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AGGLUTINATION STUDIES

Thesis for Degree of M. S.

Ferns F. Loomis

1926

 

 

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I: SLUT 713;“ C321 STUDIES

Agglutination Studies

Thesis

Submitted to the Faculty of the Hichigcn State
College of Agriculture and prlied Science in Partial
Fulfillment of'the Requirements for the fiegree of

Master of Science.

Ferns F. Lgomia

June 1926

J'H Esm-

III
IV

VI
VII

2.

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Introduction.

Review of Literature.

Technio Employed.

Experimentnl flork.

\

n. Croee Agglutinutione.
8. Salt and acid nglutinntione.
C. The Effect of Innubetion on the Eydrogen-ion
Concentration.
D. The Effect of Omittine Fhenol from the Antigen
of B. pullorun.
E. The Effect of Organic Coepeunae on Agglntinetion.
F. Application of the Lbcve Findings in Increasing
tee Sensitivity of.nntigene.
G. Routine use of Antigen Prepared as above.
1. In Testing Cloudy Sera.
2. In the Stenderfiizeticn of Bacterium
Bullorun entirene.
Summary.
acknowledgments.
References.

102335

5.

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ILG'AS-Cdvuit “a

The :gglutination test is now generally recognized
as having great significance in laboratory diagnosis.
Only comparatively recently has active research been con-
ducted along this line, and when all the findings ere com-
pleted the results seem astonishingly meagre as computed
with.the Eisnificence of the probless involved.

In general, further work on the agglutination tust
is Justified by the feet thst the subject is not complete,
and that the test is widely used in the identification of
diseases in.;nn.und animul. Any improvement that can he
made may be of considerable value in the system now used,
and further mey mute the test adeptable to other diseases.

In taking for-the subject of this thesis ”Agglutineticn
Studies" we recognized the brosdness of the field, and the
necetsity for concentrating on a few definite phases of the
subject. Accordingly the work has been divided into the
fOIIOW1ug sections:

1. Cross agglutinations of the four organisms
studied. namely: Bacillus sunguinsrium, Esoterium‘pullorum.
and Bacillus typhosts, selected becnuee of their close re-
lationship, and Pseudomonss pyoeysnee, chosen as a repre-
sentative of another distirct group, totally different from
the other three organisms.

2. Salt end said syrlutinnticns of the three

organisms.

4.

3. The effect of incubation on the hydrogen-
ion concentration of the tubes.
4. The effect of the preservative phenol on
the sgflutinetion te:t for Bacterium pullcru:. This was
instigated by the report of Rebrnssier (69) that pseudo-
egglutinctions with this organism might be avoided by the
omission of phenol from the tests.
5. The effect of orglnic substcnoes on the
agglutination.
6. The application of the shove findings in in-'
crossing the sensitivity of the antigen.
7. Routine use of the antigens prepared so above.
A. In testing cloudy sore.
B. In the standardization of B. pullorum
antigen.

In this laboratory the problem of cloudy reactions has
proven a distinct annoyance in the routine tests for fluc-
illary Shite dicrrhes in chickens, since the standard ssglu-
tinetion text does not function with cloudy sore. In order
to avoid cloudy earn it is necessary to starve the birds
for 48 hours Before bleeding. This definitely interferes
with production, and is greatly objected to by the owners.
Hence the introduction of a method for testing cloudy corn
may be considered a distinct addition to lsborstory tech-
nique; both from the stsndpoint of the laboratory worker,

and for the convenience of chicken breeders.

5.

The standardization of antigens is of great importance,
especially in testing for B. ullorum. Kullnann (l) in
conducting rescarch over a period of tee peers found the
variability of difgerent strains of this organism and of
the suns strain at different times, to be very greet. This
necessarily hcs resulted in inaccurate tests, depending
upon the particular strain used for the antigen suspension.
This difficulty has been overseas, in part, by asking up
large quuntitics of enitgens from a known strain and preserv-
ing for use in the tests. Fowever, a method of etnndcrdiz-
lug antigens ageinst a known preserved serum, in such a say
that the results would be censtunt and dependable, weuld

be a decided i-iprevearnt upon the present method.

RIIVIII -' C 3‘ LIT? 317577;?

Kolmer (2) defines agfilutinins as "antibodies that
possess the power of causing bacteria, red blood corpuscles,
and 80:8 protozoa, suspended in a fluid to adhere and form
clumps.

The first obseerticns of the agelutination reaction
were made by ietchneloff, Cherrin and Rogers (2) who in
1889 noted unusual devc10pqents of Pseudononas pyocfcneus
when grown in immune serum.. They did not recognise the
importance of the reaction and went no further with the
experimr: nt.

Schn years later Gruber’and Furham (3) and GordetIZ)

while investigating the Tfeiffer phenomenon discovered the

agelutinution reaction as such and beli'ved it to be a

6.

particular function of gonna serum. In addition, archer
obaerved that noeolute specificity did not held, since
agglutinine would react with antigens of allied rpeciee of
bacteria.

In 1895 Ffuurdler discovered the threed reaction or
Trundler'e ThCHOJfiflOfl, in which beateriu developed long
thread-like growths when ere n in immune serum. at this
time the reectien was ceneieered en important diagnostic
test, but Wee later eupglented by the ecelutination test.

During the Eu 1‘: yeer, '{Iiddl and. Grunbaum *(2; favored
the theory that ugflutiains are dell receptors having a
haptoehore grO'p WHICfi oexbines with the erglutinoren of
the bacterial prot0p1een *nd a zyuoehore groyp which is
directly responsible for agglutination. Due to their
complexity he designated then at receptors of the second
order.

Grucbeum {2) made proctieml LPDIIGdtIOU of the
agglutination test in eiegnoeinr tyyhoid fever. They dis-
covered thet agelutinine for 3. tyrhoeie developed in the
serum of typhoid patients in the eerly Etufisfi of the die-
eare, and applied this Knowledge as a diagnocitc test
inown as the Grober-Jidol reaction.

Free the first there have been VuTiOU? theories as to
the :nchenie; of agglutination.

Gruber (2) believed that the ugzlutin in acted upen.the
bacterial meqbrane increasing its viscosity which in turn
canted clu.ping of the becteriu.

relteuf's (2) idea was that the egglutinogen united
*Ehrlioho

7.

Iith the agglutinin and was precipitated on the surface
of the bacteria.

Bordet's(2) View was that of molecular physics and
consisted of two phases: first, the preparation of the
cells by the agglutinin which alters the molecular
attraction between then and the fluid in which they are
suspended, and second, produced by the addition of salt
which increases the surface tension of the combined
bacteriumragglutinin particles so that they form clumps,
thus decreasing the surface tension.

In 1901 Jcos (5) in summarizing a series of experi-
ments with Bacillus typhosus concluded that salt was
necessary for agglutination, and that in the absence of
salt the serum became inactivated through the action of
bacilli, without decreasing the vitality of the latter.

In 1902 Nicolle and Trenel (4) arrived at the con-
clusion that all free cells possessed the power to agglu-
tinate, and sensitiveness to agglutination, particularly
bacterial cells. They anneidered motility and the presence
of the bacterial membrane to be essential factors for
pronounced agglutination. At this time also, Defalle (5)
stated his theory that agglutinins were formed in the
organism.through the action of the bacterial membranes.
Hence the degree of agglutination would depend upon the
develoPment of the membranes.

Smith (4) adsanced the idea that agglutination re-
sulted from the action of Salts on the precipitate. pre-

sent on the bacteria.

 

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Cne year later, Jose (7) made the statement that
the role 0: salt in agglutination was that of actively
assisting the combination of the agrlutinable and agglu-
tinatir~ as t .nc es. J1Le was succeneful in substituting
other salts for EL‘ diun chloride.

In 1903 Lnith and cash {3) distinguished two classes
oi a~~lutinin., one act ng upon flagella and the other
upon the body of th' bactsrLa and found that the flagellar
agglutinins eight be denonstr ted with a much lower deéree
of imaunit? than those which act upon the body of the
bacilli.

Upon contirucd research (9) these men also found
certain agglutination relationships between related
bacteria.

Tiurin3 the sap Mye.r finescrman (10) ap lied the
knowledge of afifllutinins and preciuitins to Vaccination
and immunity.

a little later Hicollc (11¢ urged a uniform.tech-
nioue for agglutination tests. to enyhasized the in-
rortance of the flagella in producing the reaction.

In a later paper Smith and Reugh {12) stated th

heory that the presence of 1.ud19 a~~1 tinins produced
an affinity between the motile and non-natile races,
making it pessible to distinfiuish between the flusella
and body agglutinins of the nmtile bacilli.

In lied arer and fissile (15) found that the applica-
tion of heat would further distinguish between flagellar

and body agglutinine.

Rossi (14) believed that the flagella were primarily
responsible for the phenomena of agglutination, possessing
great sensitivity toward the agglutinating substances of
specific sera.

In 1907 nichaelis and Iona (15) found that when an
indicator is used in determining the pH of a solution,
the color is affected by the jresence of neutral salts
and the results may be wrongly interpreted due to an
apparent decrease in acidity.

In 1908 tech (16) made a study of anslutination con-
cluding that this phenomenon resulted from a softening
of the surface of the organism and the subsenuent cohesion
of use particles, as differentiated from the precipitation
of colloids.

Shortly after, tichaelis {17) published a contrary
statement claiming that the agrlutinating agent acts princi-
pally through its ability to increase the surface tension
of the agslutinable material. the change in consistency
being a secondary consideration.

In 1908 Bechold {18) demonstrated that the positive
ion of the added salt is responsible for agglutination,
and believed the variations in the effect produced by
different catiens to be sue to four things: first, the
extent of dissociation of the salt; second, the Valence
of the ion; third, the motility of the ion; fourth, the

v

electro-afiinity of the ion.

10.

In criticizing Bechold's work, Buxton and Shaffer
(19) showed that the dissociation of the salt is negligible
giving as an example sulphates, which although always less
dissociated than chlorides produce the same results.

These men agreed with fieltham's theory that the coagulating
powers of th; ions increases geometrically with the in-
crease in valence. They also maintained that there was
little evidence in favor of the motility theory. They
showed the definite coagulating powers of heavy metals,
especially fOr normal bacteria; also that the resulting
metallic hydroxide and the hydrogen ions worked in opposite
directions. They observed the phenomenon of "irregular
series" in which a given salt will cause coagulation in

the highest concentrations, this followed by a region of

no action, with a lower concentration, and at still lower
concentration is again brought about, ceasing at the min-
imum concentration.

