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4 5 6

ON THE DIFFERENCE BETWEEN SERUM AND BLOOD SOLUTIONS, THE

CONDITION OF THE TEST CULTURE AND THE SIGNIFICANCE

OF BACTERIUM COLl INFECTION IN RELATION TO

T7PH0ID DIAGNOSIS.

WYATT JOHNSTON, M.D., AND D. D. McTAGGART, M.D.,

(From the Laboratories of the Board of Health of the Province of Quebec- and the

Montreal General Hospital.

Reprinted from the Montreal Medical Journal, March, 1897.

\,ti .

^'' '■

(

ON THE DIFFERENCE BETWEEN SERUM AND BLOOD
SOLUTIONS, THE CONDITION OF THE TEST CUL-
TURE AND THE SIGNIFICANCE OF BACTERIUM
COLI INFECTION ' IN RELATION TO
TYPHOID DIAGNOSIS.

Wyatt Johnston, M.D., and D. D. MacTaggabt, M.D.

Montreal.
(From the Laboratories of the Board of Health of the Province of Quebec and the

Montreal General Hospital.) ,

We wish to report some details concerning technique which we find
necessary in order to insure successful results in Serum Diagnosis
by the dried blood method, with which we have now tested over
500 bloods.

We mention the facts only in so far as they have a direct practical
bearing on diagnostic work.

Our results already published were as follows :

1. Out of 129 cases, which we had good reason to regard as true
typhoid, 'if we exclude a few cases where the first samples were
taken at a very early stage and no re-examinations could be obtained,
and also a few cases first examined late in convalescence, we have
met with but one apparently genuine case of severe typhoid, which,
when re-examined under satisfactory conditions, did not give a decis-
ive reaction by the dry blood method and this one also gave no reac-
tion by the serum method. Occasionally the first appearance of the
reactioii is delayed leyond the end af the first week.

2. We have never met with a well marked reaction under conditions
where there were not strong reasons for V»elieving it to be due to
typhoid.

3. In a few cases wherj the result of the blood examination
remained in doubt the mild type of the fever made an accurate
clinical diagnosis impossible. In such cases, we believe bacteriologi-
cal examination to be the most exact method of procedure.

4. We have not yet met with a case of typhoid where a decisive
reaction was obtainable by the serum method and not by the dried
blood method.

5. We found that pseudo reactions may be avoided by attention to
the character of the culture media. We have found that by using
an attenuated or quiescent stock culture grown at room tempera-

^wnBWiji

ture, and transplanted at intervals of about one month, a suitable
degree of sensitiveness was obtained. From such stock cultures a 24
hour bouillon at 37°C., with a moderately diluted blood solution, or
serum would give prompt and decisive reaction within a few minutes
in the case of typhoid patients, while concentrated solutions of non-
typhoid blood or serum were found to give no reaction, even at the
end of 24 or 48 hours, hence estimation of the amount of dilution is
not necessary for ordinary diagnostic work. (See circular Board of
Health, Prov. of Quebec, Jan. 7th, 1897.)

The reaction, although specific in degree, is now generally con-
sidered to be quantitative, and small amounts of the agglutinative
substances are admitted to be present in varying amounts in non-
typhoid bloods. The specific substances are, however, a hundredfold
more abundant in typhoid blood.

With virulent cultures the presence of agglutinative substances
in non-typhoid bloods may lead to pseudo reactions occurring which
can usually be excluded by estimating quantitatively the intensity of
the reaction. These pseudo reactions we have found to be charac-
terized by a rapid clumping, without the corresponding WSt of motion
so characteristic of the true reaction. If watched for some hours
these clumps tend to break up.

Quantitative estimation is now generally done by diluting the
typhoid serum, but may also be doue by modifying the virulence of
the culture.

The degree of dilution which can be employed with a given blood
solution or serum, while still producing a decided reaction, will depend
entirely on the activity (virulence) of the culture employed. This
factor has been too much left out of the reckoning in much of the
work already published, and it probably affords a natural explanation
of the widely different results obtained by competent observers.

