NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
To face pate 7
CHART No. 2
Chart showing the effect of
e q u i - m o l e c u l a r s o l u t i o n s o f d i f f e r e n t
s a l
t
s o n l y s i s b y a s p e c i f i c s e r u m .
To face page 12
CHART No. 3
Showing the variations in the lysin content of the serum of a normal sheep for the B os
of Horse, Man and Rabbit from April 4th to July 1st 1910.
13'
Horse...
•••
..
...
•••
•••
•••
•••
1.75
Rabbit
...
...
...
...
1 •83
Guinea-pig
•••
...
•••
•••
•••
•••
•••
2.41
•••
•••
...
•••
•••
•••
•••
• • 1.
• • .
2 .4 8
MMan MMM
(cf. list on p. 5.)
Specific gravity, coagulation by heat and hypotonic lysis are, however, all
concerned with the physical conformation of RBCs, whereas serum lysis is a
chemical reaction ; and there is no obvious indication that there should be any
relation between physical characters such as these and chemical composition.
To return to the action of normal serums on washed RBCs : of the eight
tested (serum from 20 individuals of each species), dog serum was unquestionably
th.e most potent, horse easily last and guinea-pig last but one.
A peculiar feature of these experiments was the appearance with considerable
frequency of natural deviation of complement, a phenomenon which we believe has
not been recorded before.
A series of tubes, each containing O •o5 cm. RBCs and altogether 2 cc. of
fluid, with serum in percentages descending from 100 to 2 . 5 would show " partial "
and " not complete" lysis in the tubes containing from iOo per cent. to Go per cent.
serum, " complete" lysis from 6o per cent.-35 per cent. and lessening lysis below
35 per cent. For example
Proportion of serum
to RBCs.
Percentage of
serum
in tubes.
Man serum versus she ep
RBCs.
Horse serum versus rabbit
RBCs.
Serum.
RBCs.
Lysis.
39
33
29
25
21
17
13
9
8
7
6
5
4
3
2
1
to
to
to
to
to
to
to
to
to
to
to
to
to
to
to
to
1
I
I
1
I
I
I
I
I
1
I
I
I
I
I
I
1 0 0
82
72
62
52
42
32
22
20
17
15
12
10
7'5
5'0
2.5
Slight.
Partial.
Not complete.
......
Not complete.
••• •••
Partial.
••• .••
••• •••
Partial.
......
Slight.
Trace.
Nil.
Nil.
Trace.
Slight.
Partial.
Partial. +
Partial.
...
Slight.
Slight.
......
Trace.
Nil.
This deviation was observed with the following :-
Man serum versus sheep, dog, hen, horse RBCs.
Rabbit „
„ hen RBCs.
Horse „
„
rabbit RBCs.
and in some others which were not recorded.
The serums which showed deviation were very variable in their lysin content ;
for instance, man serum 7 . 5 per cent. (3 serum to I RBCs) would sometimes
lyse sheep RBCs completely, but occasionally 100 per cent. (39 serum to
RBCs) would give no lysis at all. This variation has already given rise to
trouble in connection with the Wassermann reaction for syphilis. If complete
lysis is not obtained by 100 per cent. serum, or 39 parts serum to i part RBCs,
it may be assumed that an infinite quantity of that serum would not lyse that
number of RBCs completely. This we put to experimental proof in several
instances.
TABLE No. 4.
14
Man serum.
Rabbit serum.
Guinea pig serum.
Goat serum.
Sheep serum.
Dog serum,
Hen serum.
Horse serum.
Max. Min.
Av.
Max. Min.
Av.
Max. Min.
Av.
Max. Min.
Av.
Max. Min.
Av.
Max. Min.
Av.
Max,
Min.
Av.
Max. Min.
Av.
Man RBCs
...
∞
62
?
∞
52
?
32
12
20
52
2
25
∞
2'5
25
∞
32
50
72
10
40
∞
∞
—
∞
—
∞
Rabbit RBCs
...
...
. 32
7.5
20
12
∞
16
SO
22
7 . 5
1 5
17
30
20
7 . 5
22
2 . 5
12
Guinea-pig RBCs
...
...
