NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
4
Now, it has been proved that, if a considerable quantity of immunising substances
be injected into an animal, this increase of immune body is preceded by a
diminution called by Wright the ' negative phase' 8. This diminution must be
due to a union of the receptors of the immunising substances and the immune
body. The mental picture, therefore, which we have formed of the process is as
follows :
—
First, a union of the total receptors injected with a certain proportion of the
immune body normally present in the plasma ; as a result of this fixation of
immune body a disturbance of the equilibrium between the free amboceptors and
those belonging to the protoplasmic molecule ; then a casting-off of fixed ambo-
ceptors to take the place of those lost ; and finally an over-production of these
bodies. If this hypothesis is correct then it follows that the power of any vaccine
to increase the quantity of immune body in the body fluids depends upon the
number, and avidity for amboceptors, of the receptors contained in an unit quantity
of the vaccine. The greater the number and the greater the avidity of the
receptors, the greater will be the power of the vaccine to fix immune body ; the
greater the power of the vaccine to fix immune body, the more immune body
will be fixed ; the more immune body fixed the more will be reproduced ; the
more immune body reproduced the greater will be the resulting immunity. It
appears to us, then, that the problem of determining the optimum dose of an
anti-typhoid vaccine will only be solved when we are able to estimate that
proportion of free immune body which can be fixed by the receptors of the
vaccine without danger to the organism. The first step towards this end will
evidently be a method of measuring the receptor content of any vaccine and this,
we take it, is the true basis of standardisation of all anti-tyhoid vaccines, no
matter how prepared. Let us for a moment consider in this light the various
methods of standardisation which have been used heretofore.
These methods only apply to the broth and agar vaccines, in which it is
assumed that a constituent of the bacterial protoplasm constitutes the essential
immunising element, and therefore the amount of bacterial protoplasm in the
vaccine will come into consideration in appraising the potency of the vaccine.
In his earliest inoculations Wright 9 used as a dose the amount of growth
which covered a 1/20 to a 1/4 of an agar slope, the virulence of the culture being
fixed. We have in this method at the very beginning an attempt, although a
crude one, to estimate the amount of bacterial protoplasm injected.
At a later date Wright and Leishman 10using broth vaccines determined
the degree of opacity of each sample of vaccine, taking the opacity as a criterion
of the number of undissolved bacteria which were contained in the unit of volume.
At the same time cognisance was taken of the toxicity of the vaccine when
injected into guinea-pigs.
5
The next step in the direction of estimating the amount of bacterial
protoplasm was made by Wright 11when he introduced the blood counting
method of estimating both the living and dead germs in a unit volume of vaccine.
Leishman and his colleagues 12were unable to obtain consistently satisfactory
results by this method. They found that in the case of broth vaccines errors
of from 50 to 100 per cent. in counts of the same film, made by different
observers, were by no means uncommon.
Harrison 13has recently introduced a modification of Wright's method for
counting the bacteria contained in a culture. In this worker's hands it has given
better results than any method he has tried, and it seemed quite possible to keep
the error below ± 10 per cent. in every case.
Finally 14 the amount of bacterial protoplasm in a vaccine has been esti-
mated by weighing the dried bacterial bodies in a measured quantity of vaccine.
This method has not, as far as we know, actually been used in standardising
anti-typhoid vaccine, but it has been suggested as likely to be of great help in
estimating the strength of a vaccine.
All these methods of standardisation aim at an estimation, as accurate as
possible, of the bacterial protoplasm contained in a unit of vaccine. They,
therefore, are quite inapplicable to the standardisation of 'filtrate vaccines.'
Further, if our ultimate aim is a determination of the receptor content of a
vaccine, these methods all fall short of an accurate estimate, inasmuch as they
take no cognisance of those receptors which are free in the vaccine either as a
result of their disassociation from the bacteria or as a result of the dissolution of
the bodies of dead germs. Probably this objection might not hold good in the
case of agar vaccines or in the case of broth vaccines prepared from young cul-
tures, that is, 24 to 48 hours old, if the enumeration or weighment be made before
any receptors have become free in the vaccine fluid. It would, however, with
our present methods of experimentation be impossible to arrive at any estimate
of when this takes place. It appears to us that we must assume that, since
bacteria in cultures are constantly dying, receptors will be constantly being set
free into the fluid medium.