Cultures which are made active and virulent by frequent (daily)
transplantation and growth at body temperature, are much more sen-
sative to the agglutinative substance than cultures which have become
quiescent and attenuated by infrequent (monthly) transplantation
and growth at room temperature.

This is apparently at variance with Pfeiffer's statement (Gent f.
Bald XIX, p. 594), that highly virulent cultures are less influenced
by typhoid and cholera sera than less virulent ones. No details
are given by Pfeiffer as to the conditions under which his non- virulent
cultures were used, Pfeiffer's statements refer to serum and not to
blood solution, he pays little attention to the agglutinative and
much to the paralytic phenomena of the reaction, and attaches most

importance to certain disintegrative changes produced by his special
method of testing in vivo. ♦ We have stated elsewhere that highly
active cultures, if left for a few hours longer than usual between the
times of transplantation, rapidly undergo involution changes, and while
in this Condition are far more liable to show agglutination than
was the case with the same cultures tested a few hours earlier. We
have found that for class purposes involution forms in cholera are as
abundant and striking in a virulent culture left unchanged for three or
four days as would be the case with a non-virulent culture, grown at
room temperature, if left without transplanting for as many weeks
or months. Bouillon cultures, which have stood long without trans-
planting, show a tendency to spontaneous partial clumping, which is
quite absent during the first 24 hours. For this reason we prefer to
use 24 hour bouillons, which are free from sediment, for the test.

The peculiar disintegration obtained by PfeifFer in typhoid cultures
place^ directly in the peritoneum of a specially immunized animal,
do not tend to occur where the serum is tested in vitro by the
hanging-drop method. With blood solution, however, this peculiar
phenomentlfif frequently witnessed. The clumped bacteria, if watched,
for an hour or so, may be seen to break up in granules, which
gradually become indistinct and vanish whilst under observation
until practically no trace remains of the clumps which shortly before
studded the entire field of the microscope. The change is more liable
to occur in cultures some days old than in young culture and more,
perhaps, with attenuated than virulent cultures. It does not occur
with all samples of typhoid blood, and i.s not well marked in very
dilute blood solutions.

This greater tendency to bacteriolytic action in blood solutions
often makes the reactions obtained with them look at first sight less
striking and intense than that obtained with serum where the clumps
usually remain intact Apparently, however, the difference indicates
that a large amount of the bactericidal substances originally found in
the plasma do not permanently remain as constituents of the serum.
This not only has an obvious bearing on serum therapeutics, but
explains how the action of serum may be modified by mechanical
mixture with the fibrin elements of the blood.

Quantitative estimation of the degree of dilution in the case of
blood solutions is possible by haemometry as well as by making direct
measurement. With samples of freshly dried blood, sufficiently ac-
curate observations can be made to express the degree of dilution in
multiples of 10 — (yV tit sV' etc.)

We have employed a cell having a depth of 85 mm. and giving

with a Fleiachl's hsuniometer a tint reading 100 p.c, with j^ dilution
of normal blood. In aniBmic cases the dilution will vary with the
degree of anteniia, which can readily be determined. Blood dried for
some time gradually yields li ss and less hsemoglobin, owing to the
change of thi.s substance into the hrematin compounds. This change
goes on rapidly in air where gas is being burned and slowly in pure
air. In any case, the error is in the direction of a less dilution
than that shown by the hremometer. As a matter of experience,
we find exact estimation of the dilution, while interesting for
scientitic purposes is not necessary for the practical purposes of
the test if attenuated cultures are used and the establishment of
fixed arbitrarytime limits, as recommended by Griinbaum seem only
of use in avoiding pseudo results, due to the use of highly virulent
cultures.

Griinbaum, being enthusiastic for exact estimation of dilution in
all cases, claims (Lancet, Sept. 19, 1896), that though most sera will
in time produce clumping, that typhoid serum can still bo specifically
identified by its being the only serum, which, with free dilution in
a ratio of 16 to 1, \iill produce a complete clumping and arrest
motion in 30 min. A fixed dilution ratio, with an arbitraryitime limit,
appears to us quite uncalled for as a routine diagnostic practice, and
has no standard value unless a culture of fixed virulence is used.

