORY COURSE IN SERUM STUDY
and the supernatant fluid obtained after centrifugation has lost
its agglutinative power both for the homologous strains and for
allied species. If, on the other hand, the serum is treated with a
suspension of the one allied species, the minor agglutinins only
are absorbed. The supernatant fluid then shows undiminished
agglutinative power for the homologous strain, but does not ag-
glutinate the allied strain used in absorption. These facts are
brought out in Experiment 2.
Experiment 2
ABSORPTION OF MAJOR AND MINOR AGGLUTININS FROM SERUM OF
AN ANIMAL IMMUNIZED AGAINST A SINGLE SPECIES
Reagents :
1. Serum of an animal immunized to Bacillus typhosus. The
serum must be known to contain group agglutinins for Bacillus coli.1
2. Two specimens of the same serum in 1-20 dilution which have
stood in contact over night with typhoid and with colon bacilli re-
spectively. These may be prepared on the previous day by the in-
structor. Heavy suspensions of typhoid and colon bacilli are first
prepared by washing off the growth of a number of agar bottles and
heating for one hour at 56° and 60° respectively. To each of these sus-
pensions is added an equal volume of a 1-10 dilution of agglutinating
serum. The tubes are incubated for one hour at 37°, shaken thoroughly
and placed in the ice box over night. They should be shaken once or
twice during the period of 24 hours. They are then centrifugalized and
the supernatant fluids removed and used in the second part of the
experiment.
3. Suspension of killed typhoid bacilli as in Experiment 1.
4. Suspension of killed colon bacilli as in Experiment 1.
5. Salt solution.
(1) Titrate the original antityphoid serum against both typhoid
and colon emulsions according to the following protocol. This gives a
basis for comparison with a similar test on the same serum after it has
been in contact with the bacteria.
1 To insure successful class work this must be previously ascertained in
each case, since individual animals may vary considerably in regard to the pro-
duction of minor agglutinins.
84 LABORATORY COURSE IN SERUM STUDY
1. Serum (1-10) 0.5 c.c. + Typhoid suspension 0.5 c.c.
2. Serum (1-10) 0.5 c.c. + Colon suspension 0.5 c.c.
3. Serum (1-50) 0.5 c.c. + Typhoid suspension 0.5 c.c.
4. Serum (1-50) 0.5 c.c. -f Colon suspension 0.5 c.c.
5. Serum (1-200) 0.5 c.c. + Typhoid suspension 0.5 c.c.
6. Serum (1-200) 0.5 c.c. + Colon suspension 0.5 c.c.
7. Serum (1-1000) 0.5 c.c. + Typhoid suspension 0.5 c.c.
8. Serum (1-4000) 0.5 c.c. + Typhoid suspension 0.5 c.c.
9. Salt solution 0.5 c.c. + Typhoid suspension 0.5 c.c.
10. Salt solution 0.5 c.c. + Colon suspension 0.5 c.c.
Incubate one hour at 37° C. and observe.
(2) Set up the following tests with the same serum, from which
in this case, however, the agglutinins have been removed by ab-
sorption :
1. Serum absorbed with typhoid 0.5 c.c. + Typhoid suspension 0.5 c.c.
2. Serum absorbed with typhoid 0.5 c.c. + Colon suspension 0.5 c.c.
3. Serum absorbed with colon 0.5 c.c. + Typhoid suspension 0.5 c.c.
4. Serum absorbed with colon 0.5 c.c. + Colon suspension 0.5 c.c.
5. Serum absorbed with colon
in the highest dilution
which agglutinated
typhoid bacilli before
absorption 0.5 c.c. + Typhoid suspension 0.5 c.c.
Incubate for one hour and observe.
Each student needs:
25 J-inch test tubes, 8 1-c.c. pipettes, test tube rack, 35 £-inch
test tubes, 4 centrifuge tubes, test tube racks.
LESSON XII
EFFECT OF HJEMOLYTIC AND H^EM AGGLUTINATIVE
SERA IN VIVO
1. PRELIMINARY TESTS
A RABBIT receives at four-day intervals four intravenous injections
of 2 c.c. of washed, cat's red blood cells. For obtaining the cells a cat
under ether is bled from the carotid or jugular. The defibrinated cat's
blood if sterile can be kept four days and thus used for two injections.
Seven to ten days after the last injection the rabbit is bird and the
hsemolytic and agglutinative power of the inactivated immune serum for
cat's cells is tested.
86
LABORATORY COURSE IN SERUM STUDY
The following test is made to determine the haemolytic power of the
anti-cat rabbit serum :
TUBE
IMMUNE SERUM
GUINEA PIG SERUM
OR CAT'S SERUM,
FRESH DILUTED
1-10
SALINE UP TO
5% WASHED CAT'S
RED BLOOD CELLS
1
Diluted 1-10 0.5
0.5 c.c.
2 c.c.
0.5 C.C.
2
Diluted 1-10 0.1
0.5 c.c.
2 c.c.
0.5 c.c.
3
Diluted 1-100 0.5
0.5 c.c.
2 c.c.
0.5 c.c.
4
Diluted 1-100 0.1
0.5 c.c.
2 c.c.
0.5 c.c.
5
Diluted 1-400 0.2
0.5 c.c.
2 c.c.
0.5 c.c.
6
Diluted 1-400 0.1
0.5 c.c.
2 c.c.
0.5 c.c.
In testing the agglutinative power set up a similar series but without
fresh serum.
II. IN Vivo EFFECT OF H^MOLYTIC SERUM
Etherize two cats and inject 1 c.c. of immune serum into one and
3 c.c. into the other. Give the injections directly into the saphenous
vein with a fine hypodermic needle. Put the first cat into a clean cage
with a dram for the collection of urine after 6, 24 and 48 hours. (The
first urine will con tain large amounts of haemoglobin, the last bile.) Half
an hour after the injection collect a specimen of the cat's serum by prick-
ing the ear vein. Centrifugalize and observe the blood-tinged serum.
Make smears of the blood, stain, and note with oil immersion lens the
phagocyted red cells in some of the leucocytes. The second cat will
probably die at once. If so, autopsy immediately and look for clumps
of agglutinated red cells in portal and pulmonary veins.
To demonstrate the in vivo effect of complement, give a rabbit an
intravenous injection of 2.0 c.c. of 25 % suspension of sensitized sheep
blood cells. (In sensitizing the cells, regard 0.1 c.c. of 25 % cells as a
unit and add two units of amboceptor for every unit of cells. After
the cells have stood in contact with the amboceptor for one half hour,
centrifugalize them and make them up to the original volume of 25 %
suspension).
After a half hour collect a specimen of serum from the rabbits'
ear and examine for dissolved haemoglobin. Make a smear of the blood
and examine for phagocytosis. Examine the rabbit's urine after 6,
24 and 48 hours.
The experiments in this lesson need not be done by the entire class
but can be done as a demonstration by a small group of students working
with the instructor.
88 LABORATORY COURSE IN SERUM STUDY
Materials:
5 Cats
2 Rabbits
Hypodermic syringe
Fresh guinea pig serum
Test tubes
4 f-inch test tubes
6 £-inch test tubes
Test tube racks
Cover slips, microscope slides
Blood stain (Wright's, Giemsa's or Jenner's)
1 Centrifuge tube
LESSON XIII
ISOKEMOLYSIS AND ISOAGGLUTINATION
TRANSFUSION TESTS
ANTIBODIES in the blood of one animal capable of acting on
the blood of another animal of the same species are called iso-
antibodies. Isoagglutinins and isohsemolysins sometimes occur
naturally and they can often be made to appear by immunization
of animals with the blood of other animals of the same species.
They appear with a peculiar regularity which indicates that there
are certain groups of individuals within one and the same species
whose blood is strictly homologous only with other members of the
same group. Among human beings there are four such groups,
sharply defined, permanent and hereditary. The existence of
these antibodies is of practical importance in connection with
blood transfusion because more or less serious results may ensue
if a patient is transfused with the blood of a donor who belongs to
another group and whose blood is therefore either haemolytic
or hsemagglutinating for the patient, or vice versa. (See table
in "Infection and Resistance," page- 238.)
Before transfusion, therefore, unless the urgency of the case for-
bids, a number of prospective donors are tested, and one is chosen
whose blood does not lake or agglutinate when mixed with that of
the patient. The serum of each donor must be tested with the
blood cells of the patient and the serum of the patient with the
cells of each donor.
There are two methods of collecting blood for these tests :
90 LABORATORY COURSE IN SERUM STUDY
(A) When possible from a vein, 3 to 5 c.c. in a test tube for serum,
10 drops in 5 c.c. of a solution containing 1 per cent sodium
citrate and 0.9 per cent sodium chloride, for the red cell emulsion.
(B) By pricking the finger with a Hagedorn needle and milking
out from 1 to 2 c.c. of blood which is collected in a Wright capsule
for serum and 10 drops in citrate-salt solution for cells.
Students make Wright capsules and collect specimens of blood
from each other. The capsules are of heavy glass tubing, 7 to 8 mm.
in diameter, about 10 cm. long, and drawn to a capillary opening of
about 1 mm. diameter at each end. They are laid on the table and drops
of blood expressed from the finger are allowed to enter them by capillary
action. When the capsule is two thirds full, the blood is allowed to clot
and the open end of the capsule is sealed in the flame, great care being
taken not to heat the blood. Serum is obtained by centrifugalizing the
capsules and cutting them with a small file.
The cell emulsions are prepared by washing the citrated blood twice
with salt solution and making up to 5 per cent by comparing the opacity
with that of a 5 per cent sheep cell suspension, or by measuring with
capillary pipette 1 volume of sedimented human cells and 19 volumes
of saline.1
Each student should make 12 Wright capillary pipettes of strong
glass tubing, 4 to 5 mm. in diameter. These pipettes are made by cut-
ting the tubing into pieces about 10 cm. inches long, heating the center of
each piece to melting and drawing out to a capillary of about 1 mm.
diameter and 10-12 cm. inches long. The capillary is then broken at the
middle, giving two pipettes. Adjust nipple to the pipettes. Make a
mark on the capillary end of each pipette near the hilt. Draw up 1
volume of serum to the mark, then a small buble of air, then another
volume of serum, and another bubble of air, and then a volume of the
cells which are to be mixed with the serum ; (the serum is used in excess
because in this way it is sometimes possible to detect a weak hsemolytic
action which would otherwise be overlooked on account of the low com-
plement activity of human blood). Draw the mixture into the body of
the pipette and seal the capillary tip in the flame. Mix the fluid by
rotating the pipette. Remove the nipple. Throughout these steps
keep the pipette approximately horizontal so that the fluid remains in
the body of it and does not flow to either end. Seal the open end of the
pipette by dipping in melted paraffine. Mark the pipette with a glass
pencil, using the number assigned to the serum as the numerator, that
assigned to the ce.lls as the denominator, of a fraction.
1 It is not necessary in practical work to wash the cells. Three or four drops of
blood in citrate salt solution furnishes satisfactory material.
92
LABORATORY COURSE IN SERUM STUDY
Each student tests one specimen of serum against 10 different indi-
viduals' red cells. Make a control of each serum with its own red cells.
Lay all the pipettes on their sides on a tray and incubate at 37.5° C.
for one hour. Then make observations for haemolysis or agglutination
and make the results of all the tests into a table to illustrate Land-
steiner's law of the groupings of human isoagglutination.
By the original classification of blood groups the groupings may be
arranged as follows on the assumption that there are two distinct ag-
glutinins and corresponding agglutinogens:
SERA I
• AGGLUTININS aft
II III VI
a & o
.Cells
I
II
III
IV
Agglutinogens
O
B
A
AB
+ + + -
From this table the student can determine the grouping of his tests.
It will be noted that any individual can be grouped by testing his cells
against sera of groups II and III. Study this out on the table above.
Every serologist should know his own group.
The students should carry out a few simplified tests by mixing a
drop of blood suspension and a drop of serum on a slide. The slide
method is sufficient for all practical purposes.
The class needs: «
Heavy glass tubing 7-8 mm. diameter.
Heavy glass tubing 4-5 mm. diameter.
Litre 1 per cent sodium citrate in 0.6 per cent sodium chloride.
Litre 0.9 per cent sodium chloride solution.
Triangular files.
Rubber nipples to fit 5 mm. tubing (one nipple for each student).
Hard paraffine in casserole.
94
LABORATORY COURSE IN SERUM STUDY
LESSON XIV
TITRATION OF PRECIPITATING SERA FROM RABBITS
SPECIFICITY OF PRECIPITINS
PRECIPITATING sera are active against proteins in high dilu-
tions. Their specificity is great but is limited by group reactions
so that a precipitating serum for the protein of any species of
animal or bacteria will give precipitation, with closely related
species in low but not in high dilutions. On this is based Nuttall's
well-known study of blood relationships.
