THE RELATIOK’SHIP BETWEEN THE BRCTERI- CIDAL POWER O F NORMAL GUINEA-PIG SERUM AND COMPLEMENT ACTIVITY.

JOHN GORDON and ARTHUR WORMALL. From the Department of Pathology and Bacteriology, and the Department

of Physiology, the University of Leeds.

THE present authors in collaboration with 33. R. Whitehead have previously shown (1926 l) that hsemolytic complement consists of a system of four components, all of which are necessary for the hsemolysis of sensitised red cells, and that destruction of any one component deprives the serum of its hamolytic power. Since methods are available for inactivating one component without material loss of the other components, it was thought that it would be possible to determine what relationship, if any, exists between the haemolytic complement of serum, the opsonic power and the bactericidal power of normal serum. It has been shown (Gordon, Whitehead and Wormall, 19213 2, that the whole of the complement system concerned with hsemolysis is not essential for opsonic action, since by the action of a small amount of ammonia a serum is obtained which has no haemolytic power but still has the same opsonic power as the original serum. Thus the fourth component of complement, a relatively heat- stable component which is readily inactivated by ammonia, is not essential for opsonic action.

Many investigators have studied the relationship between the bactericidal powers of normal and immune sera against various bacteria and the hzmolytic power of the same sera for sensitised red cells, and in certain instances a general relationship between the two systems concerned has been noted. In this work we have dealt with the bactericidal power of normal guinea-pig serum for B. dysenterice (Flexner) [Eberthella paradysenterice] and have found that the process is one which bears a very close relationship to hamolysis of sensitised red cells by the same serum. In an extensive study of the bactericidal power of normal serum, Muir and Browning (1909) review the earlier literature and reach the conclusion that the results of earlier workers cannot be brought into harmony. These authors find, however, that treatment of a normal guinea-pig serum with increasing amounts of an emulsion of dead bacteria usually produces a diminution in the bactericidal power, first for the same bacteria and later for other bacteria, and finally there is a diminution in hsmolytic complement.

763

754 1. GORDON AND A. WORMALL

On occasions, however, there is a marked loss of bactericidal power for one organism by treatment of the serum with other dead organisms without simultaneous loss in hzmolytic complement and it is concluded that there is a special fraction of the serum which is concerned in bactericidal action ; this fraction is termed I‘ bacteriophilic comple- ment” by Muir and Browning, but this, to quote the authors, “does not mean that bactericidal complement is distinct from. the haemolytic complement.” The explanation given to account for the phenomena involves the assumption that in serum there are present complement molecules of different degrees of activity and that those of greatest activity are first absorbed by bacteria in general. Addition of an artificial immune body will, however, bring into combination a weakly acting complement, and under these conditions bactericidal power can be demonstrated. Some of these phenomena are similar to those which we have observed in our experiments with the bactericidal power of normal guinea-pig serum, measuring the bactericidal power against B. dysenteria: (Flexner), but the resiilts appear to indicate that a very close relationship exists between hzmolytic complement and bactericidal complement ; indeed no significant differences have been observed and the conclusion is reached that one complement is responsible for the two processes. This is a view which has been expressed by several workers but no conclusive evidence appears to be available that the two systems are identical.

Liefmann and Stutzer (1910) investigated the bacteriolysis of the cholera vibrio [ Viibrio comma] and found that this process was unlike the process of haemolysis since it required only the “ end-piece ’) or albumin fraction of serum ; Braun (1911) was unable to confirm these results and claimed that ‘‘ bactericidal complement,” like hsmolytic complement] is a function of two serum constituents, one in the globulin fraction and the other in the remainder of the serum. Liefmann (1911) reaches a somewhat similar conclusion. Boehncke (1912) carried out investiga- tions similar to those of Braun, using different methods of “ splitting ” serum, to determine the rBle of the globulin and albumin fractions of serum (“ mid-piece ’I and “ end-piece ” respectively) in bactericidal processes. These two fractions of guinea-pig serum, obtained by the HCl method of Sachs and Altmann (1909) or by the CO, method of Liefmann (1909), had separately no bactericidal power against cholera vibrios in the presence of an immune serum (amboceptor), but if the two fractions were added together, the bactericidal power of the serum was restored. This similarity in structure between hsmolytic complement and bactericidal com- plement was supported by the weakening of both functions, bactericidal and haemolytic, when the serum was diluted with water and kept a t 37” for 12 hours. Boehncke also found that cobra venom has a destructive action on bactericidal power similar to that on hsmolytic complement, and that the cobra venom- inactivated serum could be activated, for bactericidal power as well as hsmolytic power, by the addition of “ end-piece ” or “ mid-piece )’ fractions, the latter usually being the more effective.

Pettersson (1927) in a review of bacterial immunity separates the bactericidal ‘‘ substances ” of serum into a-lysins and p-lysins, the former syystem consisting of complement plus immune body. The j3-lysins of serum consist, according to Pettersson, of an “activating” substance, which is inactivated by heating a t 63” to 65°C. for +hour but which is more stable than hsmolytic complement, and

BACTERICIDAL POWER A N D COMPLEMENT 765

an ~activable ” substance which is bound to the bacteria on which the “activating” substance acts. The two systems appear to be of very similar nature, viz.-

a-lysins = complement plus immune body. p-lysins = “ activating )’ substance plus ‘‘ activable )’ substance.