Since writing the above we find that Griinbaum has now stated on
theoretical grounds " that possibly the use of attenuated cultures
would enable us to dispense with the dilution" {Lancet, Dec. 19,
1896.)

We had anticipated a 'priori that the solution obtained from the
dried blood would be less sensitive as a reagent than the fresh liquid
serum. We find the blood solution on the contrary to be apparently
more potent than the serum, in causing the agglutination though not
as to the paralytic effect, and perhaps to give the reaction at a
somewhat earlier stage of the disease. This view agrees with the
researches of Widal, who found that the agglutinating substance was
contained in the globulins and fibrinogen, and that the serum albumin
and corpuscles contained none. Thus the blood serum contains only
a part of the agglutinative substance. Dr. A. H. Appel of the U. S.
Army has also recently made studies and observations showing the
greater agglutinative properties of solutions of the whole blood as com-
pared with that of the serum. A decided agglutination can be obtained
from weak solutions of the entire blood when none is produced by
stronger solutions of the serum. While Widal places the limits of
dilution with serum below 1 to 200, R. Stern who employed solutions

of tho entire blood in bouillon reports reactions with dilutions of 1 to
2000.'

Owing to the greater sensitiveness of blood solutions as compared
with typhoid serum, there is a greater tendency to pseudo-reactions if
active virulent cultures are used, than is the case in v/orking with
serum. This difficulty is, however, completely obviated by employing
attenuated cultures for testing. Cultures which exhibit darting
movements in hanging drops are too sensitive for the dry blood test.
Those cultures having a quiet but rapid gliding motion in hanging
drops have given us uniformly good results. If the movements of the
culture become sluggish, one or two daily transplantations at body
temperature will make it more active and .sensitive. One or two cc,
of the living bouillon cultures injected into the pentoneum of a guinea
pig produce immunity and a marked blood reaction without injur-
iously affecting its health.

Clean preparations containing very little fibrin can readily be
obtained if care is taken not to stir up the film of blood clot and to
use plenty of water for dissolving.

We find that the blood dries in a few minutes sufficiently to be
enclosed in an ordinary letter.

Our routine method of testing is to place a large drop of water
from capilliary pipette, on the film of dried blood and let it stand for
a minute or two. A loop full of the solution so obtained is taken
frovii the top of the drop and mixed with a loop full of the bouillon
culture, or may, if desired, be diluted further.

For the re-examination of cases giving a negative reaction, a .some-
what more virulent culture can be used or a quantitative estima-
tion also made by the serum method. We have not succeeded however
in obtaining a decided reaction by the serum when the result with
the dried blood was inconclusive and now attach equal importance to
a negative result by the dried blood test.

Our published observations (N. Y. Med. Journal, Oct. 31, 1896,
British Medical Journal, Dec. 5, 1896), on the dry blood method

1 We observe that Widal, who was the first to show that dried blood could pro-
duce the reactions, and already, in June, 1896, obtained reactions from serum after
four months drying, has recently {Semaine Med. Jan. 13, 1897), reported that he
has been able to obtain successful results by the dried blood method in the earliest
stages of the disease and that the blood after six months drying retained the power
of producing the rea^-tion. The dried blood also gave him positive reactions late in
convalescence in cases where agglutination had become very feeble. We are glad
to find our published results on these points agree with those of so high an
authority. We have found that with those who have had difficulties with the dried
method, these have been due to their having acted upon the erroneous idea that
the blood solution was much weaker than the serum whereas, even with attenuated
cultures, we have got a reaction readily with it in dilutions as high as 1 to 125.

BHj^^

1*':

were made with attenuated cultures, and pseudo reactions were prac-
tically never encountered.