Another objection which can be raised to all these methods of standardisation
is that they take no cognisance of the virulence of the organism, the cultures of
which are used in the preparation of the vaccines. Now, working with the vibrio
choleræ Pfeiffer and Friedberger 15showed that in the case of killed cultures of this
organism the immunising effects were proportional to the virulence of the inocu-
lated strain. They concluded that the virulent and the avirulent organisms differ
in the number or degree of affinity of their haptophore groups ; they demonstrated
this conclusion by experiments in which it was shown that a virulent organism
bound many more times the number of amboceptors than certain avirulent ones.
6
Strong 16 has recently confirmed and extended the work of Pfeiffer and
Friedberger. This observer in a research which had as its object the determi-
nation of the exact relationship between the virulence of an organism and its
power of binding amboceptors and of producing immunity arrived at the follow-
ing conclusions :—
The virulent cholera spirillum possesses a greater number of bacteriolytic
haptophore groups, or these groups are endowed with a greater binding power for
the amboceptors than the avirulent organism ; the number or the avidity of the
bacteriolytic receptors possessed by a bacterium is directly proportional to its
virulence ; the binding power of the free receptors of the organisms for bacterio-
lytic amboceptors ' in vitro' is proportional to the bactericidal immunity produced
in animals by each, which latter is in turn proportional to the virulence of the
organisms from which the receptors were extracted ; the bactericidal immunity
obtained by means of the inoculation with the dead organisms of different virulence
or their extracts (obtained by autolytic digestion) is proportional to the virulence
of the living strains of the bacteria employed.
It is evident, therefore, that the virulence of the organism used in the pre-
paration of a vaccine is of the greatest importance and must be taken into account
in any method of standardisation. Further, it would appear that as virulence is
in direct proportion to the number, or avidity for immune body, of the receptors,
an estimation of these latter in any vaccine will take cognisance of the virulence of
the organism from which it was prepared. If, therefore, we are able to determine
the absolute receptor content of a vaccine, we shall have complied with the
first proposition of the definition of standardisation which we have set before us.
Dr. Wright17 was the first to suggest that anti-typhoid vaccines might
possibly be standardised on this principle. He showed that these vaccines are
capable of exerting an anti-bactericidal effect on a serum normally bactericidal
to typhoid bacteria, and suggested that by progressive dilution of the vaccine this
fact might be made use of to standardise vaccines. He did not, however, persevere
with the working out of this suggestion, and his original methods of experiment
could never have been used for the absolute standardisation of vaccines. To devise
a technique for such an estimation would not be difficult, in fact we have already
done so. But when we came to consider the second proposition of Wright's
definition of standardisation, namely, the adjustment of the dose administered to
the resisting powers of the man or the animal subjected to the inoculation, we
saw that a better and easier method, a method of relative standardisation, which
would answer all practical purposes, could be devised. While reserving for a
future communication the application of this principle to the problem of the
absolute standardisation, we propose now to deal fully with its application to
the problem of the relative standardisation of anti-typhoid vaccines.
7
If the dose of vaccine administered is to be adjusted to the resisting powers
each individual inoculated, this would involve a careful determination of the
amboceptor content of the individual's serum on several occasions before the
inoculation. Such an investigation is of course impossible when a large body of
men is to be inoculated, but an average, such as has been done recently by
Leishman and his collaborateurs,18can always be obtained, by the system devised
by them of pooling equal measures of the different sera and testing the bactericidal
power of this mixture. As we are dealing here only with an average, this
determination may be ignored in practice.