Students working in groups bleed serum-treated rabbits (Lesson I)
from the carotid 10 days after the last injection, taking about 20 c.c.
of blood in small centrifuge tubes. The animal should be in a fasting
state in order that the serum will be clear. Allow the blood to clot and
centrifugalize.
Experiment 1
One half of the students in the class make dilutions in large test
tubes of sheep serum or human serum respectively (according to par-
ticular serum with which rabbit has been treated) as follows: 1-10, 1-20,
1-50, 1-100, 1-200, 1-500, 1-1000. In order to test the specificity of the
reaction the other half of the students make similar dilutions of serum from
other species of animal — guinea pig, dog, cat, sheep, horse, as available.
Then set up the following mixtures in small "precipitation" tubes
(tubes of about 5-8 mm. diameter) :
TUBE
IMMUNE RABBIT SERUM
ANTIOEN (PRECIPITINOOEN)
1
0.1 c.c.
Undiluted 0.1 c.c.
2
0.1 c.c.
1-20 0.1 c.c.
3
0.1 c.c.
1-50 0.1 c.c.
4
0.1 c.c.
1-100 0.1 c.c.
5
0.1 c.c.
1-200 0.1 c.c.
6
0.1 c.c.
1-500 0.1 c.c.
7
0.1 c.c.
1-1000 0.1 c.c.
8
0.1 c.c.
Salt solution 0.1 c.c.
9
Salt solution 0.25 c.c.
Undiluted antigen 0.1 c.c.
In making these mixtures, put the anti-serum into the tubes first
and then run the dilution into the tubes with a nipple pipette in such a
way that the latter is layered over the former as in a Heller's test for
albumin in urine. This is the so-called "ring test." . The precipitum
shows as a white line at the contact of the two fluids. After this ring
96 LABORATORY COURSE IN SERUM STUDY
has formed, the tubes can be shaken up and the ordinary precipitin reac-
tion (i.e., turbidity and flocculation) can be observed. Note the results
after one half hour and one hour at 37° C. and again after 12 hours in
the ice box. Is there a "pro zone"? •
Experiment 2
EFFECT OF HEAT ON PRECIPITINS
Heat slowly inactivates precipitins. They cannot be reactivated
by fresh serum (alexin, complement), in this regard differing from haemoly-
sins and bactericidal antibodies.
Heat 0.5 c.c. of precipitin serum for two hours at 60° C. Mix
0.1 c.c. of the heated serum with 0.1 c.c. of that dilution of the antigen
which was found in Experiment 1 to give the optimum precipitation.
Mix 0.1 c.c. of the heated serum with 0.1 c.c. of fresh normal rabbit
serum and test this mixture also with the optimum dilution of antigen.
Make observations. after 1 hour at 37° C. and again after 12 hours in ice
box.
For each student:
2 c.c. Immune serum (rabbit whose preparation was begun in
Lesson I).
1 c.c. Antigen serum (human, sheep or horse serum for half the
class, other animals' sera for the other half).
Salt solution.
12 Precipitation tubes. Test tube rack. Nipple pipette.
6 one-c.c. graduated pipettes.
0.1 c.c. normal rabbit serum, fresh,
8 f-inch test tubes.
Precipitins can also be produced against proteins heated to
70° or over. For the principles of such experiments see Infection
and Resistance, page 260.
LESSON XV
FORENSIC PRECIPITIN TEST
THE precipitin reaction is used in medicolegal work to identify
blood stains and to detect adulterations or substitutions in
meat or other food products. Since the specificity of the precipi-
tin reaction is subject to limitations similar (though not as
marked) to those discussed in the case of agglutination, the pre-
cipitating serum used in forensic tests must be very potent.
Preliminary test should show that the serum gives distinct cloud-
ing with its homologous antigen diluted 1 to 1000, within five or
ten minutes at room temperature.
An effort is made to get the suspected protein into approxi-
mately this dilution.
1
Each student gets :
2.0 c.c. antihuman serum.
2.0 c.c. antisheep serum.
0.4 c.c. normal rabbit serum.
Two specimens of material with dry blood to be identified. (To some
of the students sheep blood is given ; to others human blood or that of
other species.)
From each of the specimens upon which blood has been dried the
blood must be soaked off in salt solution. This can be done by placing
the material in a test tube with 5 c.c. salt solution and shaking for
10 or 15 minutes until the solution presents a lasting foam. This is
evidence that the protein is in solution in a concentration of at least 1 part
in 1000. Old specimens must often be left in salt solution in the refrig-
erator for 24 hours or longer. Such prolonged soaking is done in the
refrigerator in order to avoid destruction of the antigen by bacterial
growth. Since the nature of the reaction makes it necessary that the
material to be examined shall be absolutely clear, it is now cleared by
filtration or centrifugation ; if there is enough material the acetic-acid-
and-boiling test can be done and the cloud compared in heaviness with
a 1-1000 dilution of serum of the suspected variety of blood, similarly
100 LABORATORY COURSE IN SERUM STUDY
treated. This gives a rough estimate of the concentration of the antigen.
If the solution seems much too concentrated, dilute it until it approaches
1-1000, — i.e. the limit of its still presenting a lasting foam on shaking.
Place the antiserum in the tube first and make a ring test. To do
this run antigen very carefully into tube with a nipple pipette so that
the two fluids may not be mixed. Watch for a white ring at the line of
junction. Be careful to compare this with the control, since a slight
opalescence is often seen at the line of junction of salt solution and
serum not due to precipitation.
2
1. Unknown solution (a) 0.5 c.c. + antihuman serum 0.2 c.c.
2. Unknown solution (a) 0.5 c.c. + normal rabbit
serum 0.2 c.c.
3. Unknown solution (6) 0.5 c.c. + antihuman serum 0.2 c.c.
4. Unknown solution (6) 0.5 c.c. -f normal rabbit
serum 0.2 c.c.
5. Known human serum (1-1000) 0.5 c.c. -f antihuman serum 0.2 c.c.
6. Salt solution 0.5 c.c. + antihuman serum 0.2 c.c.
Observe the tubes for 15 or 20 minutes at room temperature. Then
incubate and observe. After 30 minutes shake up and observe for
cloudiness and flocculation.
3
Set up a similar series with antisheep instead of antihuman serum.
Let each student report whether either of the specimens he received is
sheep or human.
In order that conclusions may be definitely drawn from such a test
it is necessary that the tubes containing the unknown solution (say it is
suspected of being human blood) and the known human blood, 1-1000
control, should show a definite reaction within not longer than 10 min-
utes at room temperature. Antisera too weak to show such a sharp
reaction with controls of the known protein are not suitable for such
tests.
Each student needs:
6 f-inch test tubes. 12 Precipitation tubes 6 mm. in diameter.
Capillary pipettes.
LESSON XVI
BORDET-GENGOU PHENOMENON— COMPLEMENT
FIXATION
THE Bordet-Gengou phenomenon is the prototype of all com-
plement fixation methods. It depends on the fact that antigens
102
LABORATORY COURSE IN SERUM STUDY
(bacterial and others) in the presence of their specific antisera
will fix complement so that the subsequently added red cells
sensitized by their appropriate hsemolytic immune-body will fail
to be laked, because no free complement is available. Neither the
antigen alone nor the antibody alone will fix complement in this
manner.
Unfortunately bacterial cell bodies (like many other sub-
stances) if in sufficiently large amounts will of their own accord,
probably for purely physical reasons, absorb complement, in a
non-specific way, i.e., without the presence of a specific antibody.
The immune sera likewise have often the power of absorbing
complement in a non-specific way in the absence of antigen and
this power must be determined by preliminary titration in order
that allowance may be made for it in the main test.
For this reason before doing a Bordet-Gengou reaction (and,
as will be seen later, in all complement fixation work) it is neces-
sary to determine the degree to which such non-specific comple-
ment fixation will take place with the given antigen and to use
in the actual test an amount too small to give such a false reaction.
Experiment 1
PRELIMINARY DETERMINATION OF THE NON-SPECIFIC BINDING POWER
OF THE BACTERIAL ANTIGEN
Emulsify a 24 hour slant of typhoid bacilli in 10 c.c. of saline solu-
tion. Heat at 56° C for 30 minutes.
TUBE
BACTERIAL EMULSION
COMPLEMENT
1
2
3
4
5
6
0.4 C.C.
0.2
0.1
1/10 dilution of above
0.8
0.5
0.2
2u
nits1
1 Determined by preliminary titration
Add sufficient saline solution to each tube so that the final volume
after adding red cells and amboceptor shall equal 2.5 c.c. Incubate one
hour in water bath. Add 0.5 c.c. of 5% reel cell suspension plus two
units of amboceptor to each tube. Incubate for one half hour in water
bath.
104
LABORATORY COURSE IN SERUM STUDY
Experiment 2
PRELIMINARY DETERMINATION OF THE NON-SPECIFIC BINDING POWER
OF THE IMMUNE SERUM
TUBE
1/0 DILUTION OF SERUM*
COMPLEMENT
1
0.5 c.c.
2 units
2
0.3
«
3
0.2
«
4
0.1
it
1 Serum from rabbit immunized to typhoid bacilli. This serum should be heated
to 55° for 30 minutes on day of using.
Follow same procedure as directed under Experiment 1.
Experiment 3
Use that amount of typhoid suspension which is next below the
amount which was found to not interfere at all with the hsemolytic action
of the complement. Also take that amount of serum which lies next
below the amount which did not interfere with haemolysis. Let us say
there was full haemolysis in Tube 3 of the first set and 2 of the second;
we set up the third as follows :
TUBE
BACTERIAL SUSPENSION
1/10 DILUTION
SERUM
1/10 DILUTION
COMPLEMENT
1
2
3
0.8 C.C.
0.8
0.2 c.c.
0.2 c.c.
2 units
2 "
2 "
Add sufficient saline solution to bring the final total up to 2.5 c.c.
and incubate 1 hour in water bath. Add 0.5 c.c. of 5% red cell suspen-
sion and two units of amboceptor to each tube and incubate 30 minutes.
Tubes 2 and 3 are necessary control tests in all complement fixation
work, to prove that under the precise conditions under which comple-
ment is fixed in Tube 1 it is not fixed by antigen alone or by antiserum
alone.
Materials for each student:
13 half-inch test tubes. Test tube rack.
4 large test tubes.
24 hour typhoid culture.
Platinum loop.
6 one-c.c. pipettes.
1.5 c.c. immune antityphoid serum (diluted).
7. c.c. 5 per cent sheep cell emulsion.
4.0 c.c. 10 per cent guinea-pig serum.
30 units amboceptor.
Salt solution.
LESSON XVII
ALEXIN FIXATION BY DISSOLVED PROTEINS AS ANTIGENS
(AND BY SPECIFIC PRECIPITATES)
THE phenomenon of fixation of alexin by the union of antiserum
and antigens is not limited to cellular antigens, but occurs when-
ever a serum containing antibodies is mixed with its corresponding
protein antigen in the presence of alexin, or when alexin is treated
with the precipitate so obtained. The binding of complement
does not, however, depend on the formation of a precipitate, for
it occurs in dilutions at which a precipitate no longer can be seen.
106
LABORATORY COURSE IN SERUM STUDY
Complement fixation is even more delicate a method of identifying
a protein than is the precipitin reaction.
Reagents :
1. Serum of rabbit immunized against human serum, diluted 1-5.1
2. Human serum, diluted 1-5.
3. Guinea pig serum, diluted 1-10.
4. Washed sheep cells, 5 % suspension.
5. Serum of rabbit immunized against sheep cells (antisheep-cell
amboceptor).
Set up the following tubes :
TUBE
ANTIHUMAN SERUM 1-5
HUMAN- SERUM 1-5
Doo, CAT, HORSE OR
SHEEP SERUM 1-5
GuiNEA-PlQ
SERUM 1-10
1
0.5 c.c.
0.25 C.C.
—
0.5 c.c.
2
0.5 c.c.
0.025 2
—
0.5 c.c.
3
0.5 c.c.
0.005 2
—
0.5 c.c.
4
0.5 c.c.
—
0.25 c.c.
0.5 c.c.
5
0.5 c.c.
—
—
0.5 c.c.
6
—
0.25 c.c.
—
0.5 c.c.
7
Normal rabbit serum
0.25 c.c.
—
0.5 c.c.
0.5 c.c.
Bring all volumes to 2 c.c.
Incubate mixtures one hour at 37° C.
Sensitize sheep cells by adding two units of antisheep-cell serum to
sach 0.5 c.c. of cells.
At the end of the hour add a dose of these sensitized cells to each
tube. (The dose is 0.5 c.c. of 5 per cent cells + the proportionate vol-
ume of amboceptor.)
Incubate until haemolysis is complete in control tubes 4, 5 and 6.
Tube 4 is inserted as a control test to show that fixation is not given
with serum in general but only with human serum. Different students
use the sera of different animals as control. If monkey serum is avail-
able, let some of the students set up control tests with it.
Compare the dilution of human serum which still gives a positive
reaction with the titer which gave a precipitin reaction with the same
antiserum in Lesson VII.