Later on, for a few weeks we tried active virulent culture trans-
planted daily at 3Y°c., but these gave us with the dried blood solution
numerous and very peculiar pseudo-reactions, i.e., reactions not due to
«xisting typhoid. For instance, the blood of one of us (W. J.) when
dissolved gave prompt and abundant agglutination with a virulent
culture, while we habitually use it as a suitable negative control
bloo^^ with attenuated cultures. A solution of the blood of the other
(D. D. McT.) gave no reaction. (W. J. had typhoid fever 16 years
ago ; D. D. McT. has never had it). W. J's blood serum gave no
pseudo-reaction with the virulent culture.

On resuming the use of the attenuated cultures described above,
the pseudo-reactions disappeared. On re-examining, the blood drops
which had given them with the virulent cultures, no longer did so
when tested with attenuated cultures, although dry blood from
genuine cases taken at the same time still reacted typically.

For practical diagnostic work it may be stated that when a blood
does not show a decisive reaction in a serious case of fever which has
lasted over a week, the fever is almost certainly not typhoid. In very
mild febricular cases the result may remain doubtful, unless investi-
gated by an early bacteriological examination of the spleen pulp or
stools. . ,

In this connection we may state that we find that Eisner medium
containing 25 per cent, gelatine instead of 10 per cent, will remain
solid at a temperature about 30 C, and give visible typhoid colonies
within 24 hours.

Reaction with the Colon Bacillus.

Very little attention has as yet been paid to the clinical significance
of serum reactions with colon bacillus. Courmont and Rodet have
stated that typhoid blood serum reacts with colon cultures, while Achard
and Chantemesse state that it does not. Widal states that he has
studied quantitatively the intensity of reaction of typhoid sera with
Coli, but has been unable to draw any important diagnostic conclu-
sions from the results.

Various observers have reported colon reaction as being present
occasionally in different chronic and acute diseases. This can readily
be understood in the light of our present knowledge of terminal infec-
tions. One case which at first strongly resembled typhoid but gave
no serum reaction, has been recorded by Vedel vho found a marked
colon reaction and looked upon it as only colon infection, this opinion
being confirmed by the subsequent events. Personally we liave found

;

reactions with the colon bacillus to be rare with typhoid blood or
serum (even in cases wlien perforative peritonitis had occurred) pro-
vided tlie typhoid reaction was well marked. On the other hand we
have been struck by the large proportion of positive colon reactions
obtained in cases Vaving step-ladder temperature and other symptoms
strongly resembling typhoid but without the typhoid serum reaction.
We think that under these circumstances the colon reaction may have
a real diagnostic importance, and indicates that the colon infection
whether (occurring alone or as a secondary complication of typhoid
may be playing an important part in the production of the patient's
condition. The whole question of associated colon infection deserves
further study.

The reaction can be tested with ease by placing a duplicate drop of
blood solution or serum on the cover slip with the drop lo be tested by
typhoid culture and mixing it with a drop of colon bacillus culture.
Pseudo-reactions can be avoidetl by using stock cultures kept at room
temperature, and transplanted infrequently. Test cultures grown in
bouillon from the stock at room temperature for 24 hours are free
from scum or sediment, and give reliable results. The conflicting
results just mentioned may have been due to pseudo-reactioi.\s having
been taken seriously.

In our case of apparently genuine typhoid without serum reaction,
(on which, by the way the test was first applied during the third week)
the blood reacted ver'' ■ jidedly to B. Coli, producing typical clump-
ing. The same held ^^od of four other blood samples referred to
us for examination as having a clinical course like typhoid, but with
negative serum reaction. A complete colon reaction we have found
to be exceptional in ordinary typhoid and its presence would indicate
a condition of Coli intoxication sufficient to explain the existence of
many symptoms giving to typhoid its ordinary clinical features.
Whether this excludes typhoid, is another question. W. H. Park has
observed a case of fever with no typhoid serum reaction, where he
was able to cultivate the typhoid bacillus by spleen puncture. Later
on in the case however a relapse occurred and the reaction appeared.
The possibility of a latent typhoid infection overshadowed by toxic
phenomena, due to concurrent action of the colon bacillus is quite con-
sistent with the generally accepted opinion that many of the symp-
toms in typhoid and especially the intestinal ones are due to second-
ary infection by B. Coli. It follows that in severe cases of typhoid
type, with no typhoid reaction, the blood should be tested with a
culture of B. Coli and a bacteriological study made by examination
of the stools or by spleen puncture.