The “activable” substance of the B-lysin may be compared therefore to the immune body necessary for the action of hzemolytic or bactericidal complement, but unlike the immune body it does not attach itself to the bacteria unless the (‘activating ” substance is present at the same time. The “activating ” substance is similar to hzemolytic complement but requires heating for 4 hour at a higher temperature for inactivation (sometimes up to 68” to 70°C.) and also it is not affected by a reduction in the salt concentration, or by treatment with ether, petroleum-ether or chloroform (c f . Pettersson, 1928). Pettersson tabulates the bacteria which are affected by the different bactericidal systems and suggests that there are chemical differences between the cells affected by a-lysins and those affected by p-lysins. It is of interest to note that B. dysenterict! (Flexner), which is the organism we have used in our experiments, is included in those which are influenced by the a-lysins, i.e. the complement plus immune body system,

The experiments described below, whilst they are not inconsistent with the view that a special bactericidal complement exists, show that the complement required for bactericidal action has similar properties to that taking part in specific hsemolysis. The destruction of complement or one of its active components results in a loss of the bactericidal power, and conversely a serum with bactericidal power and no hzmolytic complement has not yet been obtained. Under certain conditions, e.g. by absorption with an emulsion of dead bacteria a serum can be obtained having no bactericidal power but having full hsemolytic action on sensitised red cells, but this is due, in our interpre- tation, to the removal by the dead organisms of the necessary immune body. The whole normal bactericidal action of normal guinea-pig serum against B. dysenterias! (Flexner) appears to consist therefore of a bacteriolytic process involving the action of an immune body and a complement, a system similar to that necessary for the hsmolysis of red cells. The complement required for this bactericidal process appears, from the experiments described below, and from the splitting experimenls of Braun and Boehncke mentioned above, to be identical with that affecting hsmolysis of sensitised red cells.

The first experiments which we carried out were concerned with the action of ammonia on the bactericidal power of normal guinea-pig serum against B. dysenteriat: (Flexner), and it was found that the ammonia - treated serum possessed no bactericidal power in the experiments where sufficient ammonia was used to inactivate the hzmolytic complement. Thus in all cases where inactivation (by ammonia) of the fourth component occurs, there is loss of bactericidal power. The hsemolytic power of such an ammonia-inactivated serum can readily be restored by the addition of guinea-pig serum which has been heated a t 56°C. for 30 minutes, and therefore to complete the parallelism between hzmolytic complement and bactericidal power it is necessary to show that the ammonia-inactivated serum could

756 3. GORDON AND A . WORMALL

have its bactericidal powers restored by the aclditioIi of heated serum. This was found to be the case (cf. table I ) and whereas neither ammonia- inactivated serum nor heated serum had any haemolytic or bactericidal powers separately, they possessed full powers in both respects when combined. From these experiments it appears that the whole of the hamolytic complement is necessary for bactericidal power. Another explanation which might be advanced to explain these results is that the immune body required for the bactericidal action is destroyed by ammonia and that the missing factor is supplied by the heated serum, but other experiments described below appear to disprove this possibility. I n order to make the correlation between hamolytic aud bactericidal processes more complete, experiments have been carried out to show that the action of the ammonia on the serum is of a "specific" nature, and that the addition of sodium hydroxide, in sufficient quantities to give the same p H values as are obtained by the addition of ammonia, has no effect on the bactericidal power just as it has no influence on hamolytic power. In the same experiments it was also shown that an equivalent amount of ammonium chloride has no significant action on bactericidal power or OR hEmolytic complement, and the destructive action in each case appears to be clue t o the presence of the free ammonia. Furthermore it was thought desirable to show, as was done for hamolytic complement, that the fourth component of complenient present in heated serum (56" C.) is destroyed when the heated serum is treated with ammonia. Serum heated a t 56" C. for 30 minutes fully activates amnionia-inactivated serum for bactericidal and hamolytic processes, but if this heated serum is itself treated with ammonia this activating function is lost.

The bactericidal power of normal guinea-pig serum against B. dysentericz (Flexner) does not depend solely, however, on hsmolytic complement. If guinea-pig serum is treated with an emulsion of the dead bacteria in suitable amounts, the bactericidal power can be destroyed, whereas full hemolytic activity remains. I n our experiments this inactivated serum can have its bactericidal power restored by the addition of the natural immune body in the form of heated serum, as was shown by Steinhardt (1905), ancl although this restoration does not appear to be complete, the loss may be attributed to some loss in complement activity by absorption of part of the complement on the dead organisms, an absorption (or adsorption) which appeals to be non-specific and which is probably similar to the adsorption of complement by such substances as kaolin, charcoal, etc. The amount of hsmolytic complement absorbed by bacteria depends there- fore on the amount of bacteria used. I n some of our experiments larger amounts of the heated bacteria have been used for the absorption of the immune body and in these instances i t has been noticed that a very marked or even complete loss of hamolytic power occurrecl. Such a serum, since it lacks both immune body and complenient,

BACTERICIDAL POWER A N D COMPLEMENT 757

should not be reactivated by heated serum, which is lacking in coniple- ment but contains the immune body. The results of these experiments showed, as anticipated, that the mixture of heated serum and serum inactivated by excess of heated bacteria had no hemolytic complement activity and no bactericidal power.