1 Where dilutions are made to economize material they must not bo taken
to have any significance of principle.
8 (Make a preliminary dilution of 1-20 and add 0.5 c.c. and 0.1 c.c.)
108 LABORATORY COURSE IN SERUM STUDY
MATERIALS
Each student needs:
3.0 c.c. Antihuman serum (1-5).
1.0 c.c. Human serum (1-5).
4.0 c.c. Guinea pig serum (1-10).
4.0 c.c. Sheep cells (1-20).
0.5 c.c. Dog, cat, horse or sheep serum (1-5).
15 units antisheep-cell amboceptor.
Salt solution.
10 one-c.c. pipettes.
15 half-inch test tubes.
1 test tube rack.
7 ^-inch test tubes
3 £-inch test tubes
6 1-u pipettes.
LESSON XVIII
FORENSIC COMPLEMENT FIXATION
THE fixation of complement by the specific action of a protein
and the serum of an animal immunized against it, is the basis
of a forensic test for the identification of blood stains and also for
the recognition of meats placed upon the market (for instance,
the detection of horse meat in sausages).
Since the injection of the protein from any organ or tissue of
an animal results in antibodies which react with extracts from
any other tissue of the identical species (i.e., antibodies which are
race-specific and only to a slight degree organ-specific), it is
not necessary to produce the antiserum for the detection of meat
by injection of meat extract. The injection of serum or of blood
of the same species gives quite as useful antibodies and is much
more convenient.
For the preparation of extracts from meat Seiffert recommends
the infusion of 30 grams of finely chopped meat in 30 c.c. salt
solution ; the mixture is to stand for 12 hours in the cold, and is
then filtered through a hard paper. Students prepare extracts
of two types of meat given out,1 extracting 5 grams of chopped
1 One of these should if possible be horse meat which can usually be ob-
tained from a board of health or a veterinary institution. The antihorse serum
is prepared in advance by the injection of three rabbits with horse serum.
(Lesson I.) If it is not possible to obtain horse flesh, the whole experiment can
be done with antisheep serum and mutton obtained from a butcher.
110
LABORATORY COURSE IN SERUM STUDY
meat with 5 c.c. of salt solution and filtering. The clear solution
represents approximately a 1-100 dilution of the protein. From
it a 1-1000 dilution should be prepared.
The following experiment should then be set up :
TUBE
ANTIGEN
ANTIHOBSE SEBUM
GUINEA PIG SEBUM 1-10
1
Extract A (1-100) 0.5 c.c.
0.1 c.c.
0.5 c.c.
2
Extract A (1-1000) 0.5 c.c.
0.1 c.c.
0.5 c.c.
3
Extract B (1-100) 0.5 c.c.
0.1 c.c.
0.5 c.c.
4
Extract B (1-1000) 0.5 c.c.
0.1 c.c.
0.5 c.c.
5
Extract A (1-100) 1.0 c.c.
—
0.5 c.c.
(Antigen control)
6
Extract B (1-100) 1.0 c.c.
—
0.5 c.c.
(Antigen control)
7
Antiserum control
0.2 c.c.
0.5 c.c.
8
Complement control —
—
0.5 c.c.
All tubes are to be brought up to a volume of 1.5 c.c. by the addition
of salt solution.
Incubate for one hour at 37.5° C.
At the end of this time 0.5 c.c. red cells, sensitized with two units
of amboceptor, are added to each tube and the tubes are again incubated
until haemolysis is complete in controls "5," "6," "7" and "8."
The purpose of the control tubes "5" and "6" is, of course, to prove
that the antigen alone without the presence of the antiserum does
not interfere with the complement action of the guinea pig serum, while
the purpose of the control number "7" is to make certain that the
antiserum alone does not interfere with the action of the complement.
Tube "8" is put in for a similar purpose so that a slight inhibitory
action (anticomplementary effect) on the part of either antigen or anti-
serum can be recognized by contrast with the complete laking which
ensues from the unhampered effect from the guinea pig serum on the
sensitized red cells.
MATERIALS
Each student requires :
5 grams of each of 2 types of
chopped meat (horse and any
other variety).
2 hard filter papers and funnels.
1 c.c. antihorse serum
5 c.c. guinea pig serum (1-10).
20 units antisheep-cell amboceptor.
8 one-c.c. pipettes.
8 half-inch test tubes.
2 large test tubes or wide mouth
bottles.
1 test tube rack
5 c.c. sheep cells.
Salt solution.
112 LABORATORY COURSE IN SERUM STUDY
LESSON XIX
WASSERMANN REACTION1
I
PREPARATION OF THE ANTIGEN
THE general principles of the technique of the Wassermann
reaction are the same as those of other forms of complement
fixation already discussed — with one notable exception : the
antigen is not specific in origin, i.e., is not made from Spirochaeta
pallida or (as it originally was) from syphilitic organs. Land-
steiner and his successors found that equally good or better re-
sults .could be obtained with lipoidal substances extracted from
normal organs by means of alcohol and other lipoid solvents.
Various lipoid mixtures are now used, the most serviceable being
alcoholic extracts of normal heart muscle, — usually beef, guinea
pig, or human heart. Sometimes cholesterin is added to the al-
coholic extract. The alcoholic solution, of course, is well diluted
with saline solution before use. These facts, empirically ascer-
tained since the introduction of the Wassermann reaction, show
definitely that this test is not dependent upon an antigen-
antibody union as is the case with other forms of complement
fixation.
No matter what lipoid antigen is used, its specificity and dos-
age have to be determined by careful preliminary titrations and
by trial on known syphilitic and non-syphilitic sera before it can
be used in diagnostic work.
As the preparation of antigen for use in the Wassermann test
takes time it had better be begun about three weeks before the
antigen is needed for the experiments below. The preparation
of three typical and useful antigens will be described. The class
should be divided into three groups of students and each group
of students should be made jointly responsible for the prepara-
tion of one or two specimens of one of the types of antigen.
1 Lessons XIX and XX should be repeated — if possible, an entire week
given to them.
114 LABORATORY COURSE IN SERUM STUDY
SIMPLE ALCOHOLIC EXTRACT OF HEART MUSCLE
Human, guinea pig, and beef heart are used by different workers
and are of practically equal value. Heart muscle is finely chopped
and extracted at 37° C. in 10 volumes of absolute alcohol, for
from three to seven days. The jars in which the heart muscle is
kept should be shaken one or more times a day. If guinea pig
hearts are used, a jar of 100 c.c. of absolute alcohol is set aside,
and the hearts are ground up and dropped into the alcohol when-
ever a guinea pig has to be killed for any laboratory purpose. It
is important to remove all blood from the heart. If beef or human
heart is used it is important to remove all fat and tendon before
the heart is extracted. At the end of the period of extraction the
alcohol is filtered and the filtrate tested as described below.
B
CHOLESTERIN REENFORCED HEART EXTRACT
The guinea pig hearts 1 are weighed before being added to the
alcohol. Thirty grams of finely ground heart are extracted at
least 14 days at 37° C. with 300 c.c. of absolute alcohol. This
is then filtered and half the filtrate is placed in a flask with an
excess (say 5 grams) of chemically pure cholesterin. This is kept
at 37° C. overnight and then in a water bath at 16° C. for three
hours (to precipitate excess of cholesterin). This saturated solu-
tion is then filtered and the other half of the original heart extract
is added to it, giving a heart extract which is half saturated with
cholesterin.
• C
NOGUCHI'S ACETONE-INSOLUBLE FRACTION OF HEART EXTRACT
Whereas in the preceding antigen cholesterin was added to
the heart extract, Noguchi obtains a highly specific antigen by
removing the cholesterin and certain other substances from the
heart extract by means of precipitation with acetone (in which
1 Some workers use human or beef hearts.
116 LABORATORY COURSE IN SERUM STUDY
cholesterin is soluble whereas lecithin and certain allied lipoids
are insoluble in it).
Extract 100 grams of mashed heart muscle with a liter of
absolute alcohol at 37° C. for several days. Filter and evaporate
to dryness with an electric fan. This usually takes 12 to 24 hours.
Dissolve the residue in ether (avoid flames). Stand the milky
ether solution in the ice box overnight and decant off the clear
supernatant portion. Evaporate this before an electric fan to a
small volume and to it add about 10 volumes of pure acetone.
Allow to stand for several hours and decant off the acetone.
Scrape the precipitate off with a spatula and preserve it under
acetone. For use dissolve about 0.3 grams of the brownish sticky
precipitate in 1 c.c. of ether and to it add 9 c.c. methyl alcohol.
The alcohol solution is fairly stable and can be used as a stock
solution.
II
TlTRATION OF WASSERMANN ANTIGEN
Alcoholic extracts of organs contain substances which are
haemolytic and substances which have the power of binding com-
plement non-specifically, much as do bacterial suspensions and
extracts. They also contain substances which in combination
with syphilitic sera have the power of binding complement.
It is important to test for all three of these properties and a suit-
able extract must show an antigenic value (that is, the power to
bind complement in the presence of syphilitic serum) much higher
than its anticomplementary or haemolytic values. The haemolytic
power of the antigen seldom interferes practically, and is some-
what interfered with by the guinea pig and human serum used in
the test.
Before standardizing the antigen, the alcoholic concentrated
antigen sliould be diluted 1-10 with salt solution, and the way in
which this suspension is made is of importance since the antigen
is in colloidal suspension and not in true solution and the anti-
complementary and antigenic value depend certainly in part on
the physical condition of the antigen. This will vary according
118 LABORATORY COURSE IN SERUM STUDY
to whether the alcoholic extract is run rapidly into the salt solu-
tion which is used as diluent or whether the extract is diluted by
dropping salt solution into it. It is not of so great importance
which of the two methods is used as that the same method should
be used throughout both preliminary titrations and tests.
Each student tests one of the three antigens made by the class.
This antigen is to be made up to a 1-10 dilution with saline. Add the
saline slowly to the antigen. Each student needs about 10 c.c. of the
1-10 dilution.
A syphilitic (Wassermann+ + + +) and a normal human serum con-
taming no or very little antisheep-cell amboceptor are chosen : they are
inactivated at 56° C. for half an hour before use.
Set up the following series of tubes :
1. DETERMINATION OF ANTICOMPLEMENTARY DOSE
Tube Antigen 1-10 Complement 1-10
1 0.2 c.c. 2 units
2 0.4 c.c. 2 units
3 0.8 c.c. 2 units
4 1.2 c.c. 2 units
5 1.6 c.c. 2 units
6 2.0 c.c. 2 units
2. DEMONSTRATION THAT ANTIGEN is NOT H^MOLYTIC OF ITSELF
Tube 7 Antigen 1-10 1.0 c.c. 5% Sheep cells 0.5 c.c.
3. DETERMINATION OF ANTIGENIC DOSE
Tube Antigen Syphilitic (++++) Serum l Complement 1-10
8 (1-40) 0.05 c.c. 0.1 c.c. 2 units
9 (1^0) 0.1 c.c. 0.1 c.c. 2 units
10 (1-40) 0.2 c.c. 0.1 c.c. 2 units
11 (1-10) 0.1 c.c. 0.1 c.c. 2 units
12 (1-10) 0.2 c.c. 0.1 c.c. 2 units
13 (1-10) 0.4 c.c. 0.1 c.c. 2 units
4. PROOF THAT ANTIGEN is SPECIFIC (NEGATIVE CONTROL)
Tube Antigen 1-10 Normal Human Serum l Complement 1-10
14 0.1 c.c. 0.1 c.c. 2 units
15 0.2 c.c. 0.1 c.c. 2 units
16 0.4 c.c. 0.1 c.c. 2 units
1 The sera used should be inactivated on day of test by heating to 55° for twenty
minutes in the water bath.
120 LABORATORY COURSE IN SERUM STUDY
5. SERUM CONTROLS
Tube Normal Serum Complement 1-10
17 0.1 c.c. 2 units
18 0.2 c.c. 2 units
Syphilitic (++++) Serum
19 0.1 c.c. 2 units
20 0.2 c.c. 2 units
The volume is made to 2 c.c. as nearly as possible in all the tubes.
All tubes are put in water bath for one hour. Then 0.5 c.c. of 5 per
cent sheep cells and two units of amboceptor are added to each tube (ex-
cept 2 tube 7) and the tubes are all returned to the water bath for
thirty minutes.
CONCLUSIONS TO BE DRAWN FROM THE ABOVE
Series 1
The anticomplementary dose is that quantity of antigen which
shows the slightest inhibition of laking. The amount of antigen
used in the test must under no circumstances exceed one half of
this amount and is usually taken as one third of it. Thus if
Tube 5 containing 1.6 c.c. of the 1 to 10 antigen shows a faint
haze of undissolved red cells, then the quantity used in the tests
should not exceed 0.5 c.c.
Series 2
An antigen which is haemolytic in itself in Tube 7 is rejected
as useless. When the extractions are made of normal heart this
occurrence is rare.
