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Journal of Experimental Medicine

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ON

THE

INFLUENCE OF THE
DESICCATION UPON

REACTION AND
OPSONINS. I

OF

BY HIDEYO NOGUCHI, M.D.
(From the Rockefeller Institute for Medical Research, New York.)
W h e t h e r the o p s o n i c p r o p e r t y o f n o r m a l a n d i m m u n e s e r u m s ,to w h i c h the w r i t i n g s o f W r i g h t a n d D o u g l a s s h a v e called r e n e w e d
a t t e n t i o n a n d e n d o w e d w i t h special significance, is a n e w a n d
h i t h e r t o u n d e s c r i b e d p r o p e r t y o f s e r u m o r a special f u n c t i o n o f
t h e u s u a l i m m u n e bodies o f s e r u m is still a q u e s t i o n o f dispute.
Bulloch and Atkin/ Neufeld and Rimpau/ Barratt,' Neufeld and T~pfer,*
Hektoen,' and Keith* share the view of Wright and Douglas that the opsonicaction is due to the presence of certain hitherto unrecognized, distinct bodies,while Savtchenko/ Besredka, ~° and Deann are inclined to consider the opsonins
as identical with the amboceptors (fixators). Quite recently Muir and Martin a*brought forth evidence that opsonization depends upon the co/Speration of two
substances, one of which, at least, closely resembles complement of serum incertain of its biological properties. Still later Levaditi and Inmann TM asserted
that opsonin are nothing but the complements of serum.
I t is n o t m y i n t e n t i o n to e n t e r into this discussion, but, f o r m y
p u r p o s e , it is n e c e s s a r y to m e n t i o n t h a t in c o m m o n w i t h c o m p l e m e n t , so-called, the o p s o n i n s a r e a b s o r b e d o r fixed b y sensitizedbacteria, b l o o d corpuscles, specific precipitates, 14 a n d i n d i f f e r e n t~Received for publication April 30, I9o7.
2 Wright and Douglas, Roy. Soc. Proc., I9o3, lxxii, 357; I9O4, Ixxiii, i28.
8 Bulloch and Atkin, Roy. Soc. Proc., I9O5, lxxiv, 379.
' Neufeld and Rimpau, Deut. reed. Woch., I9o4, xxx, I458.
* Barratt, Roy. Soc. Proc., I9o5, lxxvi, 524.
* Neufeld and T6pfer, Centralbl. [. Bakt., etc., I9o5, xxxviii, 456.
Hektoen, Jour. of Infect. Diseases, I9O6, iii, 434.
s Keith, Roy. Soc. Proc., I9o6, lxxvii, 573.
• Savtchenko, Ann. d. l'Inst. Pasteur, I9o2, xvi, IO6.
io Besredka, Ann. d. l'Inst. Pasteur, I9o4, xviii, 363.
~ Dean, Roy. Soc. Proc., I9o5, lxxvi, 5o6.
1, Muir and Martin, Brit. Med. Jour., I9o6, Pt. ii, I783.
,8 Levaditi and Inmann, Compt. rend. d. I. Soc. d. Biol., I9o7, lxii, 725.
1, Muir and Martin, Brit. Med. /our., I9o6, Pt. ii, I783.
455

456

Influence of Reaction upon Opsonins.

bodies 1~ as well as non-sensitized bacteria, TM and exhibits thermolability and susceptibility to deterioration by age, similar to thatwhich complement exhibits. Besides the liability to undergo adsorption and the high degree of thermolability, no other characteristics of ferments have been ascribed to opsonins. Were opsonins
recognized as special ferments certain facts of theoretical andpractical import should follow. Among other things we shouldlook for an optimum of activity to be exhibited in the presence ofa certain reaction which might or might not be that of the sample
of serum which is being studied.
If the degree of activity depended upon a given reaction then to obtain the optimum indication, upon which alone a measure of the quantity of opsonin could
rest, this reaction would have to be secured.
I have, therefore,
made a study of the influence of the reaction upon the opsonie
power of several blood serums for B. typhosus, B. dysenteriae,
Streptococcus, and Staphylococcus aureus.
The results of these experiments harmonize so well that thosealone relating to Staphylococcus aureus will be given in this paper.
T H E I N F L U E N C E OF T H E REACTION UPON OPSONINS.