The failure of a serum treated with dead organisms to show bactericidal activity, whilst retaining its full hBmolytic activity, must be attributed to some factor which is not part of the hzmolytic complement system. One explanation which has been advanced and which is supported by our results, is that an immune body for certain bacteria, a t least for B. dysenterice (Flexner), is present in normal guinea-pig serum. This immune body is absorbed when the serum is treated with an emulsion of dead organisms (B . dysenterice, Flexner) and unless excess of organisms is used the complement is unaffected ; the serum so treated has therefore no bactericidal power but has full complement activity towards sensitised red cells. If this explanation is correct, a serum deprived of its bactericidal power by dead organisms should be reactivated for bactericidal activity by the addition of any solution which contains the natural immune body ; thus ammonia- treated guinea-pig serum, which has lost its complete complement power, should, i f it still contains the normal immune body-and other evidence outlined below tends to show that this is true-be able to supply the missing factor. I n all our experiments this result has been obtained and the two sera (a) that obtained by treatment of guinea-pig serum with dead organisms and (6) that obtained by treat- ment of guinea-pig serum with small amounts of ammonia and subsequent neutralisation, have separately no significant bactericidal power but when combined they give a solution with strong bactericidal properties. From these experiments the conclusion is reached that besides hzemolytic complement another factor or component is required for bactericidal action and this factor, which is not destroyed by the small amounts of ammonia which are sufficient to inactivate comple- ment and which is not destroyed by heating a t 56°C. for 4 hour but which is removed by dead organisms, is presumed to be an immune body present in the normal serum. I n order t o prove that this factor removed by dead bacteria (the immune body) is not destroyed by ammonia to the same extent as the fourth component of complement, we have treated heated serum (56" C.) with amounts of ammonia equivalent to those used for the other experiments and have then tested this ammonia-treated heated serum for immune body by adding it to a serum which has had its immune body removed by treatment with dead bacteria. The immune body present in the heated serum was found to be unaffected to any appreciable extent by the ammonia and the conclusion is reached from these and other experiments that the amounts of ammonia used have no great destructive action on the natural immune body.

3 c JOURX. OF PATH.-VOL. XXXI,

758 J. GORDON A N D A. WORMALL

These results do not preclude absolutely, however, the possibility that various complements exist, but all the evidence available indicates that an extremely close relationship exists between the complement required for bactericidal acLion and that required for hzmolytic action. The two systems, if they are different, have very similar or identical constitutions since both of them can be split into globulin and albumin fractions (Braun, 1911 ; Liefmann, 1911 ; and Boehncke, 1912), they are both inactivated by cobra venom (for hsmolytic complement by Sachs and Omorokow, 1911 ; Ritz, 1912 ; for bactericidal complement, Boehncke, 1912), and furthermore we have found that ammonia which in suitable amounts has a more or less specific action on one component only (a relatively heat-stable factor) of h2emolytic complement (Gordon, Whitehead and Wormall, 1926, I), has a similar action on bactericidal Complement. There appears to be no reason therefore to suggest that two distinct but similar systems are required for similar processes, and indeed no results so far obtained are incompatible with the view that one complement only exists and that this together with the more specific immune body effects hsmolysis of red cells or destruction of bacteria.

General methods.

Normal guinea-pig serum was used in all these experiments ; to obtain sterile serum the necks of the animals were shaved, treated with an alcoholic solution of crystal violet and brilliant green, washed with alcohol and cut with a sterilised razor, the blood being collected in a sterile vessel. The serum obtained after incubating the blood for 1 to 2 hours was found to be sterile in every case, and we are indebted to Mr F. Haigh of the department of bacteriology for his valuable assistance in the preparation of this sterile serum. B. dysenterice (Flexner) was grown on agar for 18 hours and a suspension of the culture made in bouillon to the strength of about 100 millions per C.C. ; 0'05 C.C. of this suspension was added to each tube of serum (normal or previously treated) and to varying amounts of serum diluted with bouillon, and all the tubes were then incubated a t 37" C. ; loopfuls were withdrawn immediately and after varying intervals and inoculated to plates of 10 per cent. heated blood-agar. The amount of growth which occurred on these plates was recorded after about 20 hours' incubation.

The hamolytic complement activities of the same sera were determined by adding various amounts of each serum to 1 C.C. of a 4 per cent. suspension of sensitised ox cells, adding 0.9 per cent. NaCl where necessary to make the total volume 2 c.c., and recording the amount of hsmolysis after incubation a t 37°C. for hour, $ hour and 1 hour. All solutions were tested and found to have no simple hsmolytic action on unsensitised red cells.

TJw influence of ammonia on the bactericidal power of serum.

Sterile guinea-pig serum was treated with varying amounts of ammonia solution (0%7 N) incubated for 1 hour a t 37" C. and the solutions neutralised to pH 7.5 by the addition of N.HC1. The bactericidal powers of these sera, (a) normal serum, (6) serum heated at 56" C. for 30 minutes, (c) the NH,-treated sera and (d) mixtures of the ammonia-treated sera and the heated serum, were then determined.

TABL

E I.

Efe

ct o

f am

mon

ia o

n. t

he b

acte

rici

dal a

nd h

amol

ytic

act

iviti

es o

j gu

inea

-pig

ser

um.

Seru

m.

Nor

mal

.

. {

NH

,-tre

ated

ser

um

(A)

{ N

H,-t

reat

ed s

erum

(B

) {

Med

ium

.

4 C.

C.

seru

m

. 2

C.C.

se

rum

+

2 C

.C.

boui

llon

. E:::

izz +

' 2

C.C.

bo

uillo

n .

l::: ::Eg

+ .

2 C

.C.

boui

llon

. 4

C.C

. se

rum

.

Bac

teri

cida

l pow

er.

Gro

wth

on

subc

ultu

re a

fter

:

(A) +

equa

l vo

l.{

heat

ed s

erum

(B)

+ eq

ual

vo~.