This is different from the anterior zone phenomenon,
in which there is a certain range producing coagulation,
while above and below there is no seagulation, the upper
zone of 00agulation being called the "anterior zone".

In comparing the value of different suspensions Buxton
and Tongue (20) later found a marked difference in coagula-
tion of (l) unorganized suspensions (2) bacteria (5) im-
mune bacteria. HaCl and Cacl2 coagulate imnune bacteria,
but not normal bacteria. Anterior zones appear with normal

bacteria and immune bacteria but not with unorganized

LL.

suspension. Coagulation begins with a lower concentra-
tion of Belt with inaune then with normal bacteria.

Feels and £1613 produce irregular eeries with unorrenized
suspensions; F9013 with immune bacteria, end not at all
with normal bacteria.

In investigating the electric charges of the above
suspensions these men (21) found that each carries a
negative charge in pure water but that in salt solutions
the sign is reversed between the zones of no coagulation.
They did not consider this neutralization of the charge
responsible for coagulation.

Their conclusion was that "bacteria show no irregular
eeriee but always anterior zones unorgenizcd suspensions
the reverse". (22).

In 1510 Cornetto (23) arrived at the conclusion that
Bucl acts upon the ugglutinins to prevent both the receptor
groups from coabining, leaving the (auctioning group free.

fihile Kreus meintmined thet if a serum preci itated
the filtrate of a given bacterial culture it would also
egglutinete the houologous bacteria. Gafihtgene (24) found
that the precipitine uppeer in the eerun before ugglutinins
and concluded that these work independently. He showed
that the precipitetLon reection is more dependable with
the layer method than when the serum and tested solutione
are mixed.

In 1913, Kicheelie (25) in a series of experiments

proved that proteinr may be identified by means of acid

12.

agglutination tests since the optimum concentration of
hydrogen ions precipitation proteins is constnnt and
characteristic for each protein. He showed also that
bacteria mey be identified in the sine manner, even the
most closely releted species heving a different hydrogen
ion optimum.

This test is even more delicate than agglutination
with specific antiseru, since in the letter method the
different strains mcy hinder the test, while with the
acid egzlutinution test, the hydrogen ion concentration is
alone res onsible end the kind of acid used does not in-
terfiere with the test.

The practicsl erplicntion of fiichuleis' test hes
proven successful. hecillus typhosus urglutinutes at a
hydrogen ion concentration of 4 to 8 x 10-5; Bacillus
para tynhosus st 15 to 1.3:: x 10‘5. Bacillus 3011 is not
agglutinated by acids.

Later in the once your Beniesk (25) corrorborutod
hichselie' eXperiments. His conclusions were as follows:
"Reither the acid, as such, nor the anion, is responsible
for the agglutination, but only the hydrogen ions. This
acid agglutindtion is produced only by hydrogen ion con-
centration within certein definite limits, chcrecteristic
for each species of bacteria. There is also an ortinun
concentration constant for eech species". This method,
has been found of particular icportcnce in differentiating
the members of the colon group of bacteria.

Still later Poppa (27) portly substantiated fiicheelis'

work, but she unable to differentiate the members of u

1 :2. 0

group, and at about the Euflb time Pouli end Blocker (28)
nude 3 study of the reletien Oi proteins to inorganic
colloids and the salts of hecvy metals. They divided
the colloids into two clesscs and called the first group
suspensoidz, while the second were nened lycocolloids.
The letter show greeter viscosity and stebilit: than t e
suspensoids, which ere very sensitive toward electrolytes
and become less stcble with age. Copper sulphate is on
ezcnnle of the suspensoids and ferric hydroxide of the
lycocolloids. In the precipitntion of proteins an excess
f the suspensoids does not interfere with precipitction,
while the udditi n of electrolytes hinders the reaction.
Lycocolloids, on the contrary, if used in excess our com-
pletely nhibit preciritetion. The addition of salts hes
a favoreble effect. Fydroxyl ion assists in the precipita-
tion with positive lycocolloids, hydrogen ions with the
negative ones. The precigitction of txcessive amounts of
protein, by either the suspensoide or lvcocolloids, is
hindered by electrolytes, only a snsll fraction of the
prot in being precipitated.

These men also noted peculerities in the behavior
of salts of hecvy'nstuls then used with solutions of
electrolyte-free proteins. chen very smell amounts of
the salts ere used precieitction results.noro concentrat-
ed solutions dissolve the precipitate enfi still larger
amounts of salts usein cuuse precipitation. The follow-
ing salts followed this rule, ferric chloride, cooper
sulphate, Eercuric chloride and zinc sulrhute. The ex-

planation offered is that in the dilute solutions,

14.

hydrolysis of the eults permits th formetion of insol-
uble compounds with the proteins. These compounds form
the precipitwte. Rhon certuin concentrations of the
salt is reached the hydroxy-protein coupound is replaced
by an ionized suit-protein compound and no procieitste
is visible. A concentrated salt solution depresses the
ioniZution, which in turn forces a preci itstion of the
undissociated compound.

Two yours later, Licheelis and Pechstein (29) were
able to determine the iso-electric point of casein.

A little later, hicheelis and ddler (30) reported
that "The acid agglutination Optimum for Bacillus typhosus
agglutinated with hydrochloric acid is at the some hydro-
gen ion concentration as with acetic or lactic acids."

In 1912 Dean (31) published a puper on agglutination
drawing the following conclusions: An agglutinating serum
contains two essential factors, these being first, the
Specific antibody, and second, a non-specific substance
which may be serum globulin. The interaction of antisen
With antibody causes an aggregution of the molecules of
the globulin, resulting in turbidity. If th: antiserum
is greatly diluted it may still contain an udenuote emoun‘
of the srecific antibody , while the globulin, or non-
specific substunce, becomes insufficient. This may be
remedied by the eddition of globulin free nornel eeruu.

stuber (32) advanced the theory that agglutination
is caused by the fatty substence within the bacteria.

He considered these substunces to be true agglutinogons,

15.

which by stimulation of the synpethetic nervous system
lead to the formation of egflutinins. The Specificity
of these fatty sub tenses is only rcletive.

In 1317 Barrow (5?) mode a comparison of the micro-
scOpic and macroscoeic methods of erglutinetion. He found
the macroscopic method to be thirty tines as sensitive es
the microscopic method, due to the different conditions
under which the tests are conducted namely: The time limit
for the microscopic test is much less than for the.mucrc-
scopio; the microscopic test is conducted at room temper-
ature and the mecroseo:ic may be incubated in a water bath
at a higher tempereture. In the letter-method convection
currents aid in the union of antigen and agglutinin.

In 1018 Topley (34) node a definite study of the
effect of convection currents on the rapidity of agelutine-
tion, and found a marked difference especially with the
antigens which showed relatively poor agglutination. He
also noted more repid flocculation in tubes in whiah the
fluid contents were onl" portly submerged in the water
both.

In 1918 hierkowskie (35) demonstrated the possibility
of obtaining an increase in the agfilutinetion titer with
Bacillus tyohosus and the vi rio of cholera, by a simul-
taneous increase in the amount of serum and suspension of
three to ten tides, in such a way that the degree of the
dilution of eeruh and other constituents remoined constant.
He obtained a further increase in titer by adding small

amounts of five to ten per cent acetic acid. the action

16.

being depressed by a stronger concentrution. He also
found that the titer nicht be increased, especially with
B. typhosus, by cultivation on media which is weakly acid
or weakly alkaline, or by Warning the bacterial suspen-
sion to 56 degrees, but not over 64 degrees.

During the same year, Bauer (36) experimented with
fetty acids and soups, concluding that cleric. steeric,
and palmitio acids have the greatest effect on agglutina-
tion, while soups and isohydric mineral acids have went-
er action. The higher fetty acids were emulsified with
sodium cholate in non-agglutineble concentrations before
use in the tests. .

In 1918 Hicolle. Jouen and Bebuins (37) were able
to produce agglutination by treating gonococcus serums,
which otherwise failed to agglutinate, with normal E01,
heating in boiling wuter, cooling them, and neutralizing
with HuOH. Several races of pneunonococci were likewise
treated and gave siuiler tests. The above results were
due, according to Forges, to hydrolysis of the mucous
capsule.

desne (38) produced agglutination of red blood cor-
puscles, suspended in a large amount of isotonic solution
of dextrose, by the addition of 0.04-5 sodium chloride,
and believed that sons hydrophil colloid played a part in
maintsining the stability of a susyension of corpuscles.

Also in 1318, b‘ means of cross agglutination ex-
periments with anti-typhoid, anti-peretyphosus A, anti-

parutyphosus B. end anti cholera sera of high titre.

17.

Caprono (59) demonstrdted that homologous species are
agglutinated to the sure degree either in saline sus-
pension or a growing broth culture, but that heterolo-
gous series show egélutinstion in higher dilutions in
the broth cultures, especially before the period of
heevy growth.

In 1.19 Buchanan (40) made the statement "Bacterial
agglutination is s colloidal phenomenon best studied in
the light of colloidal and physiccl chemistry".

During this same yedr Bergel (41) studying the mech-
anism of heuurglutinstion and hemolysis dedided that the
lipoid of the red cells is the true antigen possessing
the huptophsse groups and that the receptor with which
it combines is a non-specific lipase, a constituent of
the mono-uncle r white cells. His explanation is that
a specific innune body is formed which reusines attached
to the cell for a time and is then set free. Re consid-
ered this inmanc body to be a spmogen, which, activated
by the complement already present, attaches itself to
the )ipoid part of the homologous red cells.

At about the game time, hensfield (32) presented
a physiologiccl explanaticn of agglutination. Kc claim-
ed thut bacteria are maintained in a permanent suspen-
sion through the action of s protectife colloid, and
that agglutination is produced by the action of a di-
gestive ferment which acts upon the colloidal substsnoe.
He offered as proof, the feet that there is a direct

proportion beteeen the c nccntruticn of the agglutinin

17.

and the speed of the reaction, as ie also true of other
ferments. The t-mpercturo curve conforms to the same
rule, He considered the presence of sodium chloride
important to the recotion.

During the yehre 1921 one 1922, nnueroue findings
were node. In 1321 Janelli (43) offered the theory thct
the passage of an electric current through agglutinating
Bern woulfl result in the division of the egélutinine
into two parts, each of which had no egjlutinetinq powers.
The agglutinnbility was re;tored when the two portions
were again coabined.

Also in 1921 Barrett {44) offered -n countion for
estimating the hctian between egglutinin and antigen,

y t 9 m, where y e the concentration of the egglutinin,
t - tie agglutination perioa, and m a a constant.