Series 3 f
The antigenic dose is the smallest amount that gives com-
plete inhibition in the presence of syphilitic serum. This is tested
in Tubes 8 to 13. In order that an antigen be of value the
antigenic dose must be considerably smaller than the anticom-
plementary quantity determined in Series 1. It is desirable in
the tests to use an amount of antigen containing at least several
antigenic units. Thus, if the antigen tested shows complete in-
hibition in Tube 11 and merely partial inhibition in Tube 10,
122 LABORATORY COURSE IN SERUM STUDY
and perhaps none in 8 and 9, then the quantity contained in
Tube 11, namely 0.1 c.c. of the 1 to 10 dilution, is the antigenic
dose or unit. It is desirable to use 4 or 5 times this unit in the
test, and if this quantity does not exceed one third of the anti-
complementary dose determined in Series 1, the antigen is suitable
for use.
Series 4
The object of Series 4 is of course to show that inactivated
normal sera do not fix complement in the presence of the antigen.
In practice no worker should be satisfied to test a new antigen
against merely one normal serum, but before using the antigen in
routine tests it should be used for some time parallel with an old
familiar antigen.
Series 5
Series 5 controls the positive and negative sera used in the
preceding series. They are carried out because under certain
circumstances sera become anticomplementary, especially if kept
for several days. If this occurs, the anticomplementary property
can usually be destroyed by heating to 56° C. However, occa-
sionally even inactivated sera may remain slightly anticomple-
mentary. Therefore these controls are indispensable.
MATERIALS
Each student needs :
1.0 c.c. antigen (undiluted).
10.0 c.c. complement (1-10).
15.0 c.c. 5 per cent sheep cells.
45 units amboceptor.
1.0 c.c. positive (+ + + +) serum.
1.0 c.c. negative serum.
Salt solution.
25 half-inch test tubes.
5 one-inch test tubes.
8 one-c.c. pipettes.
1 two-c.c. pipette.
1 five-c.c. pipette.
1 test tube rack.
Electric fan.
124
LABORATORY COURSE IN SERUM STUDY
LESSON XX
WASSERMANN REACTIONS
II
TECHNIQUE OF TESTS
t
I. PRELIMINARY COMPLEMENT TITRATION
ON account of the complement variability of guinea pigs'
serum and of the desirability of using a uniform amount of com-
plement, a fresh titration of the complement is done on each
occasion when Wassermann tests are performed. In order to
allow a margin of safety, twice that amount of guinea pigs ' serum
is used in the tests which in the preliminary titration gives com-
plete laking with 2 units of amboceptor in 15 minutes.
(The quantities of all the ingredients given here are known as
"half Wassermann" quantities; that is, for the sake of economy and
convenience half the amount of patients' serum, guinea pig serum,
sheep cells, etc., described in the original Wassermann test are used
and the total volume is made to 2.5 c.c. instead of 5 c.c. Some workers
carry economy farther and use " tenth Wassermann" quantities.
There are also some workers who instead of redetermining the dose
of complement each time, keep the dose of complement constant —
0.5 c.c. of 10 per cent serum for half Wassermanns — and redetermine
the amboceptor unit. The net result is not very different, but since the
guinea pig is the variable factor it seems more reasonable to vary the
amount of guinea pigs' serum.)
Into each of 7 tubes put 0.5 c.c. of 5 per cent sheep cells, two units
of amboceptor and 10 per cent complement (fresh guinea pig serum) in
the following amounts: 1
TUBE
SHEEP CELLS
SENSITIZER OR AMBOCEPTOR
COMPLEMENT 10% IN SALT
SOLUTION
1
0.5 c.c.
2 units
0.1 c.c.
2
0.5 c.c.
2 units
0.15 c.c.
3
0.5 c.c.
2 units
0.2 c.c.
4
0.5 c.c.
2 units
0.25 c.c.
5
0.5 c.c.
2 units
0.3 c.c.
6
0.5 c.c.
2 units
0.4 c.c.
7
0.5 c.c.
2 units
0.5 c.c.
8
0.5 c.c.
—
0.5 c.c.
Bring the volume of all the tubes up to 2.5 c.c. by addition of
saline solution.
1 The following is the preferable order of adding the reagents:
First pipette the complement, second the amboceptor, third the saline solution
and last the red cells, shaking thf tubes thoroughly while the cells uro being :idded.
126 LABORATORY COURSE IN SERUM STUDY
Set up one tube (complement control) with 0.5 c.c. of sheep cells
and 0.5 c.c. of complement only, in order to make sure that the guinea
pig serum alone is not at all haemolytic.
For the tests twice that amount of complement is used which just
gives complete haemolysis in 30 minutes. With average guinea pigs
0.25 c.c. gives this and 0.5 is used in the test.
II. PREPARATION OF SERUM
Inactivate at 56° C. for one half hour a known syphilitic and a
known normal serum and one or more sera to be examined.
III. SETTING UP THE TESTS. PRIMARY INCUBATION
Test tube racks with two rows of holes are used so that each serum
may have a " front" tube and a corresponding "back" tube.
(a) Into each of a pair of 5 c.c. test tubes measure 0.1 c.c. of each of
the sera.
(6) Into one (the front tube) of each pair measure a dose of diluted
antigen. (The dose is one third of the largest amount which by
previous titration has been found not to interfere with the action of the
amount of complement used in the test.)
Into an extra tube measure two doses of antigen (antigen control
tube).
(c) Into all tubes measure the predetermined dose of complement.
Shake each tube at once. Add enough saline to make the contents
of all the tubes approximately 2 c.c.
(d) Incubate in water bath for one hour at 37° C. (With certain
antigens, as for instance with alcoholic heart extracts, somewhat more
delicate results can be obtained by a four hour preliminary incubation
in the ice box at 8°-12° C. For class work, however, this is
impractical.)
IV. ADDITION OF ILEMOLYTIC SYSTEM. SECONDARY INCUBA-
TION. READING OF RESULTS
Into all tubes measure 0.5 c.c. of 5 per cent sheep cells to which
2 units of amboceptor have been added. Incubate at 37° C. Read the
results when the double antigen tube and all the serum control tubes
(i.e. those containing no antigen) are completely laked.
Fill out a protocol of the degree of inhibition, if any, in the various
tubes :
128
LABORATORY COURSE IN SERUM STUDY
INHIBITION SHOWN
IN TEST
INHIBITION SHOWN
IN CONTROL (WITH-
OUT ANTIGEN)
RESULT OF
WASSERMANN TEST
Positive serum
Negative serum
Unknown serum 1
Unknown serum 2
Unknown serum 3
Double dose of antigen
Each student needs:
One known syphilitic serum. One normal serum.
7 c.c. of 10% complement. 20 |-inch test tubes.
Five f-inch test tubes. Racks. 10 one-c.c. pipettes.
LESSON XXI
COMPLEMENT FIXATION WITH BACTERIAL EXTRACTS
ON account of the rather high anticomplementary strength of
the insoluble portion of bacterial bodies themselves, in practical
complement fixation tests the effort is made to obtain the anti-
genie substance of the microorganisms in more or less pure solu-
tion. This is partially accomplished by extracting with distilled
water, by mechanical breaking up of the bacterial bodies, etc.
Bacterial complement fixation with antigens of this type is of
practical diagnostic use in gonorrhea, glanders, typhoid fever,
whooping cough and other diseases.
Cultures of the same species of organism isolated from differ-
ent sources often show considerable biological differences, and
an immune serum which fixes complement with one such strain
often gives only partial fixation with another. For this reason,
in diagnostic tests, polyvalent antigens are used, i.e., antigens
made of numerous different strains of the bacteria in question.
On account of the prolonged manipulations required students
will not make bacterial antigens for these tests,1 but will be pro-
1 The technique of preparing the Gonococcus antigen as practiced at the
New York Board of Health and supplied to us by the kindness of Miss M. P.
Olmstead is as follows :
The strains used in the polyvalent antigen are ten of those shown by Torrey
to be serologically distinct — A, B, C, G, K, L, N, O, Q, S.
Stock transplants are kept on glucose ascitic agar, prepared as follows :
Bob veal, lean, chopped fine 1 Ib.
Distilled H2O 1 liter.
Mix, stand overnight at room tentperature. Heat to 45° C. one hour.
130 LABORATORY COURSE IN SERUM STUDY
vided with extracts already made which require only to be
properly diluted with saline before use, and with these extracts
will do fixation tests for Gonococcus and for Glanders.
Bring to a boil. Strain through cheese cloth.
Bring to original volume. Add agar 1$ per cent, Witte's peptone 2 per cent,
NaCl £ of 1 per cent.
Titrate. Reaction should be neutral to phenolphthalein.
Filter through a filter made with a layer of cotton, one of filter paper, another
layer of cotton. Filter several times.
Autoclave \ hour at 15 Ib. pressure.
Glucose 2 per cent.
10 grams of glucose dissolved in 50 c.c. distilled H20. Sterilize three days
for 20 minutes in Arnold.
Ascitic fluid 20 per cent.
Ascitic fluid is filtered through Berkefeld, sealed in sterile flasks, incubated
at 37.5° several days before using. Glucose solution and ascitic fluid
are mixed before adding to tubed and sterilized agar. Incubate tubes.
Keep two days before using.
Transplant the stock cultures every 48 hours. The gonococcus must always
be kept at 37.5° C. Any inequality of temperature will cause a poor growth of
the organism.
For the antigen transplants use veal agar prepared as above, except that salt,
glucose and ascitic fluid are omitted. The reaction should be very carefully
adjusted ; it should be neutral at the last titration before autoclaving, and when
ready to use 0.1 to 0.2 per cent acid, preferably 0.1 per cent. Medium is titrated
hot with N/20 NaOH. No change should take place when phenolphthalein is
added. One or two drops of NaOH produce a faint pink color which disappears
on adding one or two drops of N/20 HC1. The agar is then bottled and auto-
claved one hah' hour at 15 Ib. pressure.
To prepare antigen take 24-hour stock cultures and transplant to potato
tubes (tube 6x1 inch) of salt free veal agar neutral to phenolphthalein. Incu-
bate 24 hours. Transfer all this growth by means of sterile cotton swabs to
wide-mouthed Blake bottles (one tube to a bottle) containing neutral salt free
veal agar, gently rubbing the swab over the entire surface of the agar. Incubate
for 24 hours. \Vash off all this growth, if good, with neutral sterile distilled water,
10 c.c. to a bottle. If the growth is poor, 5 c.c. is sufficient. The bottle should
be gently tipped back and forth two or three times after the water is added and
the growth scraped off lightly with a bent glass rod. Do not allow the water
to remain on the agar more than a few seconds. The resulting emulsion is auto-
lyzed for one hour in a water bath at 56° C. and at 80° C. for one hour. Filter
the autolyzed emulsion through a Buchner funnel which has been well packed
with paper pulp and then through a sterile Berkefeld filter of N or V porosity.
As new niters are very alkaline they are taken to pieces before use and boiled
in distilled water at least three times, five minutes each time, being scrubbed
thoroughly with a small brush in fresh water after each boiling. Then the filter
is set up and hot distilled water allowed to stand in it for five minutes. Hot
neutral distilled water is then run through it under gentle pressure until fluid is
clear and neutral when tested with phenolphthalein. After a filter has been used
132
LABORATORY COURSE IN SERUM STUDY
GONOCOCCUS COMPLEMENT FIXATION
The antigen provided is a distilled-water extract. It is to be diluted
1-10 with 0.9 per cent saline solution. The preliminary titration is
done exactly as with the typhoid emulsion in Lesson XVI in order to
determine the "anticomplementary dose" of the antigen. That amount
which just shows beginning inhibition of haemolysis when two units of
amboceptor and two units of complement (0.5 c.c. of 1-10 dilution) are
used is designated the anticomplementary dose, and one quarter of this
dose is used as the dose of antigen in the test. Supposing that this anti-
complementary dose is found (with 0.5 c.c. of 1-10 guinea pig serum,
0.5 c.c. of 5 per cent sheep cells, and two units of amboceptor) to be 2.0
c.c., then the tests are set up as follows:
TUBE
ANTIGEN
GUINEA PIG
SERUM 1-10
1
Positive serum 0.1 c.c.
0.5 c.c.
2 units
2
3
Negative serum 0.1 c.c.
Positive serum 0.2 c.c.
0.5 c.c.
2 units
2 units
4
Negative serum 0.2 c.c.
—
2 units
5
1.0 c.c.
2 units
6
2.0 c.c.
2 units
Bring up to 2.0 c.c. with salt solution.