{ he

ated

ser

um

(c) +

equa

l vo

~.{

heat

ed s

erum

Hea

ted

seru

m

(56°

C. f

or 3

0min

s.){

imm

edia

tely

_

__

- ++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

4 C

.C.

seru

m

. E:::

~~~~

o~

. 4

C.C

. se

rum

.

:::: E:f

onf

. 4

C.C

. se

rum

.

:::: c,z

o: .

Seru

m

'

Ezgn

f .

16 1

1oun

.

1 0 0 2 + + + + 4 2 2 0 3 2 ++

++

44 h

ours

.

0 0 0 0 + + + + 0 0 0 0 0 1 ++

++

i +

Hea

vy g

row

th.

+ G

ood

grow

th.

0 N

o gr

owth

. T

he n

umbe

rs r

efer

to

the

num

ber

of c

olon

ies w

here

thes

e co

uld

be c

ount

ed.

8 ho

urs.

0 0 0 0 + ++

+ ++

0 0 0 0 0 0 ++

++

Incu

bati

on

peri

od.

4 hou

r

I ho

ur

$ ho

ur

1 ho

ur

4 ho

ur

1 ho

ur

;b ho

ur

1 ho

ur

g hou

r

1 ho

ur

3 ho

ur

1 ho

ur

hour

1 ho

ur

4 ho

ur

1 ho

ur

-~

Hse

mol

ytic

com

plem

ent

pow

er.

Hre

mol

ysis

of

sens

itise

d ce

lls b

y di

lute

d se

rum

(1 :lo

).

1.0

C.C.

xx

xx

xx

xx

x

xx

xx

xx

Y

xx

- -

xx

xx

xx

xx

x

xx

xx

xx

x

xx

xx

xx

- -

0'6

C.C

.

xx

xx

xx

xx

x

x

xx

xx

- X

- - x

xx

x

xx

xx

x

x

xx

xx

x

x

xx

xx

- -

0'2

C.C

.

xx

xx

xx

xx

X

xx

x

- -

-

X

xx

xx

x

x

xx

x

xx

xx

x

- -

x x

x x

Com

plet

e ha

emol

ysis

. -

No

hem

olys

is.

x x

X,

x x

and

x In

term

edia

tegr

ades

ofh

emol

ysis

. N

H t

reat

ed s

era

(A, B

and

C):

15

C.C

. gu

inea

-pig

ser

um +

0.87 N

. NH

, (0.10

c.c.

, 0.20 C

.C.

and 0'30 C

.C.

for

A,

B a

nd C

resp

ectiv

ely)

-inc

ubat

ed

Not

e.-T

he

dete

rmin

atio

n of

the

bac

teri

cida

l po

wer

was

mad

e in

all

case

s by

sub

cultu

ring

aft

er ti

mes

inte

rmed

iate

bet

wee

n th

ose

reco

rded

. T

he

at 3

7" C!:

for

1 ho

ur a

nd th

en n

eutr

alis

ed b

y th

e ad

ditio

n of

N . H

CI (

appr

ox. 0

'08

c.c.

, 0'16 C

.C.

and

0'25

C.C

. fo

r A

, B a

nd C

resp

ectiv

ely)

.

resu

lts o

btai

ned

wer

e si

mila

r to

thos

e fo

r 1;

hour

s, 4

?, h

ours

and

8 h

ours

.

760 J. GORDON AND A. WORMALL

The results of these experiments and the complement powers of the same sera are given in table I. From this table it will be seen that ammonia-treated sera, B and C, have lost all their bactericidal power and all, or practically all, their complement; activity ; the very slight complement activity of ammonia-treated serum B in this particular experiment is apparently not sufficient to exert any noticeable action on the relatively large amount of bacteria used in the bactericidal tests, and indeed it is not sufficient to give any significant ha3molysis of sensitised red cells i f the amount of cells is increased or if the extent of sensitisation of the red cells is diminished. The addition of serum heated a t 56" C. for 30 minutes, which alone has no bactericidal or specific hEmolytic action, to ammonia-treated sera 73 and C regenerates almost completely the original bactericidal and 1iEmolytic powers. Still smaller amounts of ammonia (ef. ammonia- treated serum A) have no apparent influence on the bactericidal power or the ha3molytic properties and in all experiments it has been found that if the amount of ammonia used is sufficient to inactivate the hEmolytic complement, loss of bactericidal power occurs.

Experiments have also been carried out to show that the power of heated serum to reactivate amnionia-treated serum for bactericidal power is lost if the heated serum itself is previously treated with ammonia. Heated serum and normal serum were treated with equal amounts of ammonia (12 C.C. serum plus 0.26 C.C. of 0.87 N . NH,). The mixtures were incubated for 1 hour and then neutralised by the acldition of the requisite amount of N . HC1. The bactericidal power and complement activity of these sera and various mixtures were tested as before and the results are given in table 11. From this table it will be seen that the component present in heated seruni which activates ammonia-treated serum for bactericidal power or complement power is destroyed if the heated serum is itself treated with ammonia.

INfiuence of sodium hydroxide and ammonium chloride on the bactericidal power of serum.