The Influence of Alkalinity.--The degree of alkalinity of several
serums were first titrated. Lacmoid paper was employed as indicator. The titration was conducted in the following manner. To
c.c. of serum ~!~ N. solution of hydrochloric acid was gradually
added until the reaction reached neutral. The amount of acidrequired for complete neutralization was taken as representing thedegree of alkalinity of each serum. The resulting fluid was thenmade up to 5 c.c. with 0. 9 per cent. salt solution. Thus the final
mixture had a strength of one fifth of the original serum. By thismeans it was possible to prepare a series of mixtures, in which o.5,
0.3, 0.2, o.I and null alkalinity were left unneutralized. These fluid
mixtures were used for opsonization, and the results are given in
Table I. The cross signs under each column represent the average
number of the bacteria taken up by a phagocyte, each cross standing
for three bacteria.
Simon, L a m a r and Bispham, .four. of Exper. Med., 19o6, viii, 651.
is Bulloch and Atkin, Roy. Soc. Proc., 19o5, lxxiv, 379.

Hideyo Noguehi.

457

The bacteria were Staphylococcus aureus, twenty-four hours
old, and the leucocytes human. The tubes were incubated at 37 °C. for thirty minutes, and the technique employed was that given
by Wright.
TABLE

I.

Varying Quantities of gt~ N. HCI Added to Leave the Indicated Degree of Alkalinity
Unneutralized. Total Volume 5 cc.
Original
Alkalinity.

Dog

Alkalinity I Alkalinity
LeA o. 5 c.c. I Left o 3 c c.

0 . 5 C.C.

Ox

+++
0.75
++++

Pig

0.8

Alkalinity
LeR o 2 c.c.

Alkalinity
L e f t o . I c c.

N e u t r a l Re,actlon.

++++

++++

+++

+++

++++

++++

++++

+++++

+++++ ++++++++++++++++

Rabbit

0.6 5

+++

+++

+++

++++4-+ ++~++++++++++ +++++++

++++

++++

++++

As the foregoing table shows, a greater degree of opsonization
is obtained at the neutral reaction than at the inherent alkaline
reactions of the serums.
For the next series of experiments the inhibitory action ofalkalinity upott opsonins was made more apparent by increasing thealkali in an ascending scale.
II.

TABLE

Amouut of ~g N. NaOH Added. Total Volume ,Original
Alkalinity.
Dog
Ox
Pig
RabbitO. 5 C.C.

0.75
0.8

0.65

13c.c.

O.5 C C.

-¢-++
++++
+++++
+++

+
+
+
-4-

++
4-++
+++
4-+

Negative

Negative
6~~C

When 2 c.c. of ~ N. sodium hydrate solution are added to I, c.c.
of a serum the opsonizing property of the latter completely disTABLE
Neutralized

Amount of ~ N. NaOH Added. Total Volume 5 c.c.

Serum.
O

Dog
Ox
RabbitIII.

++÷++++++++++++++
++++

x--2
CmC--

x 4 c.c. c .~.6
c__

!

I

X, 7 C.C.

L 8 C.C.

Negative

Negative

{

+4-

+

~

c¢
l~

I ++

L

+

/

*-

/

"

458

Influence of Reaction upon Opso~ins.

appears; I c.c. of this solution added to I c.c. of serum reduces the
opsonie activity to the minimum.
Table III shows the maximum
alkalinity in which opsonins are still able to act.
An alkali content approaching 1.6 c.c. suppresses the activity of
opsonins. It is rather remakable that this degree of alkalinity is
only about twice as high as that possessed by the majority ofnormal serums.
In Table I. it is shown that the activity of opsonin is greater at
the neutral point than at the alkalinity possessed by normal serums,
but the difference was not a marked one. In the following experiment the inhibitory influence of the native alkalinity is brought tolight by means of dilution. The normal serum of the pig was
neutralized with hydrochloric acid and divided into two portions.
To one portion sodium hydrate solution was added to make it contain just enough alkali to reproduce the original degree of alkalinity, namely, o.8 c.c. of ~ N. To the second portion no alkali wasadded, but it was employed in the neutral reaction. These two
portions were used for opsonization in ascending dilutions.
T A B L E IV.
F

Dilution of the Neutral- Alkalinity dg N. N a O H Alkalinity ~ N. N a O H o.8 c.c.
ized Serum (Pig).
0.8 c.c.
Neutralized Back withi~ N. HCI 0.8 c.c,

x:5
1:7

i

I

+++++++ ++

I:IO

+÷+

x:x5

+

I:20

1:25
x:3o
1:35
1:4°
1:5o
1:60

++++++
+++++
+++
+++
++
++
++
+
+

Controlin Saline with
Neutralized Serum
Alone,

4-+++++
+++++
+++
+++
+~
++
+5
+
+

1:8o

As Table IV. shows, the alkali restrained the opsonic action
markedly and the minimum opsonization with the alkalinized portion was between I : I5 and I : 2o, while the neutral portion was
still active at 1 : 3 o to 1 : 4 o dilutions. This restraining effect ofalkali was not so evident in the concentrated state of the serum,
but was rapidly developed as the dilution was increased. It may

Hideyo Noguehi.