Later in the some your, Boll.end,Tineley (45) made
the statement, "Chencee in agglutinobility of organisms
are associated with Variations in culture media".

In 1921 Gates (46) one able to produce agglutination
mechanicnlly by the use of the centrifufie, the results
coinciding with theie obtained with the etcndurd test.
The aaVentepe is the elimination of the inconetent tire
factor.

it about the £819 tin. Echochenneier (47) was able
to flenonstrete the use of purified bacterial fete as
agglutinogcne, with on‘y relative Specificity.

Soon after, Fcbry (4?) wee eble to chow that by

attenuating microorgnniens (3. typhoeie) by growing them

18.

on media to which was added increasing amounts of
phenol, the agglutinating powers became greater as the
strength of the organism lessened.

During the same year, Ishii (49) recorded his ex-
periments with the colon-typhoid group of bacilli. He
found that acid culture media favored agglutination while
alkaline media retards. In using formalin he found the
most favorable concentration to be 0.011%, 0.05 %,to 0.2%
showed a retarding action on agglutination. Traces of
sodium chloride were sufficient for agglutination, while
the addition of 0.1 % phenol, or 0.01 % mercuric chloride
had no effect. He considered 37° the optimum temperature.

Soon after Eersch (50) in substituting immune serum
for a fraction of the saline used in regular tests, found
a resulting increase in aldalinity which he believed to
be due to the differences in the dissociation constants
of the reacting substances and their products.

Gunn (51) was able to produce agglutination of
cholesteral suspensions with ricin. He considered this
to be a non-specific action due to the precipitation of
one colloid by another of the opposite sign.

Von Szent Gyrmgi (52) classified pathogenic micro-
organisms into tow groups. In the first group he placed
those organisms which are concerned with local disease
processes, and produce no immunity, as Bacillus pastes,
Bacillu: mallei, and Bacillus anthrasis. These organ-

isms have a tendency to flocculate when grown in fluid

1’3.

medium, and give a weak agglutination reaction. The
second group comproses these organisms causing acute
diseases resulting in immunity, as Bacillus typhosus
and vibrio cholerae. These remain in suspension'when
cultivated in fluid media and give a strong agglutin-
ation reaction. I

In 1922 Horthrup and Defiruif (53) in studying
the agglutination by electrolytes found that electrolytes
' in low concentration affect the potential primarily while
higher concentrations decrease the attractive force be-
tween the organisms.

In a similar report (G4) the same man, experiment-
ing with agglutination in the presence of proteins
normal serum and immune serum, found that when proteins
or serum were added to bacterial suspensions, the zone
of acid agglutination broadened, and the iso electric
point shifted to that of the added substance. Proteins
used were egg albumen and globulen.

At the same time, Eggerth and Bellows (55) obtained
practically the same results using egg albumin, gelatin,
protalbumen, hemoglobin, edestin, and heteralbuminose,
adding them to suspension of B. coli. They found that
at reactions more acid than the iso electric point the
bacteria had a positive charge and failed to flocculate.
This lessened stability was also shown in suspensions
of eellusose, nitrate, cellulose acetate and oil em!
ulsions when similarly treated with proteins.

Northrup and DeKruif (56) upon further study with

Bacillus typhosus with regard to the concentrations of

2C.

variou; Salts required to produce agglutination, found
it convenient to divide electrolytes into two classes.
Ehe first calss comprising those in which the agclutin-
sting concentration is not influenced by the concentrat-
ion of the suspen;ion, and which do not roversethe sign
of the change. Class tia includes those whose arglutina-
ting conc'ntrction is increcrrd with an increased tur-
bidity of the suspension. Those with the exception of
zinc sulphate reverse the charge. To quote from the
paper, ”These results are contrary to the idea that the
cenbinaticn is caused by a d ffercnce in the sign of the
harge carried by the immune body and the organism.

They agree with the assumption that the immune body forms
a filn on the surface of the organism E0 that the effect
of the charge is the result of this film.

Wolf {57) advanced the revolutionary theory that
antigens are fatty in nature.

Defiruif and Horthrup (58) found that by repeated
Ia hing with distilled water, they were able to complet-
01y remove the imuune body from sensitised bacteria.
Contrary to their original belief this removal was as
complete at pH7 as at FY3.

Hohn (59) in 1323 studied the effect of culture
media upon the agglutinalility of B. typhosue. He
found that when green on clear sgnr the bacilli loose
the power to agglutinate mueh more reseily than when the

mediuu has not born cleared or when mannitol, galactose

21.

or dextrose are added to the cleared agar. 0.1 g gel-
actose is especially valuable for use with cleared media.

In 1922 Ehionoye (60) investigated the effect of
ions on egrlutinstion, and found that us the Valency of
the cation increased, the limiting concentretion at which
agglutination would occur became lessened, the relation
being such that the curve of the Valenciee and limiting
concentrations equalled a streight line.

During the same year, Yumsguchi (61) reported that
the increased viscosity resulting from heating emulsions
o! Bacillus typhosus lessened the sgslutinebility. These
could be restored to almost the criminal viscosity and
agglutinebility by the addition of acids. Ea elso (62)
treated emulsions of the same bacillus with a 1.05 sol-
ution of son. When neutralized, these suspensions showed
a lessened titre with serum imwunized with live cultures
but no change in sgrlutinebility Was noted when seruus
from uninuls immunized with killed cultures of the organ-
ism were used.

Also in 1923 Kine (63) found that the egrlutinin-
antigen complex became dissociated upon etcnding at room
temperature. Khen normal serum was substituted for im-
mune serum only partial dissociation occured. The organ-
ises used were stuphlococci.

In the same year Krumwiede, Cooper and Provost (64)
stated that " the absorpticnof specific ezglutinins must
be used to determine agglutinatire identity.

DD
I‘D

Evans and Small (65) found that rerun dilutions of
Bacillus influensee were made in beef infusion broth at
pd 7.6 instead of nor s1 sult solutien the results were
more satisfactory sepeciellr with the less sensitive en-
tigens.

In 1:24 Duets (66) discovered that earn alksli with
a concentration of .01 normal minht be used in the sep-
aration of antigen end egélutinin, 42° bEIH? the favored
temperature. He found a 10 Z euccroee solution more
favorable than a solution of Eacl.

Lendsteimer (67) and Sander Ccheer, also in 1924 by
means of partial absorption methods were successful in
differentiating the blood of different species, even when
the precipitins were much alike. They inclined to the
belief that the essentiel differerce between sgglutinins
and preciritine is a chemical one shieh determines the
ageoificity of untigcns.

Jacobite (GB) differentieted between ertificiel acid
agglutination used by fiicheelir, and bioloricel eeid anglu-
tinution induced by the acid formed by the bacteria dur-
1ng their growth in anger solutions. then the former
method is used the bacterzs die euictly while in biological
acid agglutination.they'rsy'live for u long while.

In 1526 Rebrussier (69) in studying the effect of
phenol in the pseudo-agglutirdtien found thut by onitting
phenol from the antigens, this trouble could be practically
all sinutud.

23.

-3m2131:$1mrb

The seru were obtained from four rabbits uhich had
been previously injected with suspensions of killed cul-
tures of Bacillus sanguinarium, Bacillus pullorun, Bec-
illus typhosus, end Pseudosonus pyoryunee respectively.
The organisms were cultivated for twnety-four hours on
liver agar slants, the growth washed off with physiol-
ogical sult solution, the suspension diluted to corres-
pond with tube 1 of x Fsrlsnd's nonheloneter end heated
in a water bath at 60° C. for one Lil? hour before use.
These suspensions were then kept it 1008.und injected
intreperitoneully in one hslf c.c. amounts at intervals
of three duys, for a period of four weeks. it this tire
the rabbits were bled iron the our and the sore tested
against the respective antigens. “ince ouch serum gnve
a positive test at a concentrstion of 1:640, the titre
we: considered t: be sufficiently high for study. The
rabbits were bled iron the heart and the sore obtsined

were preserved at 10° C. for future use.

asses Letssrxngrxis ssurxrs
is a preliminary to the agglutination studies,
cross agglutinations of the four organisms selected for
study were made. These cross egglutineticns were con-
ducted with the usual antigen prepared by washing the

growth from liver agar cultures, incubeted at 37° for

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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24.

48 hours, with physiological salt solution to which

0.5 3 phenol had been added as a preservative. The

antigen was diluted to a turbidity of 1 according to
:e?drlend's nepheloneter.

The tests were set up using earns-antigen dilutions
of 1:20, 1:40, 1:80, 1:100, 1:320, and 1:640, physiolo-
gical salt solution being used in making the dilutions.
The results obtained (Table I) were as follows:

Pseudonones pyongenee serun tested with its home-
logous entigcn sure u titre of 1:640, while with B.
typhosus antigen 3 + test one obtained at 1:40, with
a 3 at 1:80. 31th 3. sanguineriun and B. pullorun an-
tigens nerntive results were obtuined in all dilutions.

71th 3. sanguinurium serum, the homologous antigen
gave a titre of 1:640, B. typhosus a + Lt 1:640, B.
pullorun a ff at 1:80, with a 3 at 1:160; while 3. pro—
Iran u untiren gets a 9 at 1:40, with a ‘ reaction at
1:80.

It will be noted thnt B. typhosus antigen geve the
highctt titre, as would be expected, while P8. pvoryenea,_
en unrelated orjunisn, hdd the lowest titre.

B. pullorum serue with it: heiologous antigen gave
a distinct positive st 1:640 with 3. typhosus untig n
a 9 at 1:320, 3. sanguinorium 9 at 1:;60 With a 1 at
1:530, while P8. proryunen gave only a 1 at 1:20.

These results are ell cooperuble, indicuting a
close relationship between 3. typhosus, B. sanguinerium
and B. ,ullhrum, and the totally different nature of PS.

pyocysnee.

STUBIEd 11?H ELL; AH‘ i313 iGCLUTIIfiTI7fl

Having determined the titres of the various sera,
both on the homologous and heterologous antigens, it was
possible to study the effect of altering the test from
the standard procedure.

The first modification to be studied was a comparison
of the effect produced by the various salts as compared to
sodium chloride .85; ordinarily used with the standard test,
incubating at 37°, 25°, and 10° C. and also according to
the rapid method.