72
5
20
72
4 0
30
35
—
∞
15
2 . 5
1 0
∞
5
—
10
∞
7 . 5
12
7 . 5
3.3
7'5
3 . 3
5
∞
32
?
∞
—
∞
∞
Goat RBCs
...
...
72
8
7.5
25
82
∞
13
∞
∞
to
∞
42
∞
∞
—
Sheep RBCs
7'5
?
8
72
∞
12
?
—
∞
5
∞ 100
1 0
6o
∞
—
-
∞
Dog RBCs
...
. . .
∞ ∞
10
?
∞
∞
—
16
∞
∞
—
∞
∞
—
-
17
33
7o
∞
—
∞
Hen RBCs
10
3.3
?
∞
∞
12
8
?
?
∞
∞
?
62
8
30
62
16
52
35
2.5
12
8
—
∞
Horse RBCs
...
...
∞
?
—
∞
62
10
35
100
75
12
2'5
1 0
∞
20
So
Explanation.—The figures express the percentages of serum required to lyse completely 0 .o5 cc. RBCs in a total volume of 2 cc. fluid.
" Max. " means the highest percentage required for complete lysis.
"Min." means the lowest
do.
do.
Av. " means the average
do.
do.
8 means that over 100 per cent., or an infinite quantity, is required for complete lysis.
in the average column means that the potency of the serum is very variable and that no average between infinity and the minimum can be given. When a minimum is given and the maximum is
infinity and infinity is entered in the average column, it means that in the majority of cases the quantity required is infinite.
17
so I cc. fresh B serum dissolved 0 .4 cc. sheep RBCs added at once, completely
in 6', but, when added in two lots, the first 0.2 cc. was completely lysed in 4', while
the second 0.2 cc. took 43'.
Similarly 0.6 cc. added altogether was completely lysed in 17', but, when
added in three lots, the third was not completely dissolved in 3 hours. So sheep
RBCs evidently absorbed, or put out of action, in this serum much more lysin
than was required for their complete lysis.
(II) It is possible for RBCs to fix at the same time amboceptors derived
from different sources, i.e., amboceptors are not mutually exclusive and RBCs
can be successively sensitized by more than one. To demonstrate this we used
the serums of three animals which had all been immunised against sheep RBCs
and which differed in potency at the time of the experiment; Goat P being the
weakest, Pony intermediate and Goat B the strongest. With fresh goat serum as
complement the highest degree of lysis was given by the RBCs which had been
subjected successively to all three serums, and the least by those which had been
in one serum only. Removal of amboceptor from each of the three serums was
demonstrated by a parallel experiment. In this case the amboceptors were all
artificial ones, but their formation having been induced by the injection of sheep
RBCs they must have been similar in nature.
When the RBCs of a normal goat were sensitized in Goat B isolytic serum,
0.1 cc. guinea-pig complement was required for complete lysis; when sensitized
in normal rabbit serum 0.075 cc. complement was required. When sensitized in
both serums separately only 0 .056 cc. complement was required, and it made no
difference whether the RBCs were put first into the goat and then into the rabbit
serum or vice versâ; the two sensitizers together gave more lysis than either
separately.
Sensitization in mixtures of varying proportions of Goat B isolysin and
normal rabbit amboceptor made it clear that goat RBCs did not absorb one in
preference to the other, but the conditions which govern the relative absorption of
the two are evidently complex and though it be known that
—
20 amboceptor A =
complete lysis.
and that 10
"
B =
" "
it cannot be deduced with certainty that 15 A + 2 . 5 B =
" "
or that 5 A + 7 .5 B
= ""
2 2
(3) Possessing three animals, which had been immunised against sheep R B C s
by repeated injections, experiments were made to determine the relative values of
various complements with these three amboceptors. The animals immunised were
Goat B, Goat K and a country bred pony. None of these three serums had any
lytic power against sheep RBCs originally.
The three serums at the time of the experiment differed in their individual
amboceptoral activities, Goat B being the most potent, Goat K next and the
pony last.