459

be remarked in passing that the inhibitory influence of the reaction on ferments is by no means a quantitative one. No matter
how large a quantity of ferment is present in a mixture no action
follows if the reaction is highly unfavorable. When the reaction
is merely such as to inhibit partially, only so much effect is obtained as the degree of optimum permits. Under such circumstances, the greater portion of ferment may remain inactive. It isonly when ferment finds itself in a fluid of optimum reaction that
real quantitative differences can be manifested. The opsonic indices can seldom be driven beyond four, and usually not beyond twoor three, in spite of repeated vaccination with bacteria. The reason
for this limit is not at once apparent, but it is not improbable thateven here the reaction of the serum may play a part. Judging frommy experiments, the estimation of the opsonic index should be madeat the neutral reaction and in a diluted serum. The advisabilityof dilution has been pointed out especially by Simon and his coworkers; and with this idea the results of Neufeld and Rimpau
with certain immune serums agree.

The Influence of Acidity.--Opsonins having been shown to be
more active in a neutral than in an alkaline medium, the next point
to be examined was the influence of the acid reaction upon the opsonization. Table V shows that opsonins are highly sensitive to theacid reaction.
TABLE V.
Neutralized
Serum,

Amouut of ~ N, HCI Added to the Neutralized Serum.

o 3 c.c.

Dog
Ox
Pig
Rabbit+4-+++++++++++++++
++++

+++
+++++
+++

++
++
+++
++

T o ta l Volume 5 c.c,Io--5 c.c.

o . 7 c c.

z

Negativec~-4-

If to the neutral serum is added 0. 5 c.c. of #~ N. hydrochloric
acid solution, no opsonic action is to be obtained. The quantityof acid indicated in the table was added to an amount of the neutralized serum corresponding to I c.c. of the original.

460

Influence of Reaction upon Opso~ins.

Are Bacteria Opsonized in the Presence of Unfavorable Reaction?
The restoration of opsonic activity.
Bacteria suspended in a serum which contains enough acid or
alkali to suppress opsonization under ordinary conditions were centrifugalized and washed in 0. 9 per cent. salt solution. The washedbacteria were then mixed with leucocytes and incubated as usual.
It was found that these bacteria were not taken up by leucocytes.
Thus it can be concluded that when the reaction is unfavorable,
opsonins do not attach themselves to the bacteria. .An analogous
phenomenon was encountered by Hektoen and Ruediger 17 in thecase of certain antiopsonic neutral salts.
In these respects, opsonins seem to differ from most bacteriolyric and h~emolytic amboceptors, for they sensitize the corresponding cells in a medium containing an inhibitory amount of acid for
opsonins. The sensitiveness of opsonins to slight degrees of acidand alkali is similar to complement, although opsonins surpass
complements in sensitiveness.
The fact was ascertained that inactivation of opsonins by meansof an unfavorable reaction is a reversible reaction. If the excessof acid or alkali is removed by neutralization the opsonins become
active once more. In this respect they resemble complements. TM.A difference is observed in that opsonins are more active in neutral
media, and complements more active in alkaline media. Further
calcium chloride reduces the action of complements but not of
opsonins, provided the concentration of this chemical used does notexceed i~ N.
Organic and inorganic acids in concentration of I N. render theserum opsonically inactive even after neutralization. The modification produced is irreversible.
Before leaving this topic, I wish to speak of an incident concerning the effect of small quantities of acids on the opsonic power ofserum. At the beginning of this study, I employed ~-~ N. solutionof various acids---hydrochloric, nitric, sulphuric, formic, acetic,
propionic, lactic, butyric, oxybutyric, citric, tartaric, oxalic, male1, Hektoen and Ruediger, ]our. of Infect. Diseases, 19o5, ii, 528.
as Noguchi, On the chemical inactivation and regeneration of complement.
Read at the annual meeting of the Society of American Bacteriologists, Dec.
28, 19o6.

Hideyo Noguehi.

461

inic, fumaric, itaconic, citraconic and glycerino-phosphoric--and as
each in turn was added in small quantities to the serum the opsonicindex was observed to rise. Contrary to my expectation, theaddition of acid gradually but perceptibly increased the opsonic powerof the serum. This first led me to think that acids might themselves act as opsonins. But the power was not further increased
as the neutral point was reached, and from that point on, the increasing acidity finally suppressed it altogether.
EFFECTOF

DESICCATION

AND

DRY

HEAT

ON

OPSONINS.