Incubate one hour in the thermostat at 37° C. or if possible four
hours in the ice box at 10° C. Then add 0.5 c.c. sheep cells sensitized
with two units of amboceptor and incubate at 37° C. until all the tubes
excepting Tube 1 are completely laked. The positive serum is either
the serum of a rabbit which has been immunized with gonococci or the
serum of a patient known by previous tests to have a positive gonococcus
fixation reaction. All the sera are of course inactivated at 56° C. for a
half hour before use.
it is boiled in distilled water and thoroughly scrubbed. It may be dried in
the air for 48 hours and put away or attached to a filter flask and sterilized to
be ready for use. Do not use for filtering gonococcus antigen a filter that has
been used for any other purpose, unless it is first boiled in 1 per cent NaOH and
reneutralized.
Bottle the filtrate with aseptic precautions. Sterilize three successive days
for one half hour each day at 56° C. and keep in the ice box. Immediately before
use the antigen is made isotnio by the addition of one part 9 per cent saline solu-
tion to nine parts antigen. The antigen is more likely to remain stable if no salt
is added until the day of use.
134
LABORATORY COURSE IN SERUM STUDY
II
GLANDERS COMPLEMENT FIXATION
This is carried out in the same manner as the gonococcus test,
using the serum of a known glanders-infected horse as a positive con-
trol.1
TUBE
ANTIGEN
GUINEA PIG SERUM 1-10
1
2
3
4
Positive serum 0.1 c.c.
Negative serum 0.1 c.c.
Positive serum 0.2 c.c.
Negative serum 0.2 c.c.
0.5 c.c.
0.5 c.c.
1C) n ft
0.5 c.c.
0.5 c.c.
0.5 c.c.
0.5 c.c.
c\ K n n
6
2.0 c.c.
0.5 c.c.
Bring up to 2.0 c.c. with salt solution.
Incubate one hour in the thermostat at 37° C. or if possible four
hours in the ice box at 10° C. Then add 0.5 c.c. sheep cells sensitized
with two units of amboceptor and incubate at 37° C. until all tubes
.excepting Tube 1 are. completely laked. The positive serum is from a
glandered horse known to be positive by previous tests.
Materials needed for each student for gonococcus fixation :
0.5 c.c. positive serum.
0.5 c.c. normal serum.
5.0 c.c. antigen (diluted).
5.0 c.c. guinea pig serum (1-10).
5.0 c.c. shesp cells 5 per cent suspension.
20 units amboceptor.
20 half-inch test tubes
Salt solution.
1 test tube rack.
lOone-c.c. pipettes (graduated).
GLANDERS ANTIGEN
1 The technique of preparing glanders antigen as practiced at the New York
Board of Health and supplied to us by the kindness of Miss M. P. Olmstead is as
follows :
Stock transplants are kept on glycerine potato agar (for formula see Park
and Williams' Pathogenic Microorganisms, 1914, page 97) from 1 per cent to
2 per cent acid to phenolphthalein. For antigen transplants the same medium
is used as in the preparation of gonococcus antigen, except for the reaction, which
should be 1.6 per cent acid. The 24 hour growth on bottles is washed off with
sterile, neutral, distilled water and heated at 80° C. for 6 to 8 hours, then filtered.
With the exceptions mentioned, the technique of antigen preparation is the same
as in the case of gonococcus.
136 LABORATORY COURSE IN SERUM STUDY
Materials needed for each student for glanders fixation :
0.5 c.c. positive glanders serum from a horse known to be infected
and giving a positive glanders complement fixation reaction.
0.5 c.c. normal horse serum.
5.0 c.c. glanders antigen.
5.0 c.c. guinea pig serum (1-10).
5.0 c.c. 5 per cent sheep cells.
20 units amboceptor.
20 half-inch test tubes.
10 one-c.c. graduated pipettes.
1 test tube rack.
Salt solution.
LESSON XXII
THE STANDARDIZATION OF DIPHTHERIA ANTITOXIN
(THE proper understanding of the work in this lesson requires
a review of the constitution of diphtheria toxins and the theoreti-
cal principles involved. The student should read again the sec-
tion on toxin and antitoxin in Infection and Resistance, in Kol-
mer's Infection and Immunity, in Paul Th. Miiller's Vorlesungen
iiber Infektion und Immunitat, in the Krause und Levaditi
Handbuch, or in some other work in which these principles are
discussed at length.)
When extensive therapeutic use first necessitated the estab-
lishment of a standard of dosage for diphtheria antitoxin, it was
hoped that a method could be developed depending upon simple
in vitro titration analogous to the titration of normal acid against
normal alkali solutions, in which the unknown antitoxin could
be measured against known amounts of a standard toxin. Since
no visible or chemically determinable reaction takes place between
the two substances in the test tube, the only method of ascertain-
ing whether or not the given quantity of toxin was neutralized
by the antitoxin was to inject the two substances, at first separate
but later previously mixed, into susceptible animals and learn
from the result whether or not free, unneutralized toxin was left.
The guinea pig, chosen as the most suitable test animal, therefore,
took the place in these reactions of the "indicator" to demonstrate
138 LABORATORY COURSE IN SERUM STUDY
the presence of free, unneucralized toxin by its death, just as
phenolphthalein in. acid-alkali titrations indicates the presence of
free alkali by a pink color. Since guinea pigs of different weights
and ages have varying resistances against the poison, a standard
weight for such measurements, namely 250 grams, was established.
The first step toward such a standardization, of course, con-
sisted in establishing standards of measurement for toxin. Beh-
ring, later together with Ehrlich, established as a toxin unit or
MLD (minimal lethal dose) the amount of a toxin solution which
would kill a guinea pig of 250 grams. Because of the importance of
the time element this was later modified to represent the amount
which would kill such a guinea pig in from four to five days.
The antitoxin unit later established mainly by the efforts
of these two workers was designated as the amount of the anti-
toxin, i.e. of the serum of a toxin immune animal, which would
neutralize 100 such MLD's (minimal lethal doses) for guinea pigs
of the standard weight. It was soon found by these workers, as
well as by others, that it was not easy to determine the exact
point of neutralization, that is, while the guinea pig might be
preserved from death from 100 such minimal lethal doses, in one
case in another slight local or systemic symptoms might easily
escape the observation of the investigator, and it seemed safer to
eliminate the personal equation entirely. This was done by es-
tablishing as an antitoxin unit not the amount that would neutral-
ize the 100 fatal doses, but a partial neutralization measured in
such a way that the toxin quantity left over in the mixture would
still kill the guinea pig in four to five days, giving the same effect
as an unneutralized single toxin unit.
Subsequent developments in the investigation of toxins, espe-
cially by Ehrlich, disclosed many difficulties in the path of such
a relatively simple method of titration. It is a comparatively
easy matter to determine the minimal lethal dose of any poison
that one may have produced. However, this quantity — or
MLD — will not be identical in one and the same toxin filtrate
if this is measured at intervals of a few weeks or months, since
the true toxin is gradually converted into a non-poisonous prod-
140 LABORATORY COURSE IN SERUM STUDY
uct, the "toxoid." The toxoid is not poisonous for guinea pigs,
and therefore the volume of original broth necessary to kill guinea
pigs of the required weight increases. Now if this deterioration
into toxoid implied at the same time diminution of neutralizing
power for antitoxin the titration could be easily adjusted by simply
remeasuring the minimal lethal dose for the particular solution
used and proceeding on this basis merely with a changed unit.
However, in old toxin solutions, although the minimal lethal
dose is larger, the neutralizing value of this poison for antitoxin
has changed very little or not at all. In other words, the toxin
derivatives or toxoids, although no longer poisonous to the
guinea pig, still retain their neutralizing power for the antitoxin.
It is obvious therefore that no constant standard could be obtained
merely by measuring the minimal lethal dose of any toxin broth
and measuring the antitoxin unit against 100 such minimal fatal
doses. The amount of toxoid would be alike in no two toxin filtrates
nor in the same filtrate at different times, and the antitoxin unit
which I determined against 100 MLD's of a toxin brothl have pro-
duced in my laboratory might be far different in antitoxic potency
from a unit similarly determined in another laboratory from an-
other toxin broth containing an entirely different proportion of
true toxin and non-poisonous antitoxin-neutralizing toxoids.
In consequence a different system has had to be worked out
largely by the laborious investigations of Ehrlich. Ehrlich
found that the antitoxin was very much more stable than the
toxin. If dried, reduced to a powder, and preserved in vacuo
in a small glass U-tube over phosphoric anhydride in the cold
and in the dark, such dried serum would preserve its antitoxic
value for a long time and no deterioration comparable to tha^ tak-
ing place in the toxin would occur. Originally Ehrlich prepared
tubes in this way, each one containing antitoxin powder of a
potency of 1700 antitoxin units to the gram, these antitoxin
units being measured against one of the toxins in his possession
representing the amount necessary to neutralize 100 MLD's of
this toxin. Against this powder then from time to time other
toxins are measured and the quantity determined which mixed
142 LABORATORY COURSE IN SERUM STUDY
with such an antitoxin unit will just kill a guinea pig of 250
grams in from four to five days, i.e. which mixed with one
standard antitoxin unit will give the effects of one free toxin unit.
This amount of toxin is known as the L + dose, and of course
if such an L + dose of a toxin measured against the standard unit
is now mixed with varying quantities of an unknown antitoxin
until a mixture of similar effect is obtained, the particular quan-
tity of antitoxin used in this mixture will be equal to the original
standard antitoxin unit. Thus the antitoxin unit established by
Ehrlich is now the standard of measurement for other antitoxins
rather than the toxin itself.
Such standard antitoxin units are preserved in the way
indicated above for America at Washington, for Germany in
Frankfort, for France at Paris, etc., and the L-j- dose of toxin
solutions can be determined against these standards and with
such L + quantities of any given toxin new antitoxin can be
standardized and portions of these in turn preserved. Con-
stancy of measurement is thereby assured if this system is kept
under proper governmental supervision without a break in con-
tinuity, and without accident. This is practically insured
against by the large number of laboratories in the world in which
this standard is being kept.
It is obvious therefore that in the investigation of antitoxic
strength three standard units are used. They may be defined
as follows :
The Minimal Lethal Dose (MLD or T) is that amount of
toxin which when subcutaneously injected invariably causes the
death of a 250-gram guinea pig in from four to five days.
Limes "Zero" Dose (L0) is that amount of toxin which is com-
pletely neutralized by one antitoxin unit so that no trace of reac-
tion, local or otherwise, ensues when it is injected mixed with
one unit of antitoxin.
Limes " Todt " Dose (L -f ) is that amount of toxin which when
mixed with one unit of antitoxin and injected subcutaneously
will cause the death of a 250-gram guinea pig in four to five days.
The quantitative relationship between the MLD, the L0 dose
144 LABORATORY COURSE IN SERUM STUDY
and the L + dose is one that is by no means regular, an irregularity
which has not yet met with an entirely satisfactory explanation
and which for the actual practical measurement of antitoxin
it is not necessary to go into at present. The student, however,
is advised again to refer to the textbooks mentioned above and to
read the explanations of this subject carefully. It is easily seen,
however, that the definition of an antitoxin unit as it is at present
used can no longer be truthfully given as the amount which neu-
tralizes 100 fatal doses for guinea pigs of 250 grams, although this
is the way it is usually put in textbooks. The original unit
measured by Ehrlich and preserved did actually neutralize 100
fatal doses of the particular toxin used. However, when the
stated amount of this original antitoxin [or of other antitoxins
based upon it and similarly preserved] is measured against many
other and unknown toxins, the L-h dose of these poisons con-
taining amounts of toxoid differing from that in Ehrlich 's
original toxin will not necessarily contain 100 minimal lethal
doses of true toxin. The antitoxin in such mixtures is neutralized
not only by the true toxin but also by the toxoid which has
different or no poisonous properties, and thus the number of
actual minimal lethal doses contained in the mixture may vary
from 50 up. An antitoxin unit, therefore, in the modern sense is
the amount of antitoxin which when mixed with the L + dose of a
standard toxin leads to death of the guinea pig in four to five days.
The L + dose of the toxin in this case must have been measured
by a previously determined standard antitoxin unit which is the
ultimate basis of measurement.
In order to allow a margin of safety this definition has been
further altered of recent years in the following way :
The antitoxin unit is the amount of antitoxic serum which
mixed with the L + dose of a standard toxin will preserve a guinea
pig of 250 grams from death.
PREPARATION OF TOXIN
Each student is given a 500 c.c. Erlenmeyer flask in which is 100 c.c.
of veal infusion broth containing 2 per cent peptone and adjusted to
146
LABORATORY COURSE IN SERUM STUDY
reaction of 0.5 per cent acidity to phenolphthalein. (Various modifica-
tions of this medium may be used. Smith recommends a broth which
has been made sugar free by inoculation with colon bacillus, and Rosenau
recommends the addition of 1 per cent glucose to the broth.) A strain
of B. diphtheria known to produce strong toxin (preferably the Park-
Williams bacillus No. 8) is used for inoculation. Before it is planted on
flasks it should be transferred by the instructor daily through a series
of several broth tubes to obtain vigorous and rapid growth. The flasks
are incubated for seven days and one or two which show the best growth
filtered through a Berkefeld filter. The filtrate is stored in a sterile
dark bottle on ice until used.