Equal quantities of washed sheep RBCs were sensitized in equal parts of
these three heated serums, respectively, and after washing were put up with serums
from a normal horse, sheep and goat as complements. Nine series were thus
obtained for comparison, namely—
Sheep RBCs, sensitized Goat B, with goat, sheep and horse complement .
,, G o a t K
, ,
,,
, , P o n y
, ,
, ,
, ,
Whichever the sensitizer, goat serum proved the best complement by far; sheep
serum complemented Goat B sensitizer much better than horse serum, Goat K
sensitizer slightly better than horse serum and pony sensitizer a trifle less than
horse serum. Horse serum therefore was far inferior to goat and slightly inferior
to sheep complement when goat sensitizer was used, but when pony sensitizer was
used horse serum showed itself to be a slightly better complement than sheep,
though inferior to goat. It is generally held that complement obtained from the
species which provides an immune serum is more potent than heterogthous comple-
ment and these experiments favour the view to a certain extent. The sheep being
more nearly allied to the goat than the horse would, other things being equal, be
expected to complement goat sensitizer better than the horse, and this proved to be
the case. Goat serum, however, complemented pony sensitizer much more strongly
than horse serum, and sheep serum was only a trifle less potent than horse serum.
Sheep serum moreover was a trifle better with pony sensitizer than it was with
Goat K sensitizer. The view that homogenous complements are more potent than
heterogenous complements is in need, therefore, of some qualification; the amount
of lysis resulting depends more on the quantity or quality of complement present
than on its source. These experiments show that goat serum contains complement
either in greater quantity or more potent than sheep serum, and that sheep serum
is better than horse serum.
It would be of interest to see if the serum of a sheep immunised against
goat RBCs would work better with sheep than with goat complement; if it does
not, the superiority of goat complement will be assured. Just at present we have
not such an animal prepared.
The following tables show the quantities of complement required in each
case:—
TABLE No. 7.
Quantities of complement in cc. required for "complete" lysis. Each tube
contained 2 cc. of liquid.
-
Goat
complement.
Sheep
complement.
Horse
complement.
Unsensitized sheep RBCs
...
...
∞
∞
∞
Sensitized in Goat B serum
...
...
< 0·0 25
0·2
> 1·' 5
Sensitized in Goat K serum
...
0
·
2
> 1·5
> 1·. 5
Sensitized in pony serum ...
„.
„.
0
·
4
> 1· 5
> 1 · 5
23
TABLE No. 8.
Quantities of complement required for " partial " lysis, 3o to 40 per cent.
Goat
complement.
Sheep
complement.
Horse
complement.
Sensitized in goat B serum
...
...
< 0.025
0.062
0.15
Sensitized in goat K serum
. . .
...
0.1
0 . 4
I.0
Sensitized in pony serum ...
...
...
0.1
0.3
0.2
Roughly the relative strengths of these three complements may be expressed
,
as under :-
Goat
. . .
Sheep...
Horse
...
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
...
...
. . .
. . .
. . .
. . .
. . .
...
. . .
...
. . .
. . .
. . .
...
...
...
1 5
23
1 5
Trials were next made with five heterogenous complements, namely, guinea-
pig, man, rabbit, dog and hen on sheep RBCs sensitized in each of the above-
mentioned immune serums. Control series of each of the complementing serums
with unsensitized sheep RBCs were put up at the same time.
The subjoined tables show the results arrived at :—
TABLE No. 9.
Quantities of complement in cc. required for " complete" lysis. Each tube
contained 2 cc. of liquid.
Guinea-pig
complement.
Man
complement.
Rabbit
complement.
Dog
complement.
Hen
complement.
Unsensitized sheep RBCs
...
...
1.0
1.0
0.75
0.25
0.75
Sensitized in goat B serum ...
...
<0.025
0.15
0.15
0.075
0.50
Sensitized in goat K serum ...
...
<0.025
0. 35
0.25
0.15
0.50
Sensitized in pony serum
...
...
0.1
o.15
0.75
0.15
0.75
TABLE No. 10.
Quantities of complement required for "partial" lysis, 3o to 40 per cent.
Guinea-pig
complement.
Man
complement.
Rabbit
complement.
Dog
complement.
Hen
complement.