In a few cases we have met with a partial typhoid reaction only, in
mild cases clinically fibricular, where the fever subsided by lysis in
within two weeks of the onset. Here, the possible presence of
typhoid appeared to indicate the prudence of keeping the patients in
bed and avoiding articles of diet which are contra-indicated in typhoid.
Our experience has been that fibriculse, with completely negative
blood reaction, get suddenly well after a few days of fever. Here, ai o,
spleen puncture, as in Dr. W. H. Park's case, miglit enable a decided
diagnosis to be made earlier than by the blood test alone. Wesbrook
recommends spleen puncture under the circumstances. The possibility
of infection by organisms resembling the typhoid bacilli must naturally
be borno in mind.

Diabetic blood has been found by Block and by W. H. Park, to
give a decided agglutination. We have examined two cases of diabeies
which both gave perfectly negative results.

Conclusions.

The difference in reaction observed between typhoid V>lood solution
and blood serum is not simply due to varying intensity, but to an
alteration in the relative prominence of the agglutinative, paralytic
and disintegrative phenomena which constitute the reaction. The
extent of this difference «kc> varies with the virulence of the culture,
but the difference probably depends also on the presence of part of
the specific substances elsewhere than in the blood serum.

Blood solution has a greater capacity than blood serum for produc-
ing the disintegrative (bacteriolytic) changes described by Pfeiffer
Descriptions of this phenomena are conspicuously absent from the
many recent accounts of the reactions with typhoid serum as observed
in hanging drops.

The paralytic effect is relatively more marked with serum than
with blood solutions.

Agglutination without stoppage of motion is more readily occasioned
in virulent cultures by blood solution than by serum, and does not
indicate existing typhoid.

It appears preferable that for the dry blood method only attenuated
cultures should be used. These have the advantage of being more
easily kept in readiness than virulent cultures, and are less sensitive
to changes of temperature. With the serum method virulent cul-
tures give prompt results. Dried blood serum can be readily obtained
and transmitted to the laboratory by pushing aside the edge of a
blood drop which has clotted for a few minutes but has not dried

and collecting the serum beneath it on the tip of an ivoty vaccine
point, etc. This does not, however, give a quantitative result.

For ordinary diagnostic purposes, the simplicity of the method as
originally described does not require modification, provided attenuated
cultures are used.

A drop of the solution obtained from a dried typhoid blood drop,
mixed with a drop of the culture, will give the reaction promptly,
without any special attention to the degree of dilution. In order,
however, to obtain the best results, it is well to dilute freely and
especially to avoid having a sticky solution of syrup-like consistency.

In cases where the clinical type strongly resembles typhoid and
where the serum does not give the typhoid reaction, a decided reaction
with cultures of the colon bacillus may explain the symptoms.

Our results with the dried blood test have been«very satisfactory,
giving uniformly positive results with genuine and well marked
typhoid cases, and not reacting with non-typhoid bloods when atten-
uated cultures were employed.

Although the use of serum undoubtedly enables the results to be
recorded and compared with greater scientific precision, we find that
dried blood answers just as well for routine diagnostic work.

The alterations in reaction, induced by very slight modifications
of the manner of testing, help to es^ktin differences in the results
reported by experienced and careful observers. With the same blood
and culture, the amount of dilution possible largely depends on
whether plain bouillon, bouillon culture or water is used for diluting.
Opinions also vary as to what should be regarded as constituting a
reaction. Personally, we do not think that anything less than com-
plete clumping and total arrest of motion obtamEOiC by the dry as
well as the moist test in a young attenuated culture, should be re-
garded as typical.

'-^»*"

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