In obnducting the stdnddrd tests for the four ergon-
isms under consideration, the set-up in each case was e32
follows: O.d§ c.c. of serum, 0.2 0.0. of antigen suspen-
sion having a turbidity of 1, and 0.85 e.c. of 0.85 5
salt solution in each tube. Dilute hydrochloric acid was,
added to obtain the desired hydrogen-ion concentration.
The serum~entigen combinatiwn of such of the four arsen-
isms wee tested Lt ph values 3.0, 4.0, 4:2, 4.4,‘4.6, 4.8,
5.0, 5.2, and 5.4 with such of the eight Belts used, nearly
copper acetate, sodium sul hate, buriun chloride, ammonium
chloride, magnesium sulphate, potassium chloride, calcium
carbonate, and sodium chloride. Duplicate sets were ins
cubuted at 87°, 25° and 10° respectively, endslso with
the rapid method.-

0f the salts used, calcium carbonate and megnIsium
sulphate were the least effective. The former, when used

Iith B. sanguindrium serum and antigen, with the incubetion

26.

at 370. effected only a + reaction at pH 3 and 4, while
the letter produced the sums results, 21th an additional
3 reaction at pH 4.2.

When used with B. pullorum serum and antigen 03303
permitted a o. reaction at hydroren pH 3.0, + at pH 4.0,
and 3 at pH 4.2, while "3304 produced only a + at pH 3.0
and 4.0 with a 1 at 4.2. With 3. typhoeue serume and anti-
gen 03003 allowed a .... ct p23, +9 at p34 and 1 st p?4.2
while £2304 ceused a 9+ et $33 with 1 at DP4 and 4.2

With P3. pyrocyenee serum and entiren Cucog gave a
. . at pH 3.0 and 3 at pH 4.0 and 4.2; egeo4 +. at pH 3.0
. at pH 4.0 end 3 at pH 4.2.

dnmonium chloride was slightly more effective, since
when used with 3. eenguinerium a +++ reaction was produced
at pH 3.0, .. at pH 4.0 and 3 at pH 4.2/

With E. typhosus e ... reaction was given at pH 3.0
. at pH 4.0 and t at pH 4.2, while with Ts. pyrocyunea
serum and antigen 3 .... reaction occured at pH 3.0, +.
at pH 4.0, + at pH 4.2 and 3 at pH 4.4.

Barium chloride and potassium chloride were next in
order of efficiency. With 3. sunguinerium the effect of
these e.lts was identical. Both gave a ++++ reaction at
pH 3.0 and 4.0, .. at pn 4.2 and 3 at pH 4.4.

with B. pullorum the degree of agglutination produced
by these t‘o suits was again the same. The reaction pro-
duced at pH 3.0 and pH 4.0 use in each case +++§ while a

... was indicated at pH 4.2, ++ at 4.4, and 3 at 4.6.

The maximum results were obtained by the use of copper
acetate, Sodium sulphute and .odium chloride. The
results of these salts being much the some.

. with B. sunguinuriun all three of the last mentioned
suits dove 3 .++. reaction at pH 5 and 4, N32804 gave 3
4+4 at pH 4.2, while cop or so tote end Ecol gave 3 ++.

Copper acetate and 30230 produced a 4 at pH 4.4 while 3

4
was indicated in the heel test.
Copper ccetute and 283304 gave a 1 reaction st pH 4.6.
with 3. pullorun serve and antisen the reiults at
pH 3 and 4 were the race, all three salts ermitting a
.... reaction. At pH 4.2, Ns2304 and H201 gave a .... ,
while Capper acetcte guve e +++ reaction. At pH 4.4, Real

gave u +++ test while Capper ucetcte and E3280 gave ++ .

4
At pH 4.6 the test WLB e for each salt. With 8. typhcsue
each of the three eults gave +1++ at pH 3 and 4. Cepper
acetcte and naCl gave +v+ at pH 4.2, while £32804 gave ++.
At pH 4.4 Heel produced 3 ++ reaction while copper acetate
guve a o, end E32304 3} Heel and Capper ecetute gave 1

at 4.6.

Tith PE. pyoryunea ...+ was indicated in each case at
pH 6 and 4, the HuCI test rhowcd ++++ at pH 4.2, while
copper ucetute and 3.2504 gave ... reaction. .At pH 4.4
Heel produced 3 ++ while the tests with £a2804 and copper
uc-f'tute indicated a 4» reaction (Table II)

The tests incubated at 25° averaged one point less

than those given for the 37° incubution.

28.

Those placed et 10° for 24 hears showed et the meet'only
doubtful reactions, but when held for 43 hours the results
compared favorably with those obtained by incubuting at 37°

f0 24 hours 0

29

TABLE II ‘ffeet of salts on Agglutinetion.

 

B. Sanguinerium

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

' 'bu. : v 1 v 1— 1 1
’ ,
pH igcetateJ 83804'321012 {IT}! «'3 W150 'KCl 'cecoq '11801
f 1" TV"
' ' ' 0 t v I
3.0; ++++ 1.****4L **+*- o++ e ++++ + ++++
- 4: Tr , T
i ’ i 3
.‘-0_*++* . ++++ ****- ++ 1 + '++++' e '++++
f ~ :t: , r. 1 .
I I O u + e
4.24_ 9+ . +++ ++ - ' - _: ++ ' u 9+
fi'r ' v v T “t f
I 4' + e
4.4: e i' + _17 - ' 1 1 - ' ' -
* Y + 'V— vi T— T r
U : 7 :— V '
BA pullorum _ A
' i 0
5.0L ++++ AA¥++++~L++++1 + ++: + :++++' ee ’ ++++
I t
f, l r Te
I ' I * g '6 + '
4.2 {+7 LA++§+ L +++ 4+ 0 L a- k ¢++I 4. +§§¢
r’ ‘“* ' " we v
I I I
4.4! 9+ - ++ ++ L :_ : ++’ ' ¢++
' v T * —v ‘T— ’ T v
t
4.6' + + I ‘ ' i 1 " ' v *
"T ' ' W— v r
‘08. t . ' ' I ‘

 

.TABLE II (
Continue ‘
d) Effect of salts on
Agglutineti
on

____:U_ B. typhoeue
f

acetate'Na$304:Be01'
28 I g mason-15° I
no 'KCl '08
CO 'NECL '
H

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a
3.0 ++ '
0+
;i++++ '
4 ' v ;++++’ +++ '
.0 * ' ++ '
- +*+ +++’ . ++++' +++ '

4 2' :; I +++ + ' + r ' *+++'

o 1 +++ ' if 4;? - +*+. ' «:
, A ++ . ' + T ++
l * .7 + - I + ri ++++'
4.4~ fl ML - . + r
‘ ' * 4* A r r +++ '
a . 1 #1. ++ '
‘r A \ '
o I . *
o v - '
_# PB ' . ' _r
o
v, ‘i . D:ocysr nee .
300’ *' . ; '
i?“ ;++AA»++++ ' ' ' j
. fir :: ++++ ++++ ' *f

4.0 * . t + . I 0

W4 ++i ‘HLH. ++++' +++'

4 o *f 4“ ! ++++'++++ ' ‘1 ‘T’ i.+*+.
.2; 00+ ' ; § '*++ . * '

r +++ ' ' ++ - '

4 0 a : ++ 4.... ' ' w' ' ’HH...
04 ... + t _. - ”9+ ' * .
~wr _i + I , * r A1 - .

w, L - - ' T ++++'

4.6. . ' i ; U i | r t

. f i o c v r , *+ '

v L‘ r

4.8’ #1 k . 1
c ' .

' : _f o I
- t .
' ' ' v I
I t
I

With the rapid method, results corresponding with
those given for incubation at 570 were obtained. Here
the buffer solutions were substituted for the colorimetric
nefhod of obtaining the desired pH, since by so doing
the time required for the set up was materially lessened.
The sane serun antigen dilution was used as in the above
trste, the tubes shaken for two minutes, and incubated for
15 m nutes at 37°.

The results would seem to indicate that physiologi-
cal sodium chloride solution is as efficient as other
salts in promoting egélutinetien of th. four organisms
studied.

In view of the feet that these results checked with
those previously obtained by other investigators, the
evident conclusion seemed to be that physiological
sodium chloride as ordinarily used in the routine agg-
lutination tests is as efficient as other salts, at
least those tested did not seem to average higher in

results produced than those given by .85 5 Heel.

Change of Hydrogen-ion Concentration During

Incubation.

The next point in question was to what extent in-
cubation at 37° would effect the hydrOgen-ion concen-
tration of the ugglutinuting mixture.

Accordingly set-ups were arranged for the four
organisms, using 0.05 0.0. of earns, 0.25 0.0. antigen
{turbidity 1), 0.5 0.0. buffer solution, 0.5 0.0. phy-
siological sult solution.

The tests were urrsnged in duplicate for each organ-
ism, at the end of thirty minutes, and 24 hours incuba-
tion indicator wee added to each tube, and the contents
compared with the pH stunderds to determine the extent
in change of th: hydrogen ion concentration. The results
were as follows: 51th the half hour incubation, the tests
averaged one point increase in slkslinity. while with the
24 hours incubation, the average change in pH Value was
1.5 toward the sideline side.

Ehile this has no particular sisnificdnce, the con-
clusion may be made that the pH of the incubeting tubes

tend to move tonsrd the alkaline side.

The Effect of emitting Phenol from
B. Pullorum.intigen
At this time an attempt was nude to substentidte
Rebrassiers (69) clein that the so-oulled pseudo-re-

actions in the ugslutinetion of B. pullcrun mirht be

In
.5...

P ‘]

avoided by the ondssion of phenol.

Routine tests were run in the usual manner, but
omitting phenol fron the antigen used. Tests were
arranged in duplicate, with incubation at 37°, and
25°C. for 24 hours, and 10° for 48 hours with the
following results:

Almost with out exception, the tubes incubated at
37° for 24 hours showed sufficient growth to interfere
with reading the tests. _

In those incubited at 2503. the growth was less
apparent, but Was evident to an undesirable degree.

Incubation for 48 hours in the ice box eliminated
the growth factor but the length of time necessary fer
agglutination is undesirable for routine work.

The antigen prepared without phenol was also tested
accordins to the rapid method, using 0.05, 0.L25, 0.0125
amounts of sera, 0.2 0.0. of antigen with a turbidity of
l, and 0.5 0.0. of physiological salt solution being
added to each tube. The tests were shaken for two min-
utes at 37° and compared wi h tests similarly conducted
but in which phenolized antigen was used.

This method was rep ated several times with vary-
ing results. at times the tests free which phcnol was
omitted seemed clearer than those in which phenol Was
used. at other times no difference was apparent in the
results produced by the two methods.