Control. Unsensitized sheep RBCs
o.25
0.075
o.25
0 . 1
0.35
Sensitized in goat B serum ...
...
< 0.025
< O.025
0.05
< 0 .025
0.15
Sensitized in goat K serum ...
...
<0.025
< 0.025
0.075
< 0.025
0.15
Sensitized in pony serum
...
...
< 0.025
< 0.025
0.025
< 0.025
0 . 1
24
Roughly, the relative strengths of these five complements may be expressed
as under :—
Gu i n ea-pig
. . .
...
. . .
...
. . .
. . .
...
. . . .
1 0 0
Dog
...
. . .
. . .
. . .
. . .
. . .
. . .
...
. . .
5o
Man
...
. . .
...
. . .
. . .
. . .
. . .
. . .
...
. . .
31
Rabbit
...
. . .
...
. . .
. . .
...
. . .
. . .
. . .
. . .
16
Hen
...
. . .
. . .
...
. . .
. . .
...
. . .
10
but the differences are really more marked than this. An accurate cal-
culation could not be made, as in the series put up no smaller quantities of
complement were used than 0 . 025 cc. and this gave "complete" lysis with the
guinea-pig and "partial" lysis with guinea-pig, man and dog complements. No
comparison can be made between the strengths of this series of complements and
those of the goat, sheep and horse, since the experiments were performed at different
times and different degrees of sensitization must have existed, and so another
experiment was instituted for this purpose in which all the eight complements were
used with sheep RBCs sensitized in goat K serum.
The results are shown in the tables subjoined:—
TABLE No. 11.
Quantities of complement in cc. required for "complete" lysis. Each tube
contained 2 cc. of liquid.
G u i n e a - p i g c o m p l e m e n t .
R a b b i t c o m p l e m e n t .
M a n c o m p l e m e n t .
D o g c o m p l m e n n t .
h e n c o m p l e m e n t .
G o a t c o m p l e m e n t .
S h e e p c o m p l e m e n t .
H o r s e c o m p l e m e n t .
Unsensitized sheep RBCs ...
...
... > 0 . 75 > 075
0 . 5
0.15
0.75
∞
∞
∞
Sensitized in goat K serum ...
...
...
0 . 0175 0 .4
0.25
0 . I5
0 . 4 > 0/5 > 0'75 > 075
TABLE No. I2.
Quantities of complement required for "Partial" lysis, 30 to 40 Per cent.
G u n i n a e - p i g c o m p l e m e n t .
R a b b i t c o m p l e m e n t .
M a n c o m p l e m e n t .
D o g c o m p l e m e n t .
H e n c o m p l e m e n t .
G o a t c o m p l e m e n t .
S h e e p c o m p l e m e n t .
H o r s e c o m p l e m e n t .
Unsensitized sheep RBCs
...
... > 0 . 75
0.75
0 . 1
0.075
0'3
∞
∞
∞
Sensitized goat K serum
...
...
...
0 . 0 1 7 5 0.15
0.075
0'0375
0.275
0 '45
075 > 075
Expressing roughly the relative values of the eight complements as before,
we get
Guinea-pi
Dog
...
Man
...
Rabbit
...
Hen
...
Goat
...
Sheep. . .
Horse
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
, ...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
1 0 0
18
II
6
5
3
2
< 2
It will be noticed that the order of the first five is the same as before, though
the values differ, and that goat, sheep and horse also preserve their order but
appear to be greatly inferior in value as complements than the rest. All the
serums used except goat, sheep and horse have some natural lytic power against
unsensitized sheep RBCs and there is no means of telling how much of their
complement is used up for the natural amboceptor and how much is available for
the artificial immune body. Experiments such as this seem to point to the view
25
that the complement normally present in a serum is able to give more lysis with
RBCs sensitized in a foreign immune body than with the unsensitized RBCs. One
would conclude, therefore, that amboceptor is deficient in a normal serum and that
there is always sufficient complement present for any one amboceptor at least. If
another amboceptor be present there is still some complement to combine with
it after the first has been exhausted. When a serum naturally lyses. RBCs it
seems likely to lead to fallacies to compare its complementing power with that of
a serum which does not naturally lyse those RBCs. It does not seem justifiable,
therefore, to conclude from the above table that goat, sheep and horse are actually
so much poorer in complement for sheep RBCs sensitized in goat K antisheep
serum as might at first sight appear, but, all things considered, it is certainly prob-
able. One thing at least stands out without doubt, and that is, that guinea-pig
serum, which normally has but little lytic power against sheep RBCs and may on
that account be rightly compared, is vastly superior in complement content to goat,
sheep or horse.