The inactivation of hamolytic complement by ammonia has been shown to be more or less specific for ammonia and closely related amines (Gordon, Whitehead and Wormall, 1926, 1) and a similar inactivation cannot be effected by the addition of sodium hydroxide in amounts sufkient to bring the pH of the serum up to that of the serum treated with ammonia. Similar experiments have been carried out with regard to the destruction of bactericidal power by ammonia and the results run parallel with those obtained previously for hamolytic complement. The destruction of bactericidal power is not due to simple alkalinisation of the serum nor is there any loss in bactericidal power when the serum is treated with an equivalent amount of ammonium chloride (table 111).

TABL

E 11.

R

eact

ivat

ion

of a

mm

onia

-tvea

ted

seru

m b

y he

ated

ser

um b

ut n

ot b

y am

mon

ia-tr

eate

d he

ated

ser

um.

Bac

teri

cida

l pow

er.

Hae

mol

ytic

com

plem

ent p

ower

.

Qro

wth

on s

ubcu

lture

aft

er:

Hae

mol

ysis

of s

ensi

tised

cel

ls b

y di

lute

d se

rum

(1 : 10

). In

cuba

tion

peri

od.

Med

ium

. [m

med

i- at

ely.

1

hour

. 2

hour

s.

S ho

urs.

5

hour

s.

7 ho

urs.

0.

8 C

.C.

xx

xx

xx

xx

-

-

-

-

-

-

xx

xx

xx

1.0

C.C

.

xx

xx

xx

xx

- - -

-

-

xx

xx

xx

xx

~

Nor

mal

(gu

inea

-pig

) 3

C.C

. se

rum

1.

5 C

.C.

seru

m +

1

*5 C

.C.

boui

llon

+t

++

++

++

+t

++

t+

++

++

++

2 1

++

++

++

+

+

++

t+

18

7 .~

1

0 ++

++

++

++

++

++

2 8

0 0 ++

t+

++

++

++

t+

2 7 ~

0 0 ++

t+

+t

++

++

+ t-

0 39

0 0 ++

++

++

++

++

++

41 +

6 ho

ur

1 h

our

4 ho

ur

1 ho

ur

4 ho

ur

1 h

our

6 ho

ur

1 h

our

4 ho

ur

1 h

our

{ N

H,-

trea

ted

seru

m (

A)

3 C

.C.

seru

m

1.5

C.C

. se

rum

+

1.5

C.C

. bo

uillo

n

3 C

.C.

seru

m

1.5

C.C

. se

rum

+

1.5

C.C

. bo

uillo

n H

eate

d se

rum

(5

6" C

.) tr

eate

d w

ith N

H, (

B)

A +

B (e

qual

amou

nts)

3

C.C

. se

rum

1.

5 C

.C.

seru

m +

1.

5 C

.C.

boui

llon

3 C

.C.

seru

m

1.5

C.C

. se

rum

+

1.5

C.C

. bo

uillo

n {

A -t h

eate

d se

rum

.

+ +

Hea

vy g

row

th.

x x

x x

Com

plet

e ha

emol

ysis

. -

No

hem

olys

is.

x x

x ,

x x

and

x In

term

edia

te g

rade

s of

hE

mol

ysis

. +

Goo

d gr

owth

. 0

Nog

row

th.

The

uum

birs

refe

r to

the

num

ber

of c

olon

ies w

here

thes

e co

uld

be c

ount

ed.

Seru

m.

Nor

mal

.

. {

NH

,-tre

ated

ser

um

NH

3-tr

eate

d se

rum

NaO

H-t

reat

ed

seru

m (C

)

NaO

H-t

reat

ed

seru

m (

D)

NH

,Cl-

trea

ted

seru

m (E

)

NH

,Cl-

trea

ted

seru

m (

F)

-I

TABL

E 11

1.

Com

pari

son of

the

actio

n of

am

mon

ia, s

odiu

m h

ydro

xide

and

am

mon

ium

chl

orid

e.

Med

ium

.

3 C

.C.

seru

m

. 1'

5 C

.C.

seru

m+

1 '5

C.C

. bo

uillo

n .

3 C

.C.

seru

m

. 1.5

C.C.

se

rum

+

1.5

C.C.

bo

uillo

n .

3 C

.C.

seru

m

. 1 *5

C.C.

seru

m +

1 *5

C.C.

boui

llon

. 3

C.C

. se

rum

.

1 5

C.C

. se

rum

+

1.5

C.C

. bo

uillo

n .

3 C

.C.

seru

m

. 1'

5 e.

c. s

erum

+ 1.

5 C

.C.

boui

llon .

3 C

.C.

seru

m

. 1 '

5 C

.C.

seru

m +

1'5

C.C

. bo

uillo

n .

3 C

.C.

seru

m

. 1'5

C.C

. se

rum

+ 1.

5 C

.C.

boui

llon

.

Bac

teri

cida

l pow

er.

Hzm

olyt

ic c

ompl

emen

t po

wer

. I

mm

edia

tely

.

++

++

++

++

++

++

++

++

++

++

++

++

++

++

Gro

wth

on

subc

ultu

re a

fter

:

1 ho

ur.

0 0 ++

++

++

-k +

0 0 0 0 0 0 0 0

3 ho

urs

0 0 ++

++

++

++

0 0 0 0 0 0 0 0

I I H

iem

olys

is o

f sen

siti

sed

cells

by

dilu

ted

seru

m (1

: 10

) In

cuba

tion

8 ho

urs.

1'0

C.C

. _

__

~ pe

riod.

1

0 0 ++

++

++

++

0 0 0 0 0 0 0 0

hour

1 ho

ur

+ hou

r

1 ho

ur

+ hou

r

1 ho

ur

+ hou

r

1 ho

ur

2 hou

r

1 ho

ur

$ ho

ur

1 ho

ur

+ hou

r

1 ho

ur

xx

xx

xx

xx

X

X -

-

xx

xx

xx

xx

x

xx

x

xx

xx

x

xx

x

xx

xx

x

xx

x

xx

xx

0.6

C.C.

xx

xx

xx

xx

- - -

-

xx

xx

xx

xx

x

xx

x

xx

xx

x

xx

x

xx

xx

x

xx

x

xx

xx

0.2

C.C

.

xx

x

xx

xx

-

-

-

X

xx

xx

X

xx

x

x

xx

xx

x

x

xx

xx

+ + H

eavy

gro

wth

. +

Goo

d gr

owth

. 0

No

grow

th.

The

num

bers

ref

er t

o th

e nu

mbe

r of

col

onie

s whe

re th

ese

coul

d be

cou

nted

. -

No

haem

olys

is.

x x

x x

Com

plet

e ha

emol

ysis

. x

x x ,

x x

and

x In

term

edia

te g

rade

s of

hae

mol

ysis

. N

ote.

-Ser

a A

and

B w

ere

activ

ated

for

hae

mol

ytic

com

plem

ent a

ctio

n by

the

add

ition

of

heat

ed s

erum

(56

" C.

for

hal

f an

hou

r) a

s in

tabl

e I.

BACTERZCIDAL POWER A N D COMPLEMENT 763

Xxperimental details (table 111). NH,-treated serum (A).-8 C.C. serum plus 0.13 C.C. 0.87 N . NH,. NH,-treated 6erum (B).-8 C.C. serum plus 0.2 C.C. 0.8'7 N . NH,. NaOH-treated serum (C).-8 C.C. serum plus N. NaOH to raise pH to