The class, working as a group, will carry out one or more of the
following determinations on one such toxin, each student injecting one
of the pigs in a given series. (In actual class work, in order to save
guinea pigs, usually only III, the determination of the antitoxic value
of an unknown serum, is carried out.)
I. DETERMINATION OF THE MLD OF A TOXIN
Guinea pigs are injected subcutaneously with varying amounts
of this toxin until the amount is determined which will regularly
cause death in from four to five days. For a satisfactory toxin the
MLD must be less than 0.01 c.c. The toxin is diluted in salt solution
so that the desired amount will be contained in 2 c.c. This amount is
injected subcutaneously, with a Rosenau syringe,1 the needle of which
is inserted in the flank of the guinea pig and passed subcutaneously in
the flank of the pig till the point is near the linea alba before the diluted
poison is injected. After injection 1 c.c. of salt solution is placed in
the syringe without removing the needle and injected to wash out the
last traces of the poison. The following example of such a determination
is taken from Rosenau's toxin No. 5. The MLD in this case was .002 c.c.
DATES TESTED
ANIMAL
TOXIN INJECTED
RESULT
5/26/04
G.P. #9
.005 O.C.
Death 1 day 18 hours
5/26/04
G.P. # 10
.005 C.C.
Death 1 day 20 hours
5/31/04
G.P. # 13
.004 c.c.
Death 3 days 12 hours
5/31/04
G.P. # 12
.003 c.c.
Death 2 days 12 hours
5/31/04
G.P. #11
.002 c.c.
Death 4 days 0 hours
7/21/04
G.P. #105
.001 o.c.
Death 5 days 5 hours
7/21/04
G.P. #107
.0008 o.c.
Death 7 days 23 hours
9/30/04
G.P. #311
.002 c.c.
Death 4 days 3 hours
12/15/04
G.P. # 1030
.002 c.o.
Death 4 days 8 hours
1 Ordinary Luer or other all-glass syringes are used in many laboratories.
148
LABORATORY COURSE IN SERUM STUDY
II. DETERMINATION OF THE L+ DOSE OF TOXIN
A dilution of a carefully standardized antitoxic serum is prepared
in sterile salt solution so that each cubic centimeter contains one unit
of antitoxin. A preliminary test with the dilutions spaced widely
should be carried out by the instructor to determine approximately
the L+ dose. A series of dilutions of toxin increasing by about TV
of the smallest amount should then be prepared, the total volume of each
dilution being brought up to 2 c.c. (The smallest amount is the amount
just below the minimal dose fatal when mixed with one unit of antitoxin
in the preliminary experiment.) 1 c.c. of antitoxin is then mixed with
2 c.c. of each dilution of toxin in a Rosenau syringe and allowed to stand
for one hour. The mixture is then injected into a 250-gram pig, as in
determining the MLD, and the syringe washed with 1 c.c. of salt solution.
If the preliminary test has shown the dose to be between 0.2 and 0.3
c.c. the test should be set up as follows :
TUBE
ANTITOXIN
TOXIN (1 IN 5)
SALT SOLXJTION
1
1.0 c.c. (= 1 unit)
1.0 c.c.
1.0 c.c.
2
1.0 c.c.
1.1 c.c.
0.9 c.c.
3
1.0 c.c.
1.2 c.c.
0.8 c.c.
4
1.0 c.c.
1.3 c.c. .
0.7 c.c.
5
1.0 c.c.
1.4 c.c.
0.6 c.c.
6
1.0 c.c.
1.5 c.c.
0.5 c.c.
If the pigs injected with the last two mixtures die on or before the
fourth day, and those injected with the first four mixtures survive but
possibly develop late paralysis, or if they die after the fifth day, the L+
dose is 1.4 c.c. of 1 in 5 toxin, or 0:28 c.c.
III. DETERMINATION OF THE ANTITOXIC VALUE OF AN UNKNOWN
SERUM
The L+ dose of a toxin having been carefully determined, this toxin
may be used for standardizing an unknown antidiphtheritic serum by an
analogous procedure. In this case the toxin is diluted so that 2 c.c.
contain precisely the L+ dose. Preliminary tests should be made to
determine approximately the unit. Then an accurate test is carried
out as follows :
If the serum is found in the preliminary determination to contain
between 200 and 400 units per c.c. the dilutions are made as follows :
A stock dilution of 1 c.c. of serum plus 19 c.c. of salt solution is prepared
150
LABORATORY COURSE IN SERUM STUDY
and the following series of dilutions accurately made from this in test
tubes :
TUBE
ANTITOXIN
SALT SOLUTION
DILUTION
1
1.0 c.c.
9.0 c.c.
1-200
2
1.0 c.o.
10.0 c.c.
• 1-220
3
1.0 c.c.
11.0 c.c.
1-240
4
1.0 c.c.
12.0 c.c.
1-260
5
1.0 c.c.
13.0 c.c.
1-280
6
1.0 c.c.
14.0 c.c.
1-300
7
1.0 c.c.
15.0 c.c.
1-320
8
1.0 c.c.
16.0 c.c.
1-340
9
1.0 c.c.
17.0 c.c.
1-360
10
1.0 c.c.
18.0 c.c.
1-380
11
1.0 c.c.
19.0 c.c.
1-100
1 c.c. of each dilution is then mixed in a Rosenau syringe with 2 c.c.
of diluted toxin, the mixture allowed to stand one hour at room tempera-
ture, injected subcutaneously into a guinea pig in the manner described
for determining the MLD, the syringe washed with salt solution and the
pigs numbered and carefully observed. Should pigs receiving dilutions
1 to 3 survive for five days, and the pigs receiving the higher dilutions
die in two to five days the serum in question would contain 260 units
per c.c. Guinea pigs which die should be autopsied and the character-
istic lesions found.
To provide a margin of safety in testing commercial sera it is cus-
tomary to consider the smallest unit which serves to protect the pig as a
unit, in this case -^ of 1 c.c., which means that there are 240 units
to the cubic centimeter.
Materials needed for III, Determination of unknown antitoxin :
Antitoxin. ,
Diphtheria toxin whose M. L. Dose and L-f Dose are exactly known.
Precision syringes.
12 guinea pigs of approximately 250 grams weight.
LESSON XXIII
TETANUS TOXIN
TETANOLYSIN AND ANTITETANOLYSIN
MANY bacteria, for instance tetanus bacillus, Staphylococcus,
various vibrios, Bacillus megatherium and Bacillus proteus,
develop, in culture media, substances which are capable of laking
red cells. These substances are relatively thermolabile and
152
LABORATORY COURSE IN SERUM STUDY
deteriorate readily in solution. They are known as haemotoxins
and their injection in suitable doses into animals leads to the pro-
duction of antitoxins whose specific protective action can be
demonstrated in vitro.
These hsemotoxins also have a hsemolytic action in the body,
although they play a smaller role than the other toxic components
(neurotoxin, for instance, in the case of tetanus). Their in vivo
hsemolytic effect is probably greatly hindered by the protective
power which normal serum possesses when in sufficient concen-
tration.
I. DETERMINATION OF MINIMAL LAKING DOSE OF TETANUS TOXIN
Tetanus toxin (either a recently prepared filtrate or a .toxin precipi-
tated with ammonium sulphate and preserved in the dry state and made
up to 1 per cent solution in 0.9 per cent saline) l is tested as follows :
TUBE
TOXIN SOLUTION
5 % RABBIT CELLS
SALINE
1
0.5 c.c.
0.5 c.c.
1.0 c.c.
2
0.2 c.c.
0.5 c.c.
1.3 c.c.
3
0.1 c.c.
0.5 c.c.
1.4 c.c.
4
0.05 c.c.
0.5 c.c.
1.45 c.c.
5
0.0
0.5 c.c.
1.5 c.c.
Incubate one hour at 37° C., and observe haemolysis.
II. TlTRATION OF ANTITETANOLYSIN IN IMMUNE HORSE SERUM
(TETANUS ANTITOXIN)
TUBE
TOXIN
IMMUNE SERUM
SALINE TO MAKE VOLUMES
UP TO 1.5 C.C.
1
2
3
4
5
One minimal laking dose
M
«i
M
It
1-1000 1.0 c.c.
1-1000 0.5 c.c.
1-10,000 1.0 c.c.
1-10,000 0.5 c.c.
1-10,000 0.2 c.c.
i
i
t
i
NORMAL HORSE SERUM
6
7
8
9
it
M
it
No toxin
1-100 1.0 c.c.
1-1000 1.0 c.c.
No serum
1-100 1.0 c.c.
it
tt
ii
it
1 A preliminary trial by the instructor is desirable before the toxin is given
out to the class, as the quantities of toxin given in the table may have to be
modified.
154 LABORATORY COURSE IN SERUM STUDY
After one half hour at 37° C. add 0.5 c.c. of 5 per cent rabbit cells
to all tubes, incubate one hour, and observe haemolysis.
Tube 8 is inserted, of course, to make certain that the toxin does
not lose its haemolytic power during the half hour of preliminary incuba-
tion, and Tube 9 to show that horse serum is not of itself haemolytic.
TETANOSPASMIN
Students should work in groups of three or four in performing
the following experiments. Fresh tetanus toxin is obtained from
a department of health or one of the commercial laboratories with
the lethal dose for guinea pigs of 350 grams worked out. Tetanus
antitoxin can be procured from the same source or in the market.
A. Inject a guinea pig of approximately 350 grams into the thigh of
one hind leg with five lethal doses of the tetanus toxin.
B. A similar guinea pig at the same time is injected with twice the
dose, i.e. ten lethal doses of tetanus toxin, which has been mixed with
one antitoxin unit,1 the mixture having been allowed to stand at room
temperature for twenty minutes.
C. Grind up five lethal doses of the tetanus toxin with half of one
cortex of fresh guinea pig brain, adding salt solution to the amount of
3 or 4 c.c. during the process. This mixture is allowed to stand at
room temperature for two or three hours. It is then centrifugalized
and the supernatant fluid injected into a third pig.
The pigs are kept under close observation for 6 days.
Materials required for each student for the tetanolysin ex-
periments: Tetanus toxin about 4 c.c.
Tetanus antitoxin about 0.1 c.c.
Normal horse serum about 0.1 c.c.
5 per cent rabbit-cell suspension 8.0 c.c.
Materials for each group of 3 or 4 students for the tetanospas-
min experiment :
3 guinea pigs of about 350 gms. each.
20 lethal doses of tetanus toxin of known toxicity.
Tetanus antitoxin, one unit.
J fresh guinea pig brain.
1 The antitoxin unit is established by the United States Hygienic Laboratory
(Bulletin 43) on principles similar to thos«- u-« •<! for diphtheria antitoxin and
represents ten times the amount of antitoxin necessary to completely neutralize
100 minimal lethal doses of a standard toxin injected into a guinea pig of 350
grams.
156
LABORATORY COURSE IN SERUM STUDY
LESSON XXIV
TOXINS OF HIGHER PLANTS AND ANIMALS
MANY of the higher plants and animals produce toxins resem-
bling in every way the toxins of bacterial origin. The property
which is essential for the identification of such substances as toxins
is the possibility of producing antitoxins for them. The other
properties, such as the extraordinary potency and thermolability,
are less essential. Most of them have more than one toxic com-
ponent, like many of the bacterial toxins (for instance, tetanus
toxin). Thus beside the effect on the nervous system and blood
vessels snake poisons are hsemolytic, and ricin, a powerful toxin
derived from the castor bean, is hsemagglutinative.
Human and guinea pig cells are susceptible to laking by
cobra venom in the absence of serum or lecithin. Sheep and ox
cells are highly resistant. The addition of lecithin or of suitable
serum, however, makes the insusceptible cells again susceptible
to laking by the venom.
Kyes at first supposed that the lecithin " activated " the hsemo-
lysin of the cobra venom by an action analogous to that of com-
plement in the case of the specific hsemolysins. Later researches,
especially those of von Dungern and Coca, showed that the venom
contained a lipoid-splitting enzyme or lipase which acted upon the
lecithin of the cell membrane or the lecithin contents of the serum
and liberated split products which possessed haemolytic action.
Set up the following tests :
HjEMOLYTTC EFFECT OF COBRA VENOM
TUBE
RED BLOOD CELLS
GUINEA Pio SERUM OR
LECITHIN EMULSION
1-2000
COBRA VENOM
1-1000
SALT SOLUTION
1
Guinea pig cells
5% 0.5
—
0.1 c.c.
0.9 c.c.
2
Sheep cells
5% 0.5
—
0.1 c.c.
0.9 c.c.
3
Sheep cells
0.5
G. P. 0.1 c.c.
0.1 c.c.
0.8 c.c.
4
Sheep cells
0.5
L. 0.4 c.c.
0.1 c.c.
0.5 c.c.
5
Sheep cells
0.5
G. P. 0.1 c.c.
—
0.9 c.c.
6
Sheep cells
0.5
L. 0.4 c.c.
—
0.6 c.c.