6. The Complementing Power of Homogenous Serums.
It appears that except in the case of the guinea-pig the fresh serum of verte-
brates can exert little or no complementing power when in contact with RBCs
of the same species which have been sensitized in heterogenous serums. In the
table given (No. 13) not all the serums employed were able to lyse the RBCs
sensitized in them in the fresh state, through deficiency of either amboceptor or
complement.
In the case of lysis by guinea-pig complement natural deviation* was several
times observed, i.e., a higher degree of lysis occurred in the middle tubes of
the series than at either end.
TABLE No. 13.
Complementing power of homogenous serums.
RB Cs sensitized in heterogenous serums and then washed.
Animals furnishing the RBCs
and complement.
Animals furnishing the serum used for sensitization.
Guinea-
pig.
Rabbit. Man.
Goat.
Sheep. Horse. Dog.
Hen.
Cat.
Guinea-pig
..
...
S
S
P
S
P
O
—
Rabbit
,..
...
...
O
O-P
O - S
O
O
O-S
O
—
Man...
...
...
O
O
O
—
O
O - T
O
—
Goat...
...
...
...
O
O-S
O
O
—
O
—
Sheep...
...
...
...
O
O-S
O
—
O
O
O
—
H orse
...
...
...
O
O
O
O
—
—
—
O
Dog...
...
..
...
O
O
—
O
—
O
—
—
Hen
O
O
O
O
—
O
O
—
Cat...
„.
."
.-
—
—
—
–
–
–
– —
* Vide p. 13.
D
26
In this and the following tables—
O
T
S
P
NC
C
O—T
O—S
O—P
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
0 . .
...
...
...
...
...
...
. . .
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
. . .
. ..
...
...
...
. . .
. . .
...
...
. . .
...
...
...
...
...
...
...
...
. . .
=
=
=
=
=
=
Nil
lysis.
Trace
,,
Slight
,,
Partial „
Nearly complete lysis.
Complete lysis.
Mean that with complement
derived from different indi-
viduals varying results occurred.
Each tube contained a total quantity of 2 cc. of fluid, including 0.05 cc. sensitized, washed RBCs.
In each series varying quantities of complement were added. A control series was put up on each
occasion containing the complementing serum and unsensitized RBCs, and the complementing power
, was judged by the difference between the controls and the series under observation.
TABLE No. 14.
Complementing power of homogenous serums when the sensitizing serum is
present in the hæmolytic mixture.
Animals furnishing the RBCs
and complement.
Animals furnishing the sensitizing serum.
Guinea- Rabbit.
pig.
Man.
Goat.
Sheep.
Horse.
Dog.
Hen.
Guinea-pig
...
...
...
...
...
S
NC
C
P
P
NC
O
Rabbit ...
...
...
...
...
O
C
O
O
O
T
NC
Man...
...
...
...
...
T
T
O
O
O
T
O
Goat...
...
...
...
...
O
T
T
O
—
T
O
Sheep...
...
...
...
...
O
O–S
O
O
O
O
O
Horse ...
...
...
..
O
O
O
P
O
P
O
Dog...
.
..
...
...
O
O
O
O
O
O
O
Hen...
...
...
...
...
O
O
O
O
O
O
O
Comparing table No. 14, which shows the results of the presence of a heated
sensitizing serum in the h æ molytic mixture, with table No. 13, which shows the
effects of adding sensitized and washed RBCs to the complementing mixture,
there is seen to be no very great difference. The experiments are troublesome
and lengthy ones to carry out and cannot be frequently multiplied on that account.