NaOH-treated serum (D).-8 C.C. serum plus N . NaOH to raise pH to

NH,Cl-treated serum (E).-8 C.C. serum plus NH,C1 in amount

NH,Cl-treated serum (F).-S C.C. serum plus NH4C1 in amount

All these solutions were incubated a t 37" C. for 1 hour, and sera A, B, C and D were neutralised to pH 7.5 by the addition of the necessary amount of N.HC1, aliquot portions being titrated to determine the exact amount of acid required.

that of serum A (approx. 0.08 c.c.).

that of serum B (approx. 0.12 c.c).

equivalent to the NH, used for serum A.

equivalent to the NH, used for serum B.

Removal of the bactericidal power of serum by treatment with dead bacteria and reactivation of this serum by the addition of RH,- treated serum.

The treatment of serum with a heated emulsion of bacteria removes the bactericidal power of this serum and if the amount of dead bacteria is suitably controlled, no appreciable loss of hzmolytic power is observed. For these experiments a heavy suspension of B, clyscnteria (Flexner) from an 18-hour culture was made in 0.9 per cent. NaCl solution to a strength of about 5000 millions per C.C. and the suspension heated at 65°C. for 1 hour and tested for sterility, when no growth occurred. 0.5 C.C. and 1.0 C.C. of this heated suspension were added to tubes containing 12 C.C. of normal guinea-pig serum and the iiubes incubated a t 37°C. for 2 hours. The serum was then centrifuged and the clear supernatant fluid pipetted off and used for testing the bactericidal power and complement activity.

Xxperinlmtal details (table IV). NH,-treated serum (A).-12 C.C. serum plus 0.1 C.C. 0.87 N.NH,-

incubated 1 hour and neutralised as before. NH,-treated serum (B).-12 C.C. serum plus 0.2 C.C. 0.81 N.NH,-

incubated 1 hour and neutralised as before. Serum (C).-12 C.C. serum plus 0.5 c c. suspension of heated B. dyselzterict:

(Flexner), incubated 2 hours a t 37" C. and then centrifuged. Serum (D).-12 C.C. serum plus 1.0 C.C. suspension of heated B. dysenteria

(Flexner), incubated a t 37" C. for 2 hours and then centrifuged.

From the results, which are tabulated in table IV, it was found that serum B had no bactericidal power and no complement activity, whereas serum A, which was treated with less ammonia, retains some

TABL

E IV

. Jf

utua

l act

ivat

ion f

or b

acte

rici

dal p

ower

of

amm

onia

-tre

ated

seru

m a

nd s

erum

tre

ated

wit

h de

ad b

acte

ria.

Bac

teri

cida

l pow

er.

Ham

olyt

ic c

ompl

emen

t pow

er.

Gro

wth

on

subc

ultu

re a

fter

: H

amol

ysis

of s

ensi

tised

cel

ls b

y di

lute

d se

rum

(1 : 10).

Seru

m.

Incu

bati

on

perio

d.

Med

ium

. m

med

iatd

y &

hour

s.

6 ho

urs.

8

hour

s.

1.0

C.C.

0.

6 C.

C.

0.2

C.C.

3

hour

s.

0 1 0 10

++

++

0 0 ++

++

0 0 0 0 0 0 0 0

3 C.

C.

seru

m

. 1.

5 C

.C.

seru

m +

1.5

C.C

. bo

uillo

n .

3 C.

C.

seru

m

. 1.

5 C

.C.

seru

m +

1.

5 C

.C.

boui

llon

. 3c

.c.

seru

m

. 1.

5 C

.C.

seru

m +

1'

5 C

.C.

boui

llon

. 3

C.C.

se

rum

.

1'5

C.C

. se

rum

+

1.5

C.C

. bo

uillo

n .

3 C.

C.

seru

m

. 1.

5 C

.C.

seru

m +

1.5

C.C

. bo

uillo

n .

3 C.

C.

seru

m

. 1.

5 C

.C.

seru

m +

1.

5 C.

C.

boui

llon

. 3 C.C.

seru

m

. 1.

5 C

.C.

seru

m +

1.5

C.C.

bo

uillo

n .

3 C.

C.

seru

m

. 1.5

C.C

. se

rum

+

1.5

C.C

. bo

uillo

n .

3 C

.C.

seru

m

. 1.

5 C

.C.

seru

m +

1.

5 C.

C. bo

uillo

n .

++

++

++

++

++

++

++

++

++

-ti

++

++

++

++

++

++

++

++

2 0 1

16

++

++

0 22

++

i

t

0 0 0 1

I 1

0 26

0 0 2 i-

++

++

0 0 ++

++

0 0 0 0 0 0 0 0

$ ho

ur

1 ho

ur

; hou

r

1 ho

ur

; hou

r

1 h

our

hour

1 ho

ur

+ hou

r

1 ho

ur

$ ho

ur

1 ho

ur

; hou

r

1 h

our

+ hou

r

1 ho

ur

8 ho

ur

1 ho

ur

xx

xx

xx

xx

x

x

xx

x

-

-

xx

xx

xx

xx

x

xx

xx

xx

x

xx

x

xx

xx

x

xx

x

xx

xx

x

xx

x

xx

xx

x

xx

x

xx

xx

xx

x

xx

xx

X x

x

-

-

xx

x

xx

xx

x

xx

xx

xx

x

xx

xx

xx

x

xx

xx

xx

x

xx

xx

xx

x

xx

xx

xx

0 0 + ++

++

+ -I-

0 -I- ++

+

+

0 0 0 0 0 0 0 0

X

xx

x

- - - - - xx

- X - x

x

- xx

X x

x

- xx

ysis

.

Nor

mal

.

. .

1 NH

,-tre

ated

se

rum

(A

)

1 NH

,-tre

ated

se

rum

(B)

. 1 1

I (C) :

12

C.C

. no

rmal

ser

um

+0*

5 C.C.

heat

ed

bac-

te

rial

em

ulsi

on

(D) :

12 C.C.

norm

al s

erum

+

la0

C.C

. he

ated

ba

c-

teri

al e

mul

sion

Equ

al v

ols.

of (

A) a

nd (C