Incubate one hour at 37° C., making observations every 15 minutes,
and note whether the occurrence of haemolysis confirms the statements
made above.
158 LABORATORY COURSE IN SERUM STUDY
HwEMAGGLUTINATIVE EFFECT OF RlCIN
Ricin agglutinates the cells of all species to a greater or lesser
degree. The presence of serum in any considerable quantity
interferes with the reaction. Specific antitoxic sera (which are
difficult to prepare) neutralize in high dilutions not only the
poisonous but also the agglutinative component of ricin.
TUBE
GUINEA Pio CELLS 5%
RICIN 1-1000
SHEEP SERUM
1
0.5 C.C.
0.1 c.c.
2
0.5 c.c.
0.5 c.c.
—
3
0.5 c.c.
1.0 c.c.
—
4
0.5 c.c.
0.1 c.c.
1.0 c.c.
5
0.5 c.c.
0.5 c.c.
1.0 c.c.
6
0.5 c.c.
1.0 c.c.
1.0 c.c.
Incubate one hour.
Make all volumes equal by adding saline.
Observe hsemagglutination.
Each student requires :
5.0 c.c. guinea pig cells 5 %.
0.5 c.c. guinea pig serum.
7.0 c.c. sheep cells.
8.0 c.c. sheep serum.
1.0 c.c. lecithin diluted 1-2000.
1.0 c.c. cobra venom diluted 1-1000.
4.0 c.c. ricin diluted 1-1000.
Salt solution.
LESSON XXV
DETERMINATION OF ANTITRYPTIC ACTIVITY OF BLOOD
SERUM
NORMAL blood contains a trypsin-like ferment. This enzyme
digests protein in neutral or weakly alkaline solution, with the
formation of amino-acids. Under ordinary conditions its action
is inhibited by the presence of substances in serum which have
been shown by Jobling and Peterson to be compounds of unsat-
urated fatty acids.
This antitryptic activity is greatly increased in cachectic
160 LABORATORY COURSE IN SERUM STUDY
individuals and tests for increase in antitrypsin have been pro-
posed as an aid to diagnosis of carcinoma. The condition, how-
ever, is not limited to patients with malignant diseases, but is
present also in pernicious anaemia, in Graves' disease, in ad-
vanced tuberculosis, and in many other infections.
A specific antitrypsin which may be quite different in its nature
from the substance present in normal serum is developed in re-
sponse to injections of trypsin into animals. The precise deter-
mination of the antitryptic activity of the serum in experimental
work is made by adding serum to mixtures of trypsin and some
protein substrate and after incubation comparing the amount of
incoagulable nitrogen in the flasks containing serum with the
amount in control flasks containing trypsin alone.
Simpler methods have been devised for clinical use. One
widely used consists in placing drops of mixtures of trypsin and
serum in varied proportion on the surface of Loeffler's blood serum
plates and determining the action of the trypsin by the formation
of a pit on the surface of the plate underneath the drop of fluid.
A more satisfactory method is that of Fuld and Gross as
follows : Mixtures of serum with varying concentrations of tryp-
sin are allowed to act on an alkaline solution of casein and after
incubation the undigested casein is precipitated by the addition
of acetic acid.
Reagents :
1. Trypsin solution. Dissolve 0.5 gram of trypsin in 50 c.c. of
salt solution containing 0.5 c.c. of normal soda. Make up to 500 c.c.
with salt solution. Samples of commercial trypsin vary greatly in their
activity and this stock solution should be diluted if necessary until the
amount required to digest 2 c.c. of casein solution is approximately
0.5 c.c.
2. Casein solution. Dissolve 1 gram of casein in 100 c.c. of N/10
NaOH by warming and neutralize the solution with N/10 HC1, using
litmus as an indicator. The volume is brought up to 500 c.c. with salt
solution and the solution filtered. It should be sterilized in the Arnold
sterilizer if it is not to be used immediately.
3. Acetic acid solution composed of glacial acetic acid 5 c.c.,
alcohol 45 c.c., water 50 c.c.
162
LABORATORY COURSE IN SERUM STUDY
A. TlTRATION OF TRYPSIN SOLUTION
In each of a series of test tubes place decreasing amounts of trypsin
solution, 1 c.c., 0.7, 0.5, 0.4, 0.3 and 0.2 c.c. Add sufficient salt solution
to bring up the volumes to 2.5 c.c. and set up a control containing 2.5 c.c.
of saline alone. To each tube add 2 c.c. of casein solution, shake, and
place in water bath for half an hour at 37° C. Remove the tubes from
the water bath and add 3 to 4 drops of the acetic acid solution to each.
The tube with the smallest amount of trypsin which shows no precipi-
tate of undigested casern contains the amount of trypsin needed for
the subsequent test.
PRELIMINARY TITRATION OF TRYPSIN
TUBE
TBYPSIN SOLUTION
SALINE
CASEIN SOLUTION
RESULT
1
1.0 c.c.
1.5 c.c.
2.0 c.c.
2
0.7 c.c.
1.8 c.c.
2.0 c.c.
3
0.5 c.c.
2.0 c.c.
2.0 c.c.
4
0.4 c.c.
2.1 c.c.
2.0 c.c.
5
0.3 c.c.
2.2 c.c.
2.0 c.c.
6
0.2 c.c.
2.3 c.c.
2.0 c.c.
7
0.0 c.c.
2.5 c.c.
2.0 c.c.
B. DETERMINATION OF ANTITRYPTIC ACTIVITY OF SERUM
For control a mixture of several normal sera is used and a 2 per cent
solution in normal saline is prepared. A 2 per cent solution of the serum
to be tested, preferably from a case of advanced carcinoma, is used for
the test.
In order to demonstrate the lipoidal nature of normal antitrypsin it
can be removed from serum by chloroform extraction. 4 c.c. of diluted
normal serum should be placed in a test tube, 0.5 c.c. of chloroform added,
the mixture thoroughly shaken and incubated for one hour, at the end
of which time the mixture is centrifugalized and the clear supernatant
fluid pipetted off. The normal serum, the carcinoma serum, and the
normal serum after chloroform extraction should be tested in parallel
as follows :
In each of six tubes place 0.5 c.c. of -the 2 per cent solution of serum.
To the first tube add the dose of trypsin which will completely digest the
casein as determined by previous titration (this is "the unit" of trypsin),
and in the successive tubes increasing amounts of the trypsin so that
Tube 6 contains four times as much trypsin as Tube 1. For example, if
164
LABORATORY COURSE IN SERUM STUDY
in the preliminary test the tube containing 0.5 c.c. of trypsin solution
was completely dissolved this series of tubes should contain 0.5, 0.75, 1.0,
1.25, 1.5 and 2.0 c.c. respectively. Sufficient salt solution is added to
bring the volumes up to 2.0 c.c., and 2.0 c.c. of casein solution added
to each tube. The tubes are incubated for one half hour and at the end
of this time the undigested casein is precipitated by the addition of a
few drops of the acetic acid solution.
In the presence of carcinoma serum from 1J times to twice the
amount of trypsin will be found necessary to completely digest the
casein that is necessary in the presence of normal serum. On the other
hand, in the serum which has been extracted with chloroform the anti-
tryptic activity will be found to have been almost completely removed.
TESTING OF SERUM
TUBE
TRYPSIN
SOLUTION
PATIENT'S
SERUM — 2%
SALINE TO BRING
VOLUMES UP TO 2 c.c.
CASEIN
SOLUTION
RESULT
1
1 unit
0.5 c.c.
it
2 C.C.
2
li units
0.5 c.c.
«
2 c.c.
3
2 units
0.5 c.c.
2 c.c.
4
2| units
0.5 c.c.
2 c.c.
5
3 units
0.5 c.c.
2 c.c.
6
4 units
0.5 c.c.
2 c.c.
7
None
0.5 c.c.
2 c.c.
LESSON XXVI
NATURE OF NORMAL OPSONIN
INTRODUCTORY REMARKS
IN the early days of immunological investigation it was sup-
posed that phagocytosis depended upon properties inherent in
the phagocyting cell. More recent investigations, starting with
those of Metchnikoff, continued by Denys and Leclef and others,
and brought to focus by Wright and his associates, have shown
that in phagocytosis, at least when carried on by the polynuclear
cells of the circulating blood, it is a function of the cooperation
of serum and phagocyte. It has been found that the serum
so acts upon the bacteria that they are rendered more easily
166 LABORATORY COURSE IN SERUM STUDY
taken up by the leucocytes, and that the serum constituent
which carries out such preparatory action is comparable to other
antibodies. The property of the serum which carries out this
preparation of the bacteria for phagocytosis has been spoken of
by Wright as the opsonic power, since he supposed it to depend
upon a specific antibody, which he called opsonin. In immune
sera this phagocytosis-aiding antibody is more heat-resistant
than it is in normal sera, and such a heat-resistant immune body
is spoken of by the Wright school as the immune opsonin and by
Neufeld and his associates as bacteriotropin.
Serum. — Obtain normal guinea pig serum by bleeding two pigs
from the carotid into centrifuge tubes and separating serum.
Leucocytes. — Wash out peritoneum of two guinea pigs injected in-
traperitoneally with 6 c.c. of aleuronat suspension l about 8 to 12 hours
before. Take 2 c.c. of suspension, centrifugalize, wash the sediment
once in saline and suspend in saline until emulsion corresponds in thick-
ness to standard tube prepared by instructor.
Bacteria. — Make suspension of staphylococcus aureus from agar
culture in salt solution until thickness corresponds to standard tube.
Draw in and out of capillary pipette until the bacteria are evenly emulsi-
fied. Allow clumps to settle and pipette off upper layers.
Experiment 1
OPSONIC ACTION OF SERUM
Set up three tubes as follows:
TUBE
GUINEA Pio SERUM
SUSPENSION OP BACTERIA
SUSPENSION OF LEUCOCYTES
1
2
3
Fresh 0.25 c.c.
(Inactivated) 0.25 c.c.
Salt solution 0.25 c.c.
0.1 C.C.
0.1 c.c.
0.1 c.c.
0.2 c.c.
0.2 c.c.
0.2 c.c.
All tubes are incubated one half hour at 37° C. Two smears are
then made of the sediment on slides and stained by Gram's method,
or with Jenner's stain. To obtain material for smears pour out the
supernatant fluid and scrape the leucocytes from the bottom of the tube
with a platinum loop. The bacteria in a hundred consecutive leucocytes
on each slide are then counted.
1 To make aleuronat suspension take
Aleuronat 5
Starch 3
Water or broth 100
Mix cold. Bring to a boil. Tube and sterilize.
For washing out the guinea pig's abdomen use sod. citrate solution in saline.
168 LABORATORY COURSE IN SERUM STUDY
Experiment 2
THE OPSONIN UNITES WITH THE BACTERIA, NOT WITH THE
LEUCOCYTES
In two small centrifuge tubes mixtures are made with the same ma-
terials employed in Experiment 1, as follows :
(1) Bacterial suspension 0.1 c.c.
Fresh guinea pig serum 0.2 c.c.
Salt solution 0.7 c.c.
(2) Leucocyte suspension 0.2 c.c.
Fresh guinea pig serum 0.2 c.c.
Salt solution 0.6 c.c.
These tubes are allowed to stand at 37° C. in water bath or incu-
bator for 30 minutes. Then centrifugalize vigorously and wash sedi-
ment once in salt solution.
To the bacterial sediment of (1) add :
Leucocyte suspension 0.2 c.c.
Salt solution 0.3 c.c.
To the leucocyte sediment of (2) add :
Bacterial suspension 0.1 c.c.
Salt solution 0.4 c.c.
Allow the tubes to stand for 30 minutes at 37° C. Then make
smears and examine as in Experiment 1.
Materials:
Serum, leucocytes, bacterial Capillary pipettes with rubber
emulsion as described. nipples.
Microscope slides. Jenner's stain.
2 Centrifuge tubes. 5 £-inch test tubes.
LESSON XXVII
DETERMINATION OF OPSONIC INDEX (WRIGHT)
PREPARE the following reagents :
1. Bacillary suspension. Make suspension of Staphylococcus
aureus culture in salt solution. Allow to stand in test tube for clumps
to settle out. Transfer upper portion to separate test tube.
2. Leucocyte suspension. Obtain 15 or more drops of blood from
finger tip in centrifuge tube containing about 10 c.c. of citrate solution.
170 LABORATORY COURSE IN SERUM STUDY
(Sodium citrate 1 gram, sodium chloride 0.5 gram, water 100 c.c.)
Centrifugalize and wash once in salt solution. With capillary pipette
remove the upper layer of the sediment which will contain most of the
leucocytes.
3. Serum. Collect about 10 drops of blood in Wright capsule.1
Seal the dry end in the flame and centrifugalize to obtain serum.
Use portion of serum to make control pool, mixing serum from 5
to 10 different individuals.