Effect of Desiccation.--Opsonins are highly labile bodies. - Theiraction disappears from the serum when it is allowed to stand for several days. In this respect they resemble complements. But noexperiment has been recorded which tells us whether opsonins are
obtainable in the dry state or not. I have tested this point. Normal
serums of dog, ox and pig were dried at 23 ° C. with the aid of
an air current, and the dissolved masses were tested again for theiropsonic power. It was found that the opsonic power of the serum
is not noticeably reduced by drying in this way. The next point
was whether the opsonins would endure longer in the dried state.
To determine this, I employed three samples of dried serum which
had been preserved in the laboratory for two years. The serums
were obtained from the rattlesnake, horse and ox, and were driedat a temperature approaching 480 C. Although I am unable tostate the original activity at the time of drying, I can state that
all three serums exhibited marked opsonic powers. Thus opsonins,once they are dried, are not labile substances.
On the other hand, dehydration "of serum by means of alcohol
renders the opsonins completely inactive. This fact would showthat opsonins differ at least in this respect from many ferments,
which stand treatment with alcohol, if not too prolonged.
Effect of Dry Heat.--The normal serum is robbed of its opsonie
power by temperatures ranging from 55 ° to 6o ° C. Even immune
serums, which are the most resistant in this respect, lost the greaterpart of their opsonic power at these temperatures, although traces
may persist in a serum heated to 65 ° C. On the whole, opsonins
are more thermostabile than complements, although less than the462

Influence of Reaction upon Opso~ins.

usual immune bodies. The slight difference in the thermal resistance obtained by different investigators may depend upon differences in the reaction or some other physical or chemical conditions
under which the tests were made.
Opsonins resemble ferments in their behavior to high temperaturein the dry state. I have subjected the dried serums of ox, rabbit
and dog to the temperature of Ioo °, I2o °, I35 ° and I5 °° C., andthen tested them for their opsonizing power. When the serums
are heated to I2o ° C. and above, they become almost insoluble inwater. To examine their activity, they must be powdered andemulsified in water. The-bacterial emulsions were added to theformer emulsions and the whole incubated at 37 ° C. for two hours.
The result is given in Table VI.
TABLE

VI.

O p s o n i c A c t i v i t y o f t h e H e a t e d or U n h e a t e d D r i e d S e r u m .

Unheated
Dried Serum.

Dried SerumH e a t e d tozoo ° c c.

Dried SerumH e a t e d tow o ° C.

i
Dried Serum Dried SerumH e a t e d to
H e a t e d to
x35 ° C.
it5o ° C.

ActiveLess active, IVery irreguActive
Activephagocytosis phagocytosis phagocytosis but positive.' lar occurrence of,,phagocytosi~
Rabbit serum I"(vaccinated)
Dog serum"0 x serum

Unheated
Dried Serum,
Redissolved
and thenHeatedt o 56 ° C.

Negative.

From the foregoing experiment a high degree of resistance ofthe dried opsonins to high temperatures may be inferred. It is
important to note that the dried serums regain their thermolability
when redissolved in water, as is shown in the last column of thetable.
Hence, it would appear that opsonins possess in common withferments the property to resist in the dry state the action of hightemperatures.
In the course of the experiments on desiccation and high temperature on opsonins, I took the opportunity of examining complements in the same manner.
It developed that complements do not disappear from the serum
when it is dried at ~3 ° C. The desiccated serum retained for several months complements unaltered in quantity and in activity.

Hideyo Noguchi.

463

Heating the dried serum to IOO°, 12o ° and 135 ° C. does notdeprive it of complementary action.
SUMMARY.

Opsonins reveal their maximum action in a medium of neutral
reaction. No opsonization takes place in a serum which contains
an amount of alkali corresponding to more than 1.6 c.c. of a ~N. solution, or acid more than 0. 5 c.c. of this concentration per ic.c. of serum. Of several normal blood serums titrated (lacmoid
used as indicator) the average alkalinity was found to be equivalent to about 0.8 c.c. of ~v N. solution.
The opsonic index obtained in the native serums is not the expression of the action of the whole content of opsonins, but only
so much as the degree of optimum of the reaction permits to comeinto action. Estimation of the opsonic power should, therefore,
be made in a medium of neutral reaction and in diluted serum.
_All serums have their opsonic power increased by diminishing
the native alkalinity.
Opsonins whose activity is suspended by an unfavorable reaction
become immediately active as soon as the reaction is brought backto the neutral point, unless the acid or alkali employed approaches
the strength of I N., at which point the alteration becomes permanent.
Treatment of a serum with alcohol robs it of its opsonic power.
The opsonic power of serum remains unaltered upon desiccation at
23 ° C. In the dry state opsonins are preserved for two years.
The temperatures of lOO°, 12o °, 135 ° and 15°o C. do not destroy
opsonins of the dried serum.
Complements of serum are also siccostabile and are preservablein that state for several months. Dry heat of I35 ° C. reduces but
does not destroy the complementary power of the dried serum.
The opsonins and complements of the dried serum regain their
original thcrmolability when they are dissolved in a proper amountof water.
Lastly, it may be emphasized that opsonins exhibit in their sensitiveness to reaction and resistance in the dry state to high temperatures certain properties characteristic of the ferments.