The collection of these data has spread over a period of two years. The opportu-
nities for sensitization were about equal in the two series, the proportion of heated
serum to RBCs having been about 10 to 1. More complement, however, was
available for the sensitized RBCs in table No. 13. Taking it all round, and
considering that stronger lysis occurred in table No. 14 than in table No. 13 and
that the strengths of the serums used must have differed, it may be concluded that
slightly better results were obtained when the sensitizing serum was present than
in its absence.
To face page 30
CHART No. 4
To illustrate the deviation of complement occurring during the early period of incubation
which in course of time becomes masked. In 7 minutes 0 . 075 amboceptor permits complete
lysis, while 0. 2 is partial. 0 . 2 amboceptor is not complete till 19 minutes.
The quantity of complement is fixed throughout.
0. {Reg: No. 1261
911 { Copies — 3 + 208
To face page 33
CHART No.7
Showing the effect of time of incubation on the degree of lysis obtained when the complement
is in high dilution.
S.O./1911{Reg: No. 1264
copies—3 +208
To face page 33
CHART No. 6
Showing the effect on lysis of dilution four
different concentrations of complement
1911 Reg : No. 1283
Copies — 3 + 208
To face page 33
CHART NO. 5
Showing the effect on lysis of the dilution of the complement.
the actual quantity remaining constant.
Time of incubation 1½ hours
S . O . { R e g : N o . 1 2 6 2
1 9 1 1 { c o p i e s — 3 + 2 0 8
To face page 34
CHART No. 8
Showing that increasing quantities of complement are required to
produce lysis as the percentage of heated serum is increased.
S. 0.
1911 Reg: No. 1265
Copies —
3 +208
To face page 34
CHART No. 9
Shows that 0.. 0 5 c o n c e n t r a t i o n o f c o m p l e m e n t e f f e c t e d c o m p l e t e l y s i s
when 0·. .
8 5 % N a C I . w a s t h e d i l u e n t . W h e n e i t h e r 4 0 % o r 7 0 % o f
sheep serum which had been heated to 55° C. for ½ hourw a s
present complete lysis was not effected with the highest concentration
of complement used; the points are outside the limits of the chart.
When 40% sheep serum which had been heated to 55° C. for 24
hours was present complete lysis was effected by 0·. 1 c o n c e n t r a t i o n
of complement; 70 % serum increased this to 0·12.
0./1911 Reg: No. 1266
copies— 3 ÷ 208
To face page 38
CHART No. 10
Showing the increase in the lysin content of the serum of rabbit 101
as a result of 6 subcutaneous inoculations of 1 cc each of goat R B Cs.
C = Complete lysis
To face page. 38
CHART No. 11
Showing the fall in the percentage of
p o l y m o r p h o n u c l e a r l e u c o c y t e s a s
t h e
immediate result of the intravenous
injection of goat R B Cs. i n t o s h e e p R .
S. 0. . / 1911 Reg : No. 1268
Copies— 3 + 208
Subcutaneous
inoculations
Intravenous
injection
To face page 38
CHART NO. 12
Showing the changes in the amount of lysin in the serum of sheep R as a result of 6 sub.
cutaneous inoculations of 7cc each of goat R B Cs.
O
e
a
ti
2
7
CJ
d
o = 0 lysis
T = Trace
P = Partial
N C = Not complete
C = Complete
S.0.
1911
Rag: No. 126 9
.Copies — 3 + 208
BULLETIN
OF THE
PASTEUR INSTITUTE
OF
SOUTHERN INDIA, COONOOR.
No. 3.
1910.
PUBLISHED UNDER THE AUTHORITY OF THE CENTRAL
COMMITTEE OF THE ASSOCIATION.
MADRAS:
PRINTED BY THE SUPERINTENDENT, GOVERNMENT PRESS,
1911.
REPORT
OF THE
Imperial Institute of Veterinary Research,
Muktesar
For the year ending 31st March 1925
CALCUTTA : GOVERNMENT OF INDIA
CENTRAL PUBLICATION BRANCH
1926
Price Rs. 1-14 or 3s. 3d.
REPORT
OF THE
Imperial Institute of Veterinary Research,
Muktesar
For the year ending 31st March 1925
CALCUTTA : GOVERNMENT OF INDIA
CENTRAL PUBLICATION BRANCH
1926