The evident conclusion is that phenolized antigen

is more dependable for routine work.

33.

THE £3333? ? €33 SIC CSLPCUXUs CH AGSLLrIHLTITH

The next step was an attempt to influence the degree
of agglutination by the addition of organic compounds.

Accordingly various organic compounds, namely,
blood albumen, edeetin, lecethin, and henoglohin were
prepared in suspensions of 1:400, 1:2000, 1:10.000,
1:50,000 and l:250,000; giving as final dilutions in the
tests 1:800, 1:4000, 1:20,000, 1:100,0CC, and l:500,000.

These varying suspensions were added in 0.5 0.0.
amounts to tubes adjusted with buffer solutions to
hydrogen-ion concentration of pH 4.4, 4.8, 5.0, 5.4, 5.6,
5.8, 6.0, 6.2 and 6.4 using B. pullorun antigen with a
turbidity of 1 and positive serum with a dilution of 1:40.
Incubation use at 37° f0 24 hours. The following results
were noted

Lecethin with a dilution of 1:800 produced a ++ re-
action at pH 6.4, 5.2, and 5.0, . at 5.8, 3 at 5.6 and
5.4, negative at pH 5.0, 4.8 and 4.4: and with a 1:4000
dilution, negative at 6.4, 0 at 6.2, 3 at 6.0 and neg-
ative for the rest of the series. Higher dilutions of
lecethin produced no effect.

The value of edestin proved slightly less than that
of lecethin. The tests were not clear, and while some
agglutination was apparent in the same tubes as those
noted with the use of lecethin, the tests were not

satisfactory.

The sane was true of blood albumen. The tests
were not satisfactory even when a dilution of 1:800
was used. For this reason the use of these substances
was not continued further.

fihen.hemoplobin use used in the same manner with
incubation at 37° £0 30 minutes, 0.5 0.0. of a 1:860
suspension produced a ++++ reaction at pH 4.4, 4.8, 5.0,
5.4, 5.6, 5.8, 6.0, and 6.2, with a ... reaction at
pH 5.4. The 1:4000 suspension {reduced 3 negative re-
action e: 6.4, .. at 5.2, . at 6.0, and 3 at 5.8, 5.6,
5.4, 5.0, 4.8, and 4.4. a l:20,000 suspension gave a
negative test at 6.4, ++ at 6.2, * at 6.0 and 5.8,
and a negative at 5.6, 5.4, 5.0, 4.8 and 4.4. a sus-
pension of 1:100,000 gave a negative test at pH 6.4,

+ at 6.2, t at 6.0, and negative for the rest of the
series, After being held at 57° for 24 hours the tube
containing hemeglobin dilution 1:4000 and buffer at

pH 6.2 incrensed to a +++ reaction. The other tubes
were not altered. (Table III)

This was a distinct increase over normal agglutir-
aticn of B. pullorun serum and antigen without the add-
ition of hemoglobin, since the previous tests had given
only a 3 reaction at p3 4.8 and above.

The previous cross agglrtinations tests with B.
pullorun antigen, and Fe. pyocyanea serum had given a
negative test in all serum—anticen dilutions.

The effect of the addition of’hemoglobin suspension

1:800 was to increase the eensitisity of the reaction

01
Ch

Table III The effect of Various concentrations of

hemoglobin in the ogflutinotion of B. pullorum serum

and antigen. Mouton-3n at 37° for 230 minutes.

 

 

 

 

 

 

 

 

r —' I T V T j I T '
PH '4.4 '4.8 '5.0 '5.4 '5.6 '5.8'6.0 '6.2 '6.4 '
T V V V 1 1 1’ V f '
Concentration' ' ' ' ' ' ' ' ' '
of Hemoelobin’ ' ' ' ' ' ' ' ' 'r
r V V T V V V V V '
a a . o - v .. u - u - I - o - u .. v - I
“—" F 1 T v v 1 v 1 “r O
1:500 000 v - o .. v .. v - a - o . v . a .. 9 . a
—'-"""_—"F 1 1 1 I 1 1 I v I
111001000 I .. i .. I - n .. I . o . I I l 4, o .. o
r Y Y T T r r V Y '
1:20Looo v .. v - v ., 0 .. I 0 3 t 3 v“, v . u
1:4 000 o .. v .. o .. o - 9 .. c 1 v , 0,, o . c
U V— 1 r 7 1 V V V '
£800 ' ++++' ++++'+++r" ++ +'+++r' ++:o'++++'++++'++++'

 

so tket with rerun dilution 1:40 upon thirty minutes
incubation at 57° 3 re otions were obtained at pH 4.4,
4.8, 5.0, 5.4, 5.6, and 5.8; e at pH 6.0; +++ at pH

6.2 and negctive at p: 6.4. The 1:4,000 snapeneion
gave negeti7e tests at pH 4.4 end 4.8, t at pH 5.0.
5.4, 6.6, and 5.8; + at 6.0; ++ at 6.2 and negotive

at 5.4 (Table IV). when held at 37° for 24 hours the
agglutinntion in the tubes containing the 1:800 suspen-
sion inerehved to + at pH 4.4, 4.8, 5.0 and 5.4; ++++
at 5.6, 5.8, 6.0, 6.2 and 6.4. The tubes in.ehich.the
1:4,0L0 suspension nos :ecd inere sod to 3 at pH 4.4,
4.8 and 5.0; ++¢+ at pH 6.2 aha 6.4. The tubes contnin-
ing a 1:20.000 suspension which bed given only negotive
tests with 50 ninutee incub_ti'n, new gore 3 at pH 6.0
end + at 6.2. rho 1:100,660 suse~hsion produce& only a
3 reaction at pH 6.2.

These results seemed very significant. In the
first place, the eddition of a suspension of homo—
globin apparently increused the sensitivity of the
antigen to rush an intent that ++.+ reactions might
be produced when nornslly negative tests would be given.

-The next step Wes the application of these find-
ings to tests with clouey sore, first using 0.05 0.6.
of serum (X), 0.2 c.o. of 3. pullorum entiren, 0.25 0.0.
amounts of hemoglobin susyensicns 1:20.000, 1:100,000
and 1:500,000 respectively. The tests were Lajnrted
to pH values 6.2, 6.4, 6.6 and 6.8 with buffer sol-

utions in 0.5 0.0. amounts. fiith 50 minutes incubation

3630

Table IV The effect of various hemoglobin suspensions
upon cross agglutination of PB. pyocyanea serum, and

B. pullorum antigens. Incubation at 57° for 60 minutes.

 

 

 

 

 

 

 

 

211 "L4 '4.8 '5.0 '5.4 '5.6 '5.8 I'5.o '5.2 «5.4 v
r V V r 1 ’1'" “I v V H
Concentration’ ' I I I I' I I
of hemoglobin' ' ' ' 0 I I I I I
T 7 ' V I T 1 v w I...
o I g, I .. I '_ I - I - I ... I - I _ I - I
' .' f T V— T’ v r“ j J.
13500.000 ' - ' - ' "" ' " C "' 'I '- l - I n . .. '
1:100.000 ' " ' " "" ' " ' " ' " I .9 I - I - v
1 T V J V “V V ‘1 ‘ T ‘1 r i"
1:30.000 I .. I - I - I I - I - I - I - I - I
r ':F ‘ ¥_V f 1 f+ r i 1—— 1 _:
1:4.000 I " I I - I - I - I - I I- I «II I - I
1f* 1 + v + T + 1 * r +‘1 1— i— ‘4

1:800

«7
U.

at 37°, the 1:20,060 suspension Troduccd s 1 reaction
at pH 6.6 and c at 6.0 the remainder of the tests
being negative. Jhen the above tests were incubated
for 24 hours at room tempercture, all tubes contcin-
ing the hemoglobin euSpension 1:20.000 showed a ++++
reaction, the tubes containing the higher dilutions
remaining negitive.

31th a second cloudy serum (Y) run according to
the'ubove eystem, the hemoglobin suspension 1:20,COC
produced a 3 recction at pH 6.2, + at 6.4 and 6.6 end
I+ at 6.8, the higher susyonsions having no effect
with a 30 minute incubation. The 24 hour incubction
produced thr seam results as those given for serum
(X).

Thses eeru were obtained from the routine inborn-
tory and had given cloudy reactions with a dilution of
1:40 on.thc previous day. Doubtlese the disappearance o.
of th» cloudy effect one due to the ice box storage durin:
the intervening 24 hours. observution to this effect
has been made by Hr. Linllnunn (1). 0n the other hand
the cenbinuticn of the constituents in the tuoes may
have affected the precipitotion to some extent. At
this time no further studies were nude to determine the
disappearance of the cloudiness.

The test with serum (X) Woe then repented, using
the cane amounts of serum, untiecn, and hemorlobin
suspensions, but decrecsine the amount of b ffer ecl-

utions to 0.26 0.0. and adding 0.25 0.0. of physiolog-

473

b

 

coo mun"

 

 

 

 

 

 

 

 

. . m .++o+. .. . 31:30.13. m . .
O L bl Ir Ll +ll Lrl b b D b? b
d 4 4 1i 4 4 . . o

. .++++. . . . . . . . . amujm.
u b “a |P b L! [DI W L 1'” I” lb!
. .++++. . .44o+.++o+ . . . . . doc mo
. t > t p . Ir: u in 0 fl 4. 1
. . . . . . . . .++++. m . 00m. vomuflfl
- P r b L IPl F vi lllb b I I51

9 1 l i !I1 A. 4 J 1 .1.
. q . . . . . . .444em 1000 00¢."
D b L P 4” I u \” ”III? HI ID; .P
. . .. . . . . . . . ..+§++. q
o I? :m L t I w M” Mr” P “ h
. . m . . . . . . . . .Houoamoacno no
. . .t t L » L ti L Li - it» comwauunooemb
. He.3. . . 3 . am . an . 3 . mm . ... . . 3 . 3&2":
- .noo.» N b L P L r L b P D

 

8.:

£5.33 .m .3 .593. 23.23 3.
3.3%.:— .§$ .5 no 833.235». "3: 3a p Sufi

icul salt solution to each tube.

hith a 50 minute incubuti n at 37° tuhee con-
taining hemoglobin snipeneion 1:20,000 showed a 1
reaction at pH 6.0 and 6.2, + at pH 6.6 and 6.8 (lele
VIII)

Upon standing at room temperature for 24 hours a
+ reaction use evidenced a pH 6.0; +t at 6.2 and ++++
at 6.6 and 6.8; (Table IX)

Lessining the un-unt of buffer solutions decreased
the tests as might be espected.