) I ,

i , E

qual

vol

s. of

(A

) and

(D)

' E

qual

vol

s. of (

B) a

nd (C

)

Equ

al v

ols.

of (

B) a

nd (D

)

+ +

Hea

vy g

row

th.

- N

ohae

m

+ G

ood

grow

th.

x x

x x

Com

plet

e ha

emol

ysis

. 0

Nog

row

th.

The

num

bers

ref

er t

o th

e nu

mbe

r of

col

onie

s w

here

the

se c

ould

be

coun

ted.

&

&.-S

era

A a

nd B

wer

e ac

tiva

ted

for

hzm

olyt

ic c

ompl

emen

t ac

tion

by t

he a

dditi

on o

f he

ated

ser

um (

56" C

. for

hal

f an

hou

r) a

s in

tab

le I

. x

x x ,

x x

and

x In

term

edia

te g

rade

s of

hre

mol

ysis

.

BACTERlClDAL POWER AND COMPLEMENT 765

of its bactericidal and hsmolytic powers. Serum D had lost all (and serum C some) of its bactericidal power, but both contained approximately the same amount of complement as the original serum and therefore the loss in bactericidal power on treatment with the heated organisms is not due to loss of complement but presumably to the loss of an immune body to this bacillus normally present in guinea-pig serum. This immune body can be supplied by serum heated a t 56°C. for 30 minutes or by ammonia-treated serum. Thus for example, sera B and D, which are lacking in bactericidal power separately, can mutually reactivate one another, since of the essential factors for bactericidal activity B is lacking in complement and D is deficient in immune body.

I n these experiments it has been noted that where the amount of dead bacteria used for the absorption of the immune body was greater than that used in table IV, there was marked loss or even total loss of haemolytic activity, and it was thought of interest to determine whether heated serum would be effective in regenerating the bacteri- cidal power of such a serum. The results of a typical experiment are given in table V, where 2.0 C.C. of the heated suspension used before was added to 12 C.C. of normal serum and the mixture incubated for 2 hours. The results indicate that where the heated organisms are used in such excess that hsmolytic complement is removed as well as the immune body, heated serum, which contains the immune body but not complement, is unable to reactivate the serum either for the hsmolytic or bactericidal processes.

Action of ammonia on the immune body. Heated serum was treated with ammonia (12 C.G. heated serum

plus 0-20 C.C. of 0%' N.NH,) incubated a t 37°C. for 1 hour and neutralised. The serum was then tested for the presence of immune body as before and the results given in table VI. The details for the treatment of serum with dead bacteria are the same as those for table I V (serum D). From the results the conclusion is drawn that treatment with small amounts of NH, does not appreciably destroy the immune body. The bactericidal power of a mixture of dead bacteria-treated serum and heated serum (or NH,-treated heated serum) is not as marked as normal serum, however, but this is probably accounted for partly by a slight loss of complement in the serum treated with bacteria, but mainly by the fact that the mixture is a serum in which the concentrations of both immune body and complement cannot be greater than half of those for normal serum.

Conclusions. 1. Small amounts of ammonia destroy the bactericidal power of

normal guinea-pig serum against B. dysmterim (Flexner) and this loss in bactericidal power runs parallel with the destruction of hsmolytic

TABL

E V.

Rem

oval

of

both

inzn

vune

bod

y an

d hc

enw

lytic

com

plem

ent

by e

xces

s of

dea

d ba

cter

ia.

4 hr.

1 hr

.

4 hr.

1 hr

.

!J hr

.

1 hr

.

xx

xx

xx

xx

- - - -

Bac

teri

cida

l po

wer

.

Nor

mal

.

. { :m

med

iate

ly.

3 cc

. ser

um

1.5

C.C

. se

rum

+

1 *5

C.C.

boui

llon

++

++

++

++

++

+-I

-

Gro

wth

on

subc

ultu

re a

fter

:

1 1l

Oll

P.

2 1 ++

++

++

++

3 ho

urs.

0 0 ++

++

++

++

7 hou

rs.

0 0 ++

++

++

++

Eae

mol

ytic

com

plem

ent

pow

er.

I I H

aem

olys

is o

f se

nsiti

sed

cells

by

dilu

ted

seru

m (

1 : 1

0).

Incu

bati

on

perio

d.

1.0

C.C

. 0’

6 C

.C.

xx

xx

xx

xx

- - - -

0.2

C.C

.

X

xx

x

- -

+ +

Hea

vy g

row

th.

+ G

ood

grow

th.

0 N

o gr

owth

. x

x x

x C

ompl

ete

haem

olys

is.

-

No

haem

olys

is.

The

num

bers

ref

er t

o th

e nu

mbe

r of

col

onie

s w

here

the

se c

ould

be

coun

ted.

x

x x ,

x x

and

x In

term

edia

te g

rade

s of

hse

mol

ysis

.

Seru

m.

-{

Nor

mal

.

.

Hea

ted

seru

m

trea

ted

with

NH

, (A

)

Seru

m tr

eate

d w

ith d

ead

bact

eria