But on further consideration of this proposition it is at once clear that the
adjustment of the dose of any anti-typhoid vaccine to the average resisting
powers of man, that is to say, the determination of the proper proportion of
immunising element to the average amboceptor content of human plasma, which
should be injected so as to give rise to the best immunity, can only be estimated
by direct experiment. Such an experiment has been begun and is still in progress
under the direction of Lt.-Col. Leishman. 19 Groups of men have been inoculated
with different doses of an anti-typhoid vaccine, the constitution and bacterial
protoplasm content of which have been carefully noted. The changes which these
inoculations brought about in the blood have been thoroughly investigated during
a period of four weeks after the first inoculation. While these original investigations
have traced the origin and development of the protective substances and the
immediate effects with different doses of vaccine, the further observations which are
to be prosecuted in India, where the inoculated men have proceeded, will show us
the fate of these substances and, what is of great importance, will give us an idea of
the degree of immunity which has been conferred on each group of men. When
this information has been obtained we shall be in a position to undertake the relative
standardisation of anti-typhoid vaccines in general on the basis of the receptor
content. For it is to be hoped that we shall then be in possession of a standard
vaccine which will be available to all laboratories which manufacture anti-typhoid
vaccine for the purpose of relative standardisation after the manner of the Ehrlich
unit in the case of diphtheria antitoxin. Being provided with such a vaccine it will be
the business of all to standardise against it every vaccine issued. We, therefore,
propose to detail a method which we have elaborated with this end in view. This
method is in reality an application of the principle upon which Wright's suggestion
was based and consists of a comparison of the anti-bactericidal power which is
exerted on a normal serum by the standard vaccine and those vaccines with which it
is to be compared. Given a certain bactericidal serum we estimate what dilution of
the various vaccines when mixed in equal parts with the serum is able to completely
remove the bactericidal power of that serum, in other words, in what dilution of
vaccine the receptors completely neutralise the amboceptor content of the serum.
8
The following is the detailed description of the technique.
The materials required are:—
(1) Wright's diluting pipette. —Any form of pipette constructed in
accordance with the principles laid down by Dr. Wright 20would serve, We have
however, found it convenient to use a pipette in which the stem is graduated in
ten divisions of five cubic millimetres each, the upper marks indicating 450 c. mm.
and 500 c. mm. respectively.
(2) A plentiful supply of sterile watch glasses. —The watch glasses are
sterilised in a covered copper vessel such as is used for the sterilisation of Petri
dishes. When wanted they are removed with sterile forceps and placed in pairs
one covering another.
(3) Series of small test-tubes in racks.—The best tubes are of small size.
They are made from glass tubing of a diameter of 0.8 cm.; they measure 4.5 cm.
long ; a small bulb is blown at the end in order to increase the capacity without
increasing the length.
They are plugged with wool and sterilised in the autoclave before use.
(4) A 24-hour broth culture of bacillus typhosus.—In order to secure a fairly
constant strength of culture a broth culture should be prepared from day to day
by planting into 10 cc. of broth 0.05 cc. of a broth culture of 24 hours' growth.
(5) Normal serum bactericidal for bacillus typhosus. —The serum which we
have used throughout our experiments, and which we recommend, is that of normal
goats. This serum exerts a marked bactericidal action on the typhoid bacillus,
the degree of this action varying in different goats and also from day to day in the
same goat.
The blood can be easily obtained from the jugular vein by means of a sterile
syringe. It will, of course, depend upon the number of vaccines to be tested
how much serum will be required, about 0.5 cc. of serum being used for each
vaccine. The blood is allowed to clot in a sterile conical glass vessel covered with
a lid. We have employed throughout our experiments only serum which had
been in contact with the clot for 24 hours.
(6) Vaccines to be tested. —No antiseptic must be added to these before
testing.
(7) Sterile normal salt solution.
(8) Sterile broth.
(9) A beaker of sterile water, which is kept constantly boiling. The pipette
is easily and quickly sterilised when necessary by washing it out with this boiling
water.
Having detailed our requirements in the way of apparatus, etc., we may now
pass on to a description of the procedure.
9
For each vaccine a series of sterile covered watch glasses numbered from
1 t o 1 0 i s p l a c e d i n f r o n t o f t h e o p e r a t o r . I n t o g l a s s N o . 1 u n d i l u t e d v a c c i n e i s
placed. From this by means of the diluting pipette, which has been previously
sterilised, different dilutions are prepared with normal salt solution, starting with
a two-fold dilution and finishing with a ten-fold dilution.