A series of teets were then run, using cloudy
chicken serum of unknown rauction, in 0.05 c.c., 0.025 c.c.
and 0.0125 c.c. amounts, 0.2 c.c. or W. pullorum sntieen
having a turbidity of 1, 0.26 0.0. of hemoglobin sus-
pension 1:20.000 and 0.5 c.c. of buffer solution of pH
5.8 and 6.8. The repid nethod was used, that is, shak-
ing the tests for 2 minutes and insubsting for 15 min-
utes at 37°. Those tubes containing 0.06 c.c. amounts
of cloudy eerun guve e ++++ reaction; 0.026 c.c. of
serum produced ++ and 0.0126 c.c. a 0 reaction at both
pH 5.8 and 6.8. I

Cholesterol use not obtainable at the time the
previous studies with or-unic coupounde were made. At
this point/::s possible to compare 1to value with
thnt of hemoglobin, which thus far bud given the most
Satisfactory results of the Various organis compounds
used in the tests.

A dilution of 1:25,660 was used arbitrcrily due to

the results Ohtnined with corresponding dilutions of

Uhen a FEifl) tion of cholesterol 1:25,600 wee
substituted for the h norlobin in the above tests, bll
tubes gave ++++ reactions. ’

These teete seemed to indicate thet both hemoplobin
and cholesterol, but especi.lly cholesterol, might be
u:ed to increase the sensitivity of th~ antigen.

However, since the sore thus for tested were not
known to be positive, the next point seemed to be
would these orginio compounds produce a similar re-
action with any eerun, whether positive or negative.

Accordinely, a series of tests were run by the
repid method, HPID? cloudy were (3),(d) end (e) in c.05,
0.025 and 0.6125 amounts, 0.2 c.c. of s suspension of
B. nullorum enticen (turbidity 1), 0.25 c.c. of buffer
solutiens of pH 6.2, 5.6 end 5.8 resuectively for such
serum Iaed , (.2: c.c. o: hemoylobin suspension l:500,000
being added to each tube.

barrel horse serum was selected be a control, since
being from a different unirml, ~gy1utinin8 for B. pullorum
would be ubsent. Therefore a satisfactory check would
be furnished for the tests.

The set-up We; duplicetad, rubsiituting cholesterol
cueueneien l;m09,200 for the hemoglobin and both sets in-
cubited in a wafer buth st 66°for 15 minutes.

All tubes conteining chicken :cruq sure ++++ reactions,

,
while the terte with horse serum wer: negutive. This might

indicate that the chicken tern tested were all positive,

40.

or that the organic substdnce used would bring down
a precipitate with any chicken serum.

It Was thought that the effect of the high temper-
ature might be in part responsible for the high degree
of agglutination. Therefore, the previous set-up was
repeated. incubating at 52° for two hours. The re-
sulting agglutination was evident only at pH 5.8/ it
this point, those tubes containing chicken serum in
0.05 c.c. amounts gave +.++ reactions, those contain-
ing 0.025 0.0. e, and those having 0.0125 0.0. gave a
3 test. All tests with horse serum were negative.

Hence, severel things were determined. First,
cholesterol eusnensien 1:8C9,200 has an effect equal
to that produced by hemoglobin :5C0,000. Second, the
temnernture at which the tests are incubated is a
definite factor in the rate of the resulting reactions.
Evidently, too, the ontinun pH veins is 5.8.

The effect of varying amounts of antigen and organic
compounds was the next point in question.

Cloudp seru from two birds suspected of being in-
fected with B. pullorum were run dg inst a tool of
cloudies previously tested end the sums horse serum used
in the previous test. The tubes were set up as follows:
{1) 0.05 c.c. of the respective sera with 0.2 c.c. of a
suspension of B. pullorum antigen, 0.05 0.0. of buffer
solution having a pH veins of 5.8 and 0.25 c.c. of hemo-

globin suspension in 1:500,000; (2) 0.05 c.c. of the sera,

41.

0.15 c.c. of antigen, 0.3 c.c. of the hemoglobin, and
0.5 c.c. of buffer solution; (3) 0.05 c.c. of the sore,
0.1 c.c.cf antigen, 0.55 c.c. of henoglobin, 0.5 c.c.
of buffer solution; (4) the set up for (l) was repented
using cholesterol suspension 1:8L9,200 insteed of homo-
globin 1:500,000; (5) was a duplicate of (2) with choles-
terol substituted for hennelobin; (6) duplicated (5)
with the substitution of cholesterol l:809,200 in place
of hemoglobin 1:500,000. The results were the same in
all cases the tests with chicken sera giving ++++ re-
actions, while those with the horse serum were negative.

Evidently, one of two conclusions must be drawn.
Either all the chicken sera tested were from birds in-
fected with 3. pullorum, or the misture Wes still too
sensitive for ecou'uto results.

To further dilute the serum-antigen mixture seemed
the logical step. Therefore two suspected sere (B) and
(L) were run with a pool of oloudies which had previously
given positive tests, and horse serum, six set-ups being
used for each earn. (1) 0.05 c.c. of the respective
sore, 0.1 c.c. of B. pullorum antigen (turbidity 1) 0.35
c.c. of hemoglobin suspension l:500,000, 0.5 c.c. of
buffer solution having pH value 5.8.. (2) 0.025 c.c. of
sero, 0.1 c.c. of antigen. 0.55 c.c. of hemoglobin, and
0.5 c.c. of buffer solution., (5) 0.0125 c.c. of sere,
0.1 c.c.of antigen, 0.4 c.c. of hemoglobin, 0.5 c.c. of
buffer solution., (4) was a duplicate of (l) substituting
a cholesterol suspension of l:819,200 for the heooglobin.,

(5) duplicated (2) substituting cholesterol for hemorlcbin;

42.

(6) duplicated (5) with the substitution of cholesterol
for hemoglobin. These tubes were incubated for 15 min-
utes at 56° and allowed to stand for 15 minutes at room
temweruturo before reading. The results were as follows:
In the forst set of tubes, serum 3 gave a negative test,
L +++., the pool negative, and horse serum negative. The
results for set (2) and (5) were identical with those of
(1). Test (4) using cholesterol 1:809,200 gave a ++++
for serum L.a d for the pool. Serum B and the horse serum
gave negative reactions. The results for (5) end (6) were
the same as those given for (2) and (3).

Th? effect of increasing the serum antigen dilution
Was a decrease in the amount of egglutinstion to the ex-
tent thut the pool of cloudy sera which had previously
given a .... reaction, now became negative. Allowing the
tubes to stand for 15 minutes at room temperature after
being removed iron the ester both resulted in the pro-
duction of a decidedly clear supernatant fluid.

To re-check the results so far Obtained, and to
further determine the effect of temperature, serum (1)
was tested in 0.05 c.c., 0.025 c.c. and 0.0125 c.c.
amounts at pH values 5.0, 5.2, 5.4, 5.8, 6.0 and 6.2,
using 0.1 c.c. of antigen {turbidity l), 0.4 c.c. of
organic Bubstunces as before, and 0.5 c.c. of the
buffer solutions. The testl were incubeted at 56° for
15 minutes with an added 15 minutes at room tempereture,
and in duplicate at 57° for 24 hours. All tubes indicat-

ed a .... reaction. however, these i~cubdted in the

43.

water bmth ct 56° guve a sharper reaction than those
held at 37° for 24 hours. Likewise, the tubes in which
cholesterol was used gave a cleerer test then those

n th hemoglobin.

The same conditions were repeated, using negctive
chicken serum (not cloudy) with negntive results in all
tubes.

The questi n unturell: drove us to whether the
agglutinatixn with cloudy sers was due to a combination
vd th the organic coupounds, not formed with cleer sera.
This was refuted by the micriscopic test which showed
a merked agglutination with serum (L) and none with the
clear negative serum. .

the nest question to arise was that of specificity 9
whether Other antigens would produce like results when
sensitized with cholesterol. Accordingly, the previous
set-up was repeated using serum (L) with B. sanguincrium,
B. typhosus, and PS. pyooysnes antigens. Positive re-
sults w=re obtained in ell cases, indicating that the
test was still too sensitive for precticticsl use in the
diagnosis of Bacillsry Shite hiurrhes in chickens.

The test with the vuri us antigens were repeated,
with decrensing snounts of serum (L) the latter being
used in 0.0125 c.c. und.0.01 c.c. amounts, the amount
of the other constituents remaining sonctunt. 3 resc-
-tions Were still evident with-B. typhosus end 3. sung-

uinerium antigens. However, when the amount of serum

44.

used was decreased to .005 c.c.. B. typhosus and B.
ssnguinsriun antigens failed to react above pH 5.4 while
B. pullorum antigen still produced ¢+++ agglutination
at pH 5.5 end 5.8, ... at pH 5.0, ++ at 5.2, 1 at 5.4,
with a negative at 6.6. Therefore the development of the
test seemed to be approaching the point where reliable
reactions might be expected. As a further proof cf the
relsibility of the test, th; turbidity of the antigen was
decreased to 1/2 and used in 0.2 c.c. amounts in testing
four known positive sera (clear) sgninst a known negative
and a guinea pig serum, using buffer solutions of 6.4 and
6.6 in 0.4 c.c. amounts, 0.4 c.c. o: cholesterol suspen-
sion 1:809,200 and 0.005 6.0. of the resp ctive sore.
Incubstion was for-15 minutes at 56° and 15 minutes at
room temperature.

The results were checked with the routine test, and
With the microscopic test, the results in all cases bein:
identiOcl.

Serus R gave a 0+ reaction, serum 213 ++++, serum
138 +++, and serum 187 ++, while the known negative
chicken serum gave a negative reaction, and the guinea
pig serum likewise failed to agglutinate.

As the cloudy sore available for the test had been limit-
ed in number, 102 chickens were bled. and the sore tested
for the purpose of substantiating the reliability of the
test. 01 the sera thus obtained, 50 gave cloudy reactions
with the routine test. Of these 30 cloudy ones (32) gave

a +9 reaction with the sensitized test and likewise showed

45.

some agglutimtion v.1 th the microscopic test. The other
(933) gave a +++e rea;tian when testod according to both
methods.

The clear ECIJ were WC‘ 1 b7 the Eonswiti ca trot,
the routine toot, and tho micr030031o test, the results
in all cases being identioxl.

when the birds CB and 9%3 were starved for 48 hours
and bled, the oloqr sera tfiua obtlined wo?e testea by the
sensitized, routine and 3'370330710 H‘tund,g1ving the some
results as those noted ”have.