(8

)

-( Se

rum

(B)

+ se

rum

(A)

Seru

m

(B) +

heat

ed

seru

m

- + +

Hea

vy g

row

th.

TA

BL

E VI.

Act

ion

of a

mm

onia

on

heat

ed s

erum

wit

h rd

eren

ce to

the

imm

une

body

.

Med

ium

.

3 C

.C.

seru

m

1.5

C.C

. se

rum

+

1.5 C.C. b

ouill

on

3 C.C. se

rum

1.5

C.C.

se

rum

+

1 *5

C.C

. bo

uillo

n

3 C

.C.

seru

m

1.5

C.C

. se

rum

+

15 C.

C.

boui

llon

3 C

.C.

seru

m

1.5 C.C. s

erum

+

1.5 C.C.

boui

llon

3 C.C.

seru

m

1.<5

C.C.

seru

m +

1.

5 C

.C.

boui

llon

Bac

teri

cida

l po

wer

.

[rnm

edi-

atel

y.

+ + + + + + + + + +

Gro

wth

on

subc

ultu

re a

fter

:

1 ho

ur.

0 5 ++

++

+ + + + + +

2 ho

urs.

0 0 ++

++

+ + 33 + 7 +

t G

ood

grow

th.

0 N

o gr

owth

.

S ho

urs.

0 0 ++

++

+ + 41 + 3 +

The

num

bers

-ref

er to

the

num

ber

of c

oion

ies w

here

the

se c

ould

be

coun

ted.

5 ho

urs.

0 0 ++

++

+ ++

+ ++

13

++

7 ho

urs.

0 0 ++

++

++

++

+ ++

92

++

Incu

bati

on

perio

d.

4 ho

ur

1 ho

ur

+ hou

r

1 h

our

+ hou

r

1 ho

ur

f ho

ur

1 h

our

4 ho

ur

1 h

our

Hm

mol

ytic

com

plem

ent p

ower

.

Hzm

olys

is o

f se

nsiti

sed

cells

by

dilu

ted

seru

m (1

: 10)

.

1.0

C.C.

__

_~

-

xx

xx

xx

xx

- -

xx

xx

xx

xx

xx

x

xx

xx

xx

x

xx

xx

0.6

C.C.

___..

xx

xx

xx

xx

- -

xx

xx

xx

xx

xx

xx

x

xx

x

xx

xx

__

0.

2 C.

C.

__

xx

xx

x

- - X

xx

- X -

X

x x

x x

Com

plet

e ha

emol

ysis

. -

No

hzm

olys

is.

x x

x , x

x a

nd x

In

term

edia

te g

rade

s of

h;e

mol

ysis

. ~ ua -a

763 /. GORDON AND A. WORMALL

complement. The action of ammonia on the bactericidal system and the haemolytic system of this serum is not due t o p H effects nor is any significant action exerted by neutral ammonium salts.

2. When suitable amounts of a suspension of B. dysenterim (Flexner) heated for 1 hour a t 65" C. are added to normal guinea-pig serum and the mixture incubated at 37" C. for 2 hours, loss of bactericidal power occurs with no appreciable loss in hamolytic complement. This loss of bactericidal power is attributed to the removal of an immune body present in the normal serum, and the missing factor can be supplied by serum which has been heated a t 56" C. for 30 minutes.

3. Ammonia-treated serum and serum treated with dead bacteria, both of which have no bactericidal power separately, have full bacteri- cidal activity when combined. Thus these two sera are deficient iii different respects for bactericidal power.

4. When larger amounts of the emulsion of dead bacteria are used for the absorption experiments, a serum is obtained which has lost all its hEmolytic power as well as bactericidal power. This serum cannot be reactivated for the hamolytic or bactericidal processes by seruni heated a t 56" C.

5. Evidence is produced that the immune body concerned in the bactericidal process studied is not affected to any significant extent by amounts of ammonia which inactivate complement.

6 . From these results, and since bactericidal complement has been split by various authors in the same way as haemolytic complement, the conclusion is reached that hamolytic complement and bactericidal complement have a similar constitution and are probably identical. Thus the bactericidal system investigated here appears to consist of an immune body (present in normal guinea-pig serum) and complement.

The author! desire to express their gratitude to the Medical Research Council for part-time grants and to Professor J. W. McLeod for his kind interest in this work and for many valuable suggestions.

BOEHNCKE . . . . . . . BRAUN . . . . . . . . GORDON, WHITEHEAD AND

WORMALL 3 , 7,

LIEFMANN . . . . . . . LIEFMANN AND STUTZER . . MUIR AND BROWNING . . . PETTERSSON . . . . . . RIrz . . . . . . . . . SACHS AND ALTNAPIX. . . SACHS AND OMOROKOW . . STEINHARDT . . . . . .

9 , . . . . . . .

,1 . . . . . .

REFERENCES. Z.fur Immunitatsf., 1912, or& xiii. 240. 2. fi ir Jmmunitatsf., 1911, orig., ix. 665. (1) Biochem. Journal, 1926, xx. 1028; J.

Immunol., 1927, xiii. 439. (2) Biochem. Journal, 1926, xx. 1044. Mu?zch. med. Woch., 1909, lvi. 2097. Central. f . Bakt., Ref., 1911,l. Beitrage, p. 149. Berl. klin. Woch., 1910, xlvii. 1929. this Journal, 1909, xiii. 76. Acta SOC. Medicorum Suecanae, 1927, vol. liii. Z. fiir Immunitatsf., 1928, liv. 292, z. fur Immunitatsf., 1911, xi. 321. Handbuch der Path. Microorganismen (Kolle

and Wassermann), 1909, ii. 476. 2. fi ir Immunitatsf., 1911, orig., xi. 710. J. illed. Res., 1905-6, xiv. 161.