In making this series of dilutions we have found it convenient, first to
measure out the normal salt solution into the respective watch glasses, and then
to add the requisite quantity of vaccine to each. The following table shows at a
glance for each dilution the most convenient amounts of the two substances:-
No. of watch glass.
Dilution.
Normal salt solution.
Vaccine.
2
•
2 fold.
1 0 0
c m .
100 cm.
3
•
.
3 ,,
100
„
50 „
4
•
•
•
•
4
„
150 „
50„
5
-
•
•
5
„
200 „
50 „
6
.
•
6
„
250 „
50
„
7
•
.
.
.
7
„
300 „
50
„
8
.
•
8
„
350
„
50 „
9
•
.
.
.
9
„
400
„
50 „
lo.
1 0 „
450
„
50
„
These dilutions being made, the small test-tubes, numbered 1 to 10, are
arranged in series in a rack. Into each tube there are measured carefully 100 cm.
of the corresponding dilution of vaccine; thus No. 1 tube receives 100 cm. of
undiluted vaccine, No. 2 tube 100 cm. of a two-fold dilution and so on.
The same process as described above is carried out in the case of each
vaccine to be tested and, also, of course, in the case of the standard vaccine against
which they are being tested. After this has been accomplished, into each tube
there are measured 100 cm. of the fresh serum of the goat, the same serum
being used for all tubes. The contents of the tubes are then well shaken up so
as to procure complete mixture. During this operation the small bulb at the
bottom of the tube prevents the soiling of the upper portion of the tube and of
the cotton wool plug. All preparations are then placed in the hot-air chamber
(37°C.) for one hour, each series of tubes being placed in a separate tin bearing
the designation of the vaccine.
While the tubes are in the incubator we proceed to make ready the dilution of
typhoid culture, a small quantum of which is to be added to each tube in order
to test as to whether or not the total amboceptor content has been removed from
the serum. For this purpose we have found it convenient to add to each tube
50 cm. of a 1,000,000-fold dilution of a 24-hour broth culture. If care be taken
to prepare this culture in the manner we have mentioned above, it is possible
10
to obtain from day to day cultures containing approximately the same number
of living bacteria. In a large number of experiments made to test this point
we have found that such a culture contains on an average 400,000,000 living
bacteria per c.c., when the enumeration was done in the manner described
by Wright 21. We can, therefore, calculate that 50 cm. of a 1,000,000-fold
dilution of such a culture will on an average contain eight bacteria, a number
eminently suitable to indicate whether or not the anti-bactericidal effect of the
dilution of vaccine on the serum has been complete. We, therefore, proceed to
prepare in sterile watch glasses and by means again of Wright's diluting pipette a
1,000,000-fold dilution of the typhoid culture, using sterile broth as the diluent.
The actual amount of this dilution which must be made ready depends of course
on the number of vaccines which are being tested. To each tube there is then
added 50 cm. of the 1,000,000-fold dilution.
The tubes are then thoroughly shaken to insure an even distribution of the
bacteria throughout the contents. They are again placed in the incubator at
37°C. and left there for from 18 to 24 hours. At the end of this time 0.5 cc. of
sterile broth is added to each tube. They are again incubated, this time for
three days. After this interval it is easy to see in which tubes a growth has
taken place and in which the contents are sterile. If there be any doubt on this
point, or if there be any irregularities, samples are planted on to agar and any
resulting growth stained and examined in the usual way.
Before passing on to put forward some examples of this method of compar-
ative standardisation we have to note several points which have to be carefully
attended to in connection with the technique above described.
(1) The vaccine, unless it be a filtrate vaccine, must be thoroughly shaken
up before testing so as to insure an even distribution of the suspended bacteria.
(2) After the serum has been added to the vaccine dilutions the tubes must
be thoroughly shaken before being incubated. If the vaccine is one containing
the bodies of the bacteria, it is well to shake the tubes once or twice during the
period they are in the hot-air chamber.
(3) While adding the 50 cm. of the culture dilution to each tube great care
must be taken to avoid soiling the sides of the tubes with the living bacteria.
The end of the pipette should be carefully introduced, without touching the sides,
to the very bottom of the tube before the 50 cm. are expelled. If this point is
not consistently attended to, the most discordant results will be obtained. The
sides of the tubes become soiled with living bacteria, on which the serum is not
able to exert its bactericidal action and which, therefore, grow after the addi-
tion of the sterile broth.