-3noo it see :od Ju ”t1 13:13 to con01u&e that the sen-
sitizod to t as oufilinod may be oonoiaered a reliable means
of diagnosis for the presence of B. pullorum in the zero of
chickens.

who fidVJntdgGS of thio te“t ma,7 be st ted as follows;
first,'the rapidity w 1h which it may he conducted, only
30 minutes 38 mg necessary for incubation, 15 minutve 1n
the Water bath at 56°C., and 15 minutes st1nding at roo;:n
togporature, to allow o:z,lotn preoiritation. Second,
only one tenth 88 inch serua 13 rrsnuirofl as in the st.ndard
test. Third, the avoidance o? the loss of rroduction cune-
ed by starving the birds to obtain clear Ecru.

The abject of runniav th tasts at both p? 6.2 and 6.4
is tnot corn v r3 1:1 tb 1r reaction with the buffer solu‘ions.
Some rfive: u oliorc=1* test at p7 5.2, while 0th rs éivo a olifiht-
1y sharper tost at pH 6.4. H-nco to obtain th: nozisun re-

sults 1t {Genoa qd715431c to an all scru at both concentrationa.

I33
Ci
.

rrfi- \Q- I‘m-,1 ~ ~ (“TI-9? ~ -‘y'fi-Tq--vv.
L4... .u:.u ..LL. .4...-_.|.-....o C.- A io,___.\,'.. .‘44‘

The lost division of the work dog on attempt to
prove .Eet entiyens Eifht be o‘enderdized, that is that
differoet etrhins of the some ergonicn siting widely
different resolts in to guundard test 3 rht be eheeied
with 3 Known orneerred cerue, and by the addition of o”-
sonic substinoee, be made to Rive a ++++ reaction.

Ten strains of B. pullorum more need in making the
uniifenr, which Yuried freq a straight ++++ when tested
with the standerd method to a negotive.

?er:infi yroportisns of eerun-antigen more need in
the attempt to otunderdise these unfifie 2. The antigens
were used in Various turbiditiae, with different amounts
of saline, with and without buf?er solutions, and with
different dilutions of cholesterol.

The ersteo giving the best results may be outlined
as follows: Each antigen wee teetéd for its reaction with
a known ++++ scrum, uzing 0.125 c.c. of the serum, 0.2 6.0.
of antigen with a turbidity c? 2, end .8 c.c. of normal
Selina, givin? a final dilution of 1:400. Of the antigens
tested § 13 alone VuTe a inerp ++++ reaction. The remain-
ing anti: no were then repeated, Uiifi? th“ sane serum-
entigen dilution, but decreeein? the «mount of saline to
0.4 c.c. and oddinv 0.4 c.c. of cholesterol oueponcien
1:4L9,BCQ. finti?:n Cfib fore a ++++ reaction, those givcn

by the other entifon: heinr UHSLtififACtflrya The remaining

49.
eight antigens were use n tested, th e tinv= Heine
0.4 c.c. of cholesterol soluti n 1:204,ROO, the other
conetitutente remaining eonetcnt. it this noint a ++++
reaction was given by antigen F 7. Tests on the rennin-
ing antigens werx repented, substitutin? chole:terol
1:1L2,4OO for the suspension yreviously ured. This
caused ++++ reactions to eppcer with nntigane 29, El, and
8. Cholesterol suspeneion 1:51,200 one then need with
the rencining entifene, resulting in a ++t+ reeetion with
$4?. It was found that by rubetituting 0.4 c.c. of
cholesterol 1:25,SUU, ++ + reactions co Id be forced with
th: remaining three antigens, 525, 40, end 11 (Teble Vf‘

Since Up to this tine, no agglutination had ever
been obtained with ontigen 5 11 and only very week re-
actions with § 47 the above results seem to indicite more
than the ever the poorest antigens may be reinforced or
sensitized by the addition of cholesterol euepeneione of
sufficient concentrcticn so that a reaction, e uul in
strength to that produced b' the more effective antigens,
umy'be secured.

Hence the conclution.nuy be erwn that it is possible
to standardize entifene for use with a known preserved
seru. :1.

This would prevent the possibility of Variations in
the standard te:t, enueed by chnnging from one strain to
another. Liaewiee, antigens prepared from th sine etrcin
mdy be checked from tine to ti;e to secure constcnt re-

Enltgo
*Followe p.ge 57

The value of such a stonderdizotion is apparent.
is noted by'fiollmunn (l) in an extensive survey of B.
pullorum antigens of various stroine, great irregularity
occurs froa time to ti 2. This irregularity is, of course
reflected in the routine laboratory tests. iceurdte
results cannot be insured, and definite stonderds cannot
be maintained, if antigens giving widely different re-

sults are used.

51.

HITHGD 0 LT m1 'R'IJ TI D O? JET 3 H

(1) ++++ serum, having a 1-400 titre, obtained by bleed-
ing chickens known to be infected with B. pullorun
is used.

(2) Irepcrs.antigen us usuul, and dilute to a turbidity
of 2. t

(3) Set up 7 tubes according to the following table.

 

 

 

 

 

 

 

 

 

 

0 0 0 0 I I 0
v} - 2' 3'4 ; 5' G‘Control
at n . . ' 0.2 ' 0.2 ’ O 2 ’ 0.2 ' 0.2 ' 0.2 ' 0.2
A ige o c ' ' . ' ' ' ___
Serum c.c. '0.025'0.025'0. 02 5'0.025'0.025'0.025' -
Th f I :I—"""'—""I; T
Sfiline 0.0. ' 0.8 ' 0.4 . 004 . 0.4. 004’ ' 0.4. 0.4
'1 v v v v T =f
Concentration ' ' ' ' ' ' '
of cholcctrrolc.b. - ' ' ' ' ' '
I T Y 1 1 v 1*
1°40? GOO ' ' 0.4 ' ' ' ' '
H 1 1 ”I r r 1 T
1:204 sco ' ' ' 0.4 ' v v v
"""""'"L VA 1 1 v r "*r 1
1:102 L400 ' ' ' ' 0.4 ' ' '
r‘ ‘v 1 1 1" I *r
:51 200 ’ ' ' ' . 004 . .
2—4: F 1 1 v r v “v
1:25.1LO ' ' ' ' ' ' 0.4 ' 0.4

 

(4) Shane and incuthe at 58° for 15 minutes and sllow to
stand at room temperature for 15 minutes.

(5) Observe and record.

The tube containing the least amount of cholesterol
giving a ++1+ reaction has the right antigen cancontru-
tion. This amount of cholesterol should be udaed to
eJoh tube when this particular antigen is used in the
routine test.

(6) Set up at lesst two known neg Jtiv and two known

positive sers ans the newly prcerod antigen Jo

F")
u».

 

 

 

 

follows:
0 I
' 1 ' 2
T 1
“1‘; c.c.' 0.2 ' 0.2
1 V 7
:1.“ ‘2??? 0000 .000025' 0.001
V" 1
432111319 0.00 ' 0.4 . 004
1 3
Cholesterol ' '
000‘ 0.4 ' 3-4

 

(7) Shake and incubate at 55° for 15 minutes in the water
bcth. Remove st the end of this period and allow to
stand at room temperature for 15 minutes.

(8) Read the results. The nonstive sera should be negative
in both tubes, and th positive sore should be positive
in at least the first tube. In this case the correct
antigen-cholesterol mixture has been determined. This
combination of antigen and cholesterol is now ready for

use in the routine test.

If dilutions 1:40 and 1:100 have been used according to the

standard.sethod, then dilutions of 1:400 and 1:1000 should

be used in this test, as indicnted by the above table. The

object of the higher diluti ns is to kae the test applicable
to general use. By increasing the sensitivity, cloudy re-
actions are avoided. The above procedure is tentative and

should be adapted to th routine of the individual laboratory.

53.

01?“? P! -
”HI-4-“-5

Sodium chloride as used in the standard agglutination
test produced meinuu efficiency. At least none of the
salts studied guve better results at the same concentration.

Incubation at temperstures below 37°C. decreases the
amount of growth when phenol is omitted, but also decreases
the amount of agglutination unless held for a decidedly
longer period.

Hydrogen-ion concentration influences the results meter-
ielly. Verying reaults were obtained.

The addition of cholesterol or hemoglobin causes en
increszed sensitivity of the antigen, resulting in higher
titres for the sore used.

Cloudy reactions can be avoided by using high titres,
mode possible by using more unstJble antigens.

Conbinstions have been obtained allowing the use of
a dilution tin tines higher than could be used with the
usual entie n, still giving a negatire and a positive test
as usuul. This ask s possible the use of an antigen ten
times as sensitive.

Th: modifiedite t any be conducted in a much shorter
time then the st ndurd toit. Fifteen minutes incubution at
56°C uni 15 minutes standing Lt room temperature is suffic-
ient to clear the aura.

Antigens may be stunderiJed ageinst a known preserved

serum. Even the weakest antigens which produce no agglutin-

ation in the stunderd test n.y be brOUjht to a ++++

reaction by the addition of a snsoension o-

of the correct concentration.

.P
J

cholesterol

* t“fr~.“' "-‘hfl-r-rfi’a
“04-3.“; ML ..Le'J- Lb 111,0

I wish to acknowledge my indebtedness to fit.
xsllmsnn, Dr. Ciltner end other members of the
Department for suggestion: and assistance re-

ceived during the course of these studies.

1)?‘ a: 131 w ~1- u‘
48*-----b—- iJ O.)

1. Hellmann, ¥.L.
"Bacterium Psllorum Studies"
Technical Bulletin Ho. 68, :griculturel Ex-
periment Etetion, Hiehigan Ftcte Collrge.

2. Kfilmer, Joio
Textbook. "Infection, Imnunity and Specific
Therany". pr. 379-282.

5. 3008. A.
“The Kechsnism of ngelutinution".
«Utsuhrift Eur ?F?iene und Infectienskrcnkheiten.
Vol. 36 (1911)

4. Kicolle, C. and Trenel, H
"ztudies on the Ihenonenu of i3”lutinatien.”
innulcs de l' institu Pas eur, Vol. 16
pp. 562-586.
D. 3::th 3.6, '57.
"The Role of the Kembrine of fincteria in
L5? utinctionfl.
Lnndles do 1' institut Pasteur, V01. 16
pp. 595-613.

6. (Lilith. ROJ.
"Concerning the fiechunism of agglutination"
Proceedinss of th' Linn Society of H.S. Wales.
Vol. 27 (1302) pp. 65-72.