TEST
Use capillary pipette l with mark about 2 cm. from the tip. Take
up first serum to the mark, then air, then leucocytes to the mark, then
bacteria. Mix the reagents in a watch glass and again draw into the
pipette and seal the tip. Incubate one half hour. Blow out contents
of capillary, mix thoroughly and make smears. Stain with Jenner's
stain 4 minutes, wash and blot.
Each student makes test with his own serum and with a control
pool which does not include his own.
The opsonic index is the ratio between the phagocytic average
of the specimen with the patient's serum and of that with the
normal pool, expressed as a decimal, i.e.
" Normal " specimen — phagocyte average 2.5
Patient's specimen — phagocyte average 3.75
Opsonic index 1.5
If the figures for normal and patient were reversed, the index
would be 0.66.
Materials :
Staphylococcus suspension.
Citrate solution.
Wright capsules and pipettes.
Jenner's stain.
LESSON XXVIII
DIFFERENCE IN HEAT STABILITY BETWEEN NORMAL AND
IMMUNE OPSONINS OR BACTERIOTROPINS
PREPARE materials — bacterial (staphylococcus) emulsion —
leucocytes and serum as for opsonin test by Wright's method.
1 See Lesson XIII, page 88.
172 LABORATORY COURSE IN SERUM STUDY
Experiment 1
HEAT SUSCEPTIBILITY OF NORMAL OPSONIN
Divide the normal serum obtained from own finger into two parts.
Heat one of these in a water bath at 56° C. for 30 minutes.
Now carry out opsonin tests in parallel (1) with unheated serum,
(2) with the heated serum — using same bacterial emulsion and leuco-
cytes in both cases.
Compare results by counting slides.
Experiment 2
HEAT SUSCEPTIBILITY OF IMMUNE OPSONIN
Staphylococcus immune rabbit serum will be given out.
Divide into two parts. Heat one part to 56° C. for 20 minutes in
a water bath.
Carry out parallel tests as above and compare the results.
Materials :
Staphylococcus emulsion.
Glass tubing for Wright capsules and pipettes.
Staphylococcus immune serum.
LESSON XXIX
TITRATION OF NORMAL OPSONIN BY DILUTION METHOD
TITRATE opsonic power of normal guinea pig serum for Staphylo-
coccus aureus by dilution method as follows :
1. Make dilutions of serum 1-5, 1-10, 1-20, 1-100.
2. Set up series of opsonic tests in Wright capillary pipette, using
Staphylococcus suspension, suspension of guinea pig leucocytes and
diluted serum equal parts. Make additional test with undiluted serum,
and also control with leucocytes, bacteria and salt solution.
3. Incubate 30 minutes, make smears and stain with Jenner's stain.
Determine by counts in which dilution there is a definite increase of
phagocytosis as compared with the salt solution control.
Materials : same as for two preceding lessons.
174 LABORATORY COURSE IN SERUM STUDY
LESSON XXX
TITRATION OF IMMUNE OPSONIN BY DILUTION METHOD
INTRODUCTORY REMARKS
THERE are several methods of titrating the opsonic power of
a serum. The most accurate method probably is that in which a
number of progressively increasing dilutions of the serum are
made and the opsonic power of each dilution is tested. In this
way the degree of dilution at which the opsonic power of the serum
disappears, that is, at which there is not more phagocytosis in
the specimen with this serum than there is in a control with salt
solution, is determined. In this way two sera can be compared
as to their opsonic strength. It is the method first used by Klein
in the case of typhoid opsonins, and the one utilized by Jobling
in the standardization of anti-meningococcus serum.
Reagents :
1. Suspension of Staphylococcus aureus from 24-hour agar slant.
2. Suspension of leucocytes obtained from peritoneum of guinea
pig injected with aleuronat.
(The method of preparing these two suspensions has been given in
a previous lesson.)
3. Serum of rabbit immunized against Staphylococcus aureus.
Before beginning the experiment 0.2 c.c. of this serum should be
placed in test tube and heated one half hour at 56° for Series III below.
4. Serum of normal rabbit.
Prepare dilutions of each of the sera, 1-5, 1-10, 1-20 and 1-100.
I
Set up a series of six tubes. In the first put 0.25 c.c. of undiluted
immune serum, in the next four 0.25 c.c. of 1-5, 1-10, 1-20 and 1-100
dilutions respectively, and in the last tube 0.25 c.c. of salt solution for
control.
II
Set up a similar series with normal serum, omitting the fifth and
sixth tubes.
176 LABORATORY COURSE IN SERUM STUDY
III
Set up a third series of three tubes containing immune serum which
has been heated at 56° for half an hour. After heating, dilutions of
1-5, 1-10 and 1-20 should be prepared, and 0.25 of each dilution added
to one of the tubes.
To each of these tubes now add 0.1 c.c. of bacterial suspension
and 0.2 c.c. of washed leucocytes. Incubate for one half hour and pre-
pare smears from the sediment of each tube. Stain the smears with
Jenner's stain and count the number of bacteria contained in 50 leuco-
cytes in each smear, calculating the average number per leucocyte.
Any slide which shows a definite increase above the salt solution should
be regarded as showing positive opsonic power in the corresponding
dilution of serum.
Materials: same as three preceding lessons.
15 £-inch t£st tubes. 5 J-inch test tubes.
LESSON XXXI
PREPARATION OF BACTERIAL VACCINE
THE preparation of bacterial vaccines consists in four steps :
I. PREPARATION OF THE EMULSION
Each student is given two agar slants of Staphylococcus aureus of
24-hour growth. To each tube add about 2 c.c. of sterile salt solution
with a sterile pipette. Remove the bacteria from the surface of the agar
by scraping gently with a sterile platinum loop, being careful not to cut
into the surface of the agar. When an even suspension is obtained this is
transferred to a small sterile bottle or test tube containing glass beads
and thoroughly shaken to break up clumps of the organisms.
II. STANDARDIZATION
The second step in the preparation of vaccine is to determine the
number of organisms contained per c.c.
(a) Hcemocytometer Method
A staining solution is prepared by adding to 20 c.c. of 1 per cent
phenol 1 c.c. of a saturated alcoholic solution of thionin. A small
178 LABORATORY COURSE IN SERUM STUDY
amount of the carefully shaken bacterial suspension is removed to a
watch glass. A 1-100 dilution is prepared in a red cell pipette with the
staining solution as diluent to the 101 mark. After carefully shaking
and after blowing out the portion of the fluid in the capillary end of
the pipette, a small drop is placed in a counting chamber and covered
with a flat cover slip. After allowing 15 minutes for the bacteria to
settle, a count is made, with a No. 5 or a No. 6 lens, of a number of
squares until 200 or more bacteria have been counted. It is best to
take this count from different portions of the ruled surface and from
two separate drops of the mixture. The small squares have an area
of ^rJ-fr of a square mm., the depth of the chamber is 0.1 mm., the dilu-
tion is 1-100. The number of bacteria may be estimated by the fol-
lowing formula :
No. of bacteria counted X 400 X 10 X 100 X 1000 _ Dumber of bac-
Number of squares counted
tend in i c.c.
(6) Wright's Method
A Wright capillary pipette is prepared with a mark about one inch
from the tip. A small puncture is made in the tip of the finger and a
fresh drop of blood obtained. Three units of salt solution are then
drawn up in the pipette, admitting a bubble of air between each two
portions of salt solution. Blood from the finger tip is then drawn up
to the mark, a bubble of air admitted, and bacterial suspension drawn
up to the mark. The mixture is then blown out on a clean slide and
drawn in and out of the pipette several times to insure even mixing of
the blood and bacteria. A drop of this mixture is placed on a second
slide and carefully spread across the slide in the manner of making blood
smears. It is important that the film be thin and even so that the red
cells are not piled in masses in any portion of the film. This film is
stained with Jenner's stain, or by any other simple method, and a differ-
ential count of the number of bacteria and red cells in a number of
fields in different parts of the slide is made. For this a ruled scale to
be inserted in the eyepiece of the microscope is very helpful. A num-
ber of fields are counted, taken at random, until 200 red cells have
been counted. The number of bacteria in the suspension may then
be estimated from the number of bacteria counted, using the following
formula (assuming that the blood of the worker contains 5,000,000 red
cells per cubic mm.) :
Number of bacteria X 5,000,000 X 1000 XT
Number of red cells (200) "= Number °f bactena per C'C'
180 LABORATORY COURSE IN SERUM STUDY
(c) Other Methods
A number of other methods have been devised for standardization
of vaccines.
One method (Hopkins) is to centrifugalize at high speed in a special
tube with graduated tip until the supernatant fluid is clear. The num-
ber of organisms for a number of species in such a closely packed
sediment has been determined, and is as follows :
Staphylococcus aureus 0.01 c.c. equals 10 billion
Streptococcus haemolyticus 0.01 c.c. equals 8 billion
Gonococcus 0.01 c.c. equals 8 billion
Pneumococcus (capsulated) 0.01 c.c. equals 2.5 billion
B. typhosus 0.01 c.c. equals 8 billion
B. coli 0.01 c.c. equals 4 billion
Another method of standardization is by the turbidity of the emul-
sion. Standard tubes of carefully counted suspensions are prepared,
and the vaccine to be standardized is then diluted until the turbidity
is equal to that in the standard tube.
III. STERILIZATION
The most reliable means of sterilization is by means of heat. For
staphylococci and streptococci 59° to 60° C. for half an hour is satis-
factory ; for typhoid bacilli 55° to 56° for an hour is usually used. In
heating, the bacteria should be placed in a sealed tube and the entire
tube immersed in the water bath. After exposure to heat test the steril-
ity of the vaccine by transferring two or three loopfuls of the suspen-
sion to an agar slant.
IV. DILUTION
The stock suspension prepared is as a rule too concentrated to allow
the accurate measurement of the dose desired, which may vary accord-
ing to the organism from 20 to 500 millions or even more. After
the vaccine has been sterilized by heat, sufficient 5 per cent phenol
or an equivalent quantity of other aromatic disinfectant should be
added to bring the content of phenol in the mixture up to 0.5 per cent.
Each student will prepare a suspension from the cultures
given out, standardize this suspension by methods (a) and (ft),
heat at 59° for half an hour, test for sterility, and prepare two
dilutions, one containing 200 million and one containing 1000
million per c.c.
182 LABORATORY COURSE IN SERUM STUDY
Materials :
2 agar slants of staphylococcus aureus.
Sterile saline solution.
Sterile pipette.
Sterile tube or bottle with glass beads.
Haemocytometer.
Thionin solution in 1 per cent phenol.
Glass slides.
Wright or Jenner stain.
Phenol.
LESSON XXXII
ANAPHYLAXIS
INTRODUCTORY REMARKS
BECAUSE of the large number of animals required in this work,
the class had better do these experiments in relatively large
groups.
I. ACTIVE SENSITIZATION
Four guinea pigs of about 200 grams weight are etherized on operat-
ing boards for intravenous injection into external jugular veins.
Guinea pigs (a) and (b) receive 0.25 c.c. horse serum diluted with
1.75 c.c. salt solution.
Guinea pigs (c) and (d) receive 0.25 c.c. sheep serum diluted with
1.75 c.c. salt solution.
This should be done two weeks before the time for Lesson XXXII.
Two weeks after the sensitizing injection the pigs are again injected
intravenously as follows, into opposite jugular vein :
(a) 0.5 c.c. of horse serum.
(6) 0.5 c.c. of sheep serum,
(c) 0.5 c.c. of sheep serum.
The results in (a) and (c) demonstrate anaphylactic shock.
The absence of symptoms in (b) demonstrate specificity.
Guinea pig (d) is given 0.01 c.c. sheep serum subcutaneously four
times, at one hour intervals. Observe carefully symptoms after each
injection. Then give 0.5 c.c. sheep serum intravenously. Symptoms
will be slighter than in (a) and (c). This demonstrates desensitization.
184 LABORATORY COURSE IN SERUM STUDY
Materials needed for each group of students :
4 guinea pigs.
1 c.c. horse serum.
1 c.c. sheep serum.
Syringe.
Scalpel, forceps, scissors, thread, for isolation and ligation of jugu-
lar vein.
Ether.
LESSON XXXIII
ANAPHYLAXIS (Continued)
II. PASSIVE SENSITIZATION
OBTAIN serum of rabbit which has been highly immunized to horse
serum.
Make preliminary titration of precipitin of this serum for horse
serum.
Inject 1.0 c.c. of this serum intravenously into each of three guinea
pigs. After 24 hours inject into the opposite jugular vein as follows :
Guinea pig (a) 0.1 c.c. horse serum
Guinea pig (6) 0.3 c.c. horse serum
Guinea pig (c) 1.0 c.c. horse serum
Observe symptoms.
In case of anaphylactic death, immediately open chest and observe
condition of lungs and also whether heart is still beating. While such
an animal is dying it is important to watch carefully the gradual altera-
1 tion of respirations, the great irregularity and slowing of breathing and
the intense inspiratory efforts just before death.
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