(4) After the addition of the living bacteria all the tubes must be again
thoroughly shaken before being incubated.
( 5 ) A t l e a s t f o u r t u b e s c o n t a i n i n g o n l y s t e r i l e b r o t h s h o u l d b e i n s e m i n a t e d
with 50 cm. of the 1,000,000 dilution of the typhoid culture, in order to control
the certain presence of living bacteria in the quantum added to each tube. In
our experience, however, we have never found that any of these tubes remained
sterile.
( 6 ) I n o r d e r t o t e s t t h e s t e r i l i t y o f t h e v a c c i n e s , o f t h e s e r u m a n d o f t h e s a l t
solution samples of these should always be planted on agar or in broth.
( 7 ) A f t e r t h e a d d i t i o n o f t h e s t e r i l e b r o t h i t i s n e c e s s a r y t o i n c u b a t e t h e
tubes for three days, as owing to the inhibitory action of the serum growth may
be considerably delayed.
As an example of the results which are to be obtained by this method, we
have now to detail comparative observations made with two vaccines, one broth
and one agar, and the sera of three different goats. The vaccines were prepared
as follows
: —
Vaccine I was a broth culture of bacillus typhosus which had been incubated
at 37°C. for four weeks. It was killed by heating in a water bath at 6o°C. for
half an hour.
Vaccine II was prepared by emulsifying agar cultures, 48 hours old, with
normal salt solution. No attempt was made to measure the amount of the salt
solution used. The bacteria were killed by heating the emulsion at 60°C. for half
an hour.
These two vaccines were tested side by side, after the manner already
described, against the sera of three different goats.
The following were the results obtained:—
4th May 1905. Experiment I. Serum of goat IV.
Dilution of vaccine.
Vaccine I.
Vaccine II.
Undiluted
Growth. Growth.
2-fold dilution
.
.
, ,
, ,
3 ,, ,,
.
.
.
Sterile
, ,
.
4 ,,
,,
.
, ,
, ,
5 ,, 7)
a
.
.
, ,
, ,
6 ,,
,,
.
.
, ,
Sterile
7 ,,
.,
.
.
.
, ,
, ,
8
,,.
.
.
, ,
,,
9 ,,
)7
.
, ,
, ,
1 0
, ,
, ,
.
, ,
, ,
C 2
It will be seen from this table that vaccine I in a two-fold dilution completely
removed the bactericidal action of the serum, while in a three-fold dilution it failed
to do so. We may, therefore, for purposes of calculation hereafter take it that
" neutralisation " between receptor of vaccine and amboceptor of serum would be
obtained when a dilution of 2.5 of vaccine was added in equal parts to the serum.
On the other hand, the neutralisation point in the case of vaccine II and the
serum would be obtained in a 5.5-fold dilution. Thus we see that, as regards
receptor content, vaccine I stands in relation to vaccine II as 2.5 to 5.5, or as
1: 2.2. In other words, vaccine 11 is 21 times 'stronger' in immunising elements
than vaccine I.
12th May 1905.
Experiment II.
Serum of goat V.
Dilution of vaccine.
V a c c i n e I .
Vaccine II.
Undiluted
.
, ,
.
Growth.
Growth.
2-fold dilution .
.
.
Sterile.
,,
3 ,,
,,
. .
,,
4 ,,
,,
.
.
.
,,
Sterile.
5 ,, ,,
•
,, ,,
6 „
.,,
.
.
, ,
,,
,,
7 ,,
,,.
.
, ,
,, ,,
8 „
,, .
•
•
,, ,,
9 ,,
,, •
•
•
,, ,,
10
„
,, .
.,
.
,, ,,
In this experiment the serum was evidently richer in amboceptor
content than was the case in the previous observation. Thus, the neutra
-lisation point in the case of vaccine I would be obtained in 1.5-fold dilution
.and with vaccine II in a 3.5-fold dilution. Thus, the relation of their receptor
contents would be as r5 to 3.5, or as 1 : 2.3. In other words, vaccine II