7. JDOE, A.
"”he Eechnniem of fgglutinutien"
Zietschrii‘t fur Hygiene und Infektionskrunkheiten.
Vol. 40. pp. 203-230.

8. Smith. T and Reagh, LmL.
”The Hon-identity of hgelutinins Acting upon the
Flagells. and Upon the Body of Ructeris” .
Journal of fiedicsl Research.Vol. X pp. 89-100.

9. Smith, To and fi833h. A013.
"The Agglutinstion of affinities of fielated
Bacteria".
Journal of uedical Research Vol IX pp. 270-300.

10. fiessermsn, A. .
'Agslutinins and Preciritins".
Jutschrift fur Hygiene und iniectienskronkheiten.
V01. 42. pp. 267-292.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

57.

Nicolle, Co
"E1periments Relating to the Phenomena of
Agglutinationfi
Annales de 1' institute Pasteur. Vol. 18 pp.209-240

Smith, T and Reagh, A. L.
"The Non-identity of Agglutinins Acting upon the
Flagella and upon the Body Bacteria".
Journal of Medical Research, Vol. X. pp. 89-100.

Beyer, H.G. and Reagh, A.L.
"The Further Differentiation of Flagella and
Somatic Agglutinins".
Journal of Medical Research, Vol XII, pp. 315-328.

Rosei, G. de.
"The Problem of Agglutination, and Particularly
the Role of Bacterial Flagella.'
Centralblatt fur Bakteriologie Parasitenkunde und
Infektionskrankheiten. Vol. 56, pp. 685-691.

hichaelis, L. and Rona.
"The Use of Indicators for Determining Hydrogen-
ion Concentration."
Zeitschrift fur Elektrochemie v01 14, pp. 251-255.

Loch, Jo
"The Mechanism of Agglutination'.
Zeitschrift fur Chemie und Industrie Kolloide.
V01 IIIopPO 113-114.

Michaelis, L.
"The Mechanism of Agglutination"
Zeitschrift fur Chemie und Industrie Kollide.
v01. 4, pp. 550

Bechold, H.
'Agglutinationfi. Zeitschrift fur thsikalische
Chemie, Stedhiometrie und Verwandschaftslehre,
Vol. 48, Page 385.

Buxton, 8.3. and Schaffer P.
Agglutination and Allied Phenomena"
Zeitschrift fur Physickalische Chemie Stochiometrie
and Verwandschaftslehre. Vol. 57. pp. 47-63.

Buxton, B.H. and Teague, 0.
“A Comparison of Different Suspensions".
ibid.. 701. 57, pp. 47-63.

Teague, 0. and Burton, 8.3.
”The Charge Carried by Suspended Particles".
ibido v01. 57, pp. 75-890

22.

23.

24.

26.

27.

28.

29.

31.

33.

58.

Tongue, 0. and Burton, B.H.
”The Phenomenon of the Anterior zone."
ibid. 701. 60, PI. 489-506.

Cagnetto
"The ction of Salt and Agylutinins."
Biochemische Central 1 tt, Vol. 8, pp 591.

Gaehtcens, W.
"The Relation of Bacterial Precipitins to ’gclu-
t: nine”. ;;ietschrift fur I; azauntatsforschuno und
Experimentalle Therapie. Vol. 4, pp. 559-74.

hichaelis, L.
"The Agglutination of Bacteria by Acids".
Folio serology. Vol. 7, pp. 1010-2.

BE M1811 , II.
"The Acid Agglutination of Bacteria".
Jeitschrift fur Innunitatsforschung und Experiment-
elle Therapie. Vol. 12, pp. 268-515.

Poppa.
"Acid nglutination of the Paratyphosus Group”.
ibid, Vol. 13, pp. 185-91.

Pauli, W. and Flecher, L.
"The Relation of Proteins to Inorganic Colloids and
Salts of Heavy fietals.
Biochemische Jutsohrift. Vol, 41. Pp. 461-512.

michaelis, L. and i‘echstein, H. A
"The Isa-Electric faint of Casein"
ibid. Vol. 47, pp. 260-8

hichaelie, L. and Adler.
' cid igrlutination by Pydrochloric Maid"
zeitschrift fur Immunitsforschtnng und prerinnntelle
"herupie. Vol. 23, pp. 327-30.

Dean, H.R.
""actors Concerned in galutination."
Proceedin~s of the Royal Society of London. Vol.
84 . pp. 416‘54.

Stuber, A.
'hgglutinins"
Biochemische Sietschrift. Vol. 77 pp. 330--40.

(Farrow, ROP o
""acroscopic versus Kicroscopic he‘lutination"
Lancet, :pril 7,19l7.

56.

38.

39.

40.

41.

42.

43.

r'9
.v
I O

Tapley, U.
"The Effect of Convection Currents on Agglutinetion"
IflDOCt, JHHB 8. 19180

Sierkowski.
"The Influence of Certain :‘hyeioal Chemical Fe ctors
on Precipitation and figglutinution".
Chemleohes gentrnlblatt. 1916 II Pp. 664.

Bauer, Elsa
"Agglutinetion”
Biochemische Leitechrift. Vol. 83, pp. 120-8

:ioolle, d., Jonen, 0., and “ebaine, E.
”Ilon Ag~1utinnhle W eteri‘".
Comptee endue des eancee de la Societe do Biologie
V01. 81. PP. 839-40;

RSdSEB, W.
"Anglutinetion for Red Blood Corpuscles and the
Hoffmeieter Leriee".
Journal of Chemical tooiety. 114 I pp. 511-2.

Cantone, C.
"Cauguletion in Growine Cultures.”
LO hperirnentale 1701. 73, pp. 429-40.

Buchanan, R.E..
" g lutinntion“.
Jon rnul of Bacteriolo:y, Vol.4 , p. 75.

Rergel, S.
"The Lechenism of flowerelutination and Henolyeie"'
Jicteohrift fur Immunituteforechung und EXperimen-
telle Therepie. Vol. 27 pp. 441-58.

RLUEfield, 25..
”A Physiological Explgnntian of nglutination'.
ibidc V01. 27. Pp. 197‘212.

Zanelli, P.
"Continued fiction of the Electric Current on
figrlutinuting Sena.
Annnli d'igiene. Vol. L0 pp. 40 0-7.

44. Barrett, 3.

"Theory of flgglutinin Estimation.
British Journal of Experimentcl Pathology.
'01. II PuT’S 216-2’2.

61.

57 . ‘3";01f. c.
"The nature of Antigens."
Eedioal Science, Vol. V pp. 401-7

58. DeKruif,l§. and Northrup, J.
”The R moval of Antibody from Sensitized Organisms".
Journal of General Physiology. Vol. V. pp. 139-42.

59. Hon-n. J.
"The Influence of the Culture medium on.the Agg-
1utinability of Bacillus Typhosus.“
Hunchener Medizinische fiochsenschrift. V01. 69,
pp. 7-10.

60. Shicnoya, T.
“The Effect of Ions on.Agglutination"
Lancet 1922, II pp. 905~7.

61. Yamaguchi, K.
"The effect of Heating on the Agglutinability of
Bacillus Typhosus."
Japan Medical World. V01. 3, pp. 51
Chemical Abstracts. Vol. 17, pp. 2142.

62. Yamaguchi, K.
"The Effect of Alkalies on the Agglutinability
of Bacillus Typhosus."
Chemical Abstracts, Vol. 17, pp. 2142.

65. Hine, T. Q.M.
"Auto Dissociation of Agglutinin-Antigen Complex".
British Journal of Experimental Pathology".
Vol. 4, pp. 231-4.

64. Krumwiede, 0.. Cooper, 6., and Provost, D.
"Agglutinin.Absorption".
Journal of Immunology, Vol X, pp 55.

65. Evans, M, and Small, J.
"Methods of Preparing and Preserving Antigens of
B. Influenzae and their Effects upon Specific
Agglutination".
ibid Vol. I pp. 613.

66. Ozcta, M.
"Studies on Bacterial Agglutinine".
Zeitschrift fur Immunitstsforschung und Experi-
mentelle Therapie. Vol. 39, pp. 270-81.

67. Landsteiner,.K. and Van.Der Scheer, J.
"Specificity of Agglutinins and Precipitins".
Journal of Experimental Medicine. Vol. 40,
PP 0 91-107 0

68. Jacobitz, E.
"Bacterial Agglutination in Seegar Solution."

Chemical Abstracts Vol. XVIII PP. 5621.

45.

46.

47.

48.

49.

50.

51.

52.

55.

55.

56.

60.

Hall, J. and Tinsley, G.
"The Effect of Certain Media Upon the Agglutin-
ation of fieningococoi".
Lancet 1921 II Pp. 494-5.

Gates, F. L.
"Agglutination with the Aid of the Centrifuge".
Journal of Experimental ‘Medicine. Vol. 35,
PP. 63-760

Schachenmeier, H.
"Agglutination Action of Bacterial Fat."
Biochemische heitschrift. Vol. 124 pp. 165-71

Fabry, B.
"The Agglutinability of Microorganisms".
Comptes Rendus des Sceances de la Societe de
31010319. V01. 38. pp. 201“20

IShii. 0.
"Agglutination in the Colon-Typhoid Group of
8&01111'0

Journal of Bacteriology, Vol. 7, pp. 59-70.

Hirsch, E.F.
"Changes in Agglutination of Bacteria by
Immune Serum" .
Journal of Infectious Diseases. Vol. 30, pp. 651-7

Gunn, 30A.
"Agglutination by Ricin".
Journal of Physiology Vol. 54, p 558.

Gyorgi, A. vonsaent.
"Physiso Chemical Considerations in Regard to
Agglutination".
Experiment Station Record, 701. 44, p. 876.

Northrup, J. and DeKruif, P.
"Agglutination of the Bacillus of Rabbit septicemia
and of Bacillus Typhosus with Electrolytes".
Journal of General Physiology. Vol. 4 pp. 659-54.

Northrup, J. and De.iruif P.
"Normal Serum and Immune Serum".
ibid pages 655-67.

Eggerth, A. and Bellows, M.
"The Floculation of Bacteria by Proteins"
ibid, p. 669-680.

DeKruif, P and Northrup, J.
"The Influence of the Concentration of’the Suspan-
sion on the Concentration of the Salt Required to
Produce Complete Agglutination". ibid Vol. I
PP. 605‘6090

62.

69. Rebraesicr, R. E.
"studies of salmonella Pullorum" ;
Jourml of: the American Veterinary ucdical
£88001dt10no V01. 68, p. 6L5.

 

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