IsMied March 12, 1907. 
 
 U. S. DEPARTMENT OF AGRICULTURE, 
 
 BUREAU OF ANIMAL INDUSTRY. BULLETIN No. 95. 
 A. D. MELVIN, CHIEF OF BUKBAU. . 
 
 THE BACTERIOLYTIC POWER OF THE 
 BLOOD SERUM OF HOGS. 
 
 BY 
 
 B. M. BOLTON, M. D., 
 
 Bacteriologist, Biochemic Division, Bureau of Animal Industry. 
 
 WASHINGTON: 
 
 GOVERNMENT PRINTING OFFICE. 
 1907.
 
 Issued March 12, 1907. 
 
 U. S. DEPARTMENT OF AGRICULTURE, 
 
 BUREAU OF ANIMAL INDUSTRY. BULLETIN No. 95. 
 A. D. MELVIN, CHIEF OF BUREAU. 
 
 THE BACTERIOLYTIC POWER OF THE 
 BLOOD SERUM OF HOGS. 
 
 BY 
 
 B. M. BOLTON. M. D., 
 Bacteriologist, Biochemic Division, Bureau of Animal Industry. 
 
 WASHINGTON: 
 
 GOVERNMENT PRINTING OFFICE. 
 
 1907.
 
 BUREAU OF ANIMAL INDUSTRY. 
 
 \ I' MIIVIV 
 
 A**i*lnnt 1'liiff: \. M. I- \aiti s. TON. 
 Chief Cl.rL I- 1 .. B. Joi 
 
 M. KOK.XKT. chief; .1 VMK* A. KMKHV, a istant chief. 
 /Ann/ I hn.t i,,n: Ki>. 11. WKK-TKI:, chief ; (' I'.. I. A SB, assistant chief. 
 Iit*lcti'i Itirisinn: KICK I'. Si KIM* >M. chief . I". .. HOICK, as-ociati- .-Im-i; M..i!i!i- 
 
 rnthul,H/icnl I)iri*',n,t: .!<iu\ K. Moin.KK. chief; HENRY. I. \V \MIIH i;\. ai-tant <-hi*-f. 
 
 tjiiiinililini Itiiition: K|CIIMM>\V. II !< K MAN, chief. 
 
 i <>f '/.>K>IIJ>I: I>. II. ll \s-4iM. chief. 
 
 HiiiitSliitiiin: I-!. ('. S< IIHOKDKH. Hi]M-rmtcn<lciit ; \V . I:. < orTux, 
 JIiitlHiii'hiKin: (tKtiKiiK M. KOMMKI.. 
 JAMK- M I'i KKN-. 
 
 KHH-IIKMIC IMVIMoN. 
 
 '7,/: M. IMK.SKT. 
 
 n,t 1'lii>f. .1 \MK> A. K.MKHY. . 
 
 Mint-in."/-, liinn Isihnrnlnr'ii.*: T. M. I'HK K. chcini>t in chariM-of central laUmitor\ : 
 
 A. K. (iltMIAM. K MTII Ho \i.l.A\Ii. ( '. II. SWAM.KH. WlI.I.IA.M I'.. SMITH. 1 \ 
 
 IH^KK. CI.AKENJ-K T. N. MAIOII. I'IIILII- C\>IIKMAN. I'.. II. IM.KK.-OI.I , I'KTKK 
 
 VAI.AKK, itssitiUiiit clu-mi-ts in Iminch laboratories. 
 IliMj-rlinli rn Iit>f#ti,/,iti<>u.<<: Cliief of Division in charge: 1'.. M. I'>OI.TO\ ami ('. N. 
 
 Mi HRYDE, Iwcterioloiri.^ts; \V. 15. NII.K-. in>|H-ctor in charp- of t'n-l<l c\|K-riii)cntM. 
 I'millri/ l> I,, rimmt.*: K. \V. HIMWX, a^intant rhiMiiir-t in charge. 
 
 ' l>i/,* <unl lHxinfi-1-tmit*: JAM !> A. MMKHY. chiMni>t; ( '. N M I'.i 
 
 r<--lirnln.n of Tiih,r<;ilin >n,>l Mntl,-in: Chief of IMvision in charge: A. M. Wf>T ami 
 II. .1. SIIOKK, ansistant ItacteriologiHts : \V. S. STAMTKH anl II. S. M- AIIKS. 
 arista nt.-. 
 
 2
 
 LETTER OF TRANSMITTAL. 
 
 U. S. DEPARTMENT OF AGRICULTURE, 
 
 BUREAU OF ANIMAL INDUSTRY, 
 
 Washington, D. C., December 27, 1906. 
 
 SIR: I have the honor to transmit for publication the accompanj'ing 
 paper entitled "The Bacteriolytic Power of the Blood Serum of 
 Hogs," by B. M. Bolton, M. D., of the Biochemic Division of this 
 Bureau. 
 
 The experiments herein described were carried out with certain 
 strains of the hog-cholera bacillus, and are a part of the general 
 investigations concerning hog cholera which have been conducted for 
 some years by the Biochemic Division. The subject of bacteriolysis 
 has claimed much attention from bacteriologists in recent years, and 
 Doctor Bolton's work is believed to be of interest on account of the 
 light it throws upon the defensive mechanism of the animal bod} 7 in 
 its fight against infectious diseases. 
 
 I recommend that the paper be published in the bulletin series of 
 this Bureau. 
 
 Respectfully, A. D. MELVIN. 
 
 Chief of Bureau. 
 Hon. JAMES WILSON, 
 
 Secretary of Agriculture. 
 
 3

 
 CONTENTS. 
 
 Introduction 7 
 
 Definition of sera used in experiments 8 
 
 Nature of the substance causing destruction of bacteria 9 
 
 Methods used in drawing blood, and description of cultures employed 14 
 
 The bactericidal potency of serum from the same hog at different times . . 15 
 
 Modifications which occur in the bactericidal power of serum upon standing . 19 
 Comparison of the bactericidal potency of the same serum for different strains 
 
 of bacteria 21 
 
 The potency of serum from arterial blood compared with that from venous 
 
 blood 22 
 
 Effects of heat upon the bactericidal power of hog serum 24 
 
 Effects of injecting cultures of B. cholerx suis upon the bacteriolytic power of 
 
 hog serum for this organism 
 
 Effects of dilution upon the bacteriolytic power of normal and immune serum. 
 
 Dilution of normal serum 
 
 Dilution of immune serum 
 
 Summary of the dilution experiments 
 
 General discussion 
 
 Summary of the conditions affecting bacteriolysis 
 
 The theory of complement diversion 
 
 Conclusions 
 
 Bibliography 
 
 5
 
 ILLUSTRATIONS. 
 
 Ki<;. 1. Illustration of tin- incrliunism uf hai-ti-rinly-i- a<v..rdinir to the Khrlii'h 
 
 hypothesis 11 
 
 1'. I>iversin of complement in niiililiit-.l immune serum 13 
 
 .".. Partial bacteriolysis arnl partial diversion of i-iiiii|>li-iiicnt in ililuifd 
 
 iiiiiniiiic H-runi 13 
 
 4. DivenkMl Of Complement in ininnun' serum, not licatcl. ami without 
 
 tin- ail- lit ion of foreign -<un|.lcni-nt 51 
 
 a
 
 THE BACTERIOLYTIC POWER OF THE BLOOD SERUM 
 
 OF HOGS. 
 
 INTRODUCTION. 
 
 Aside from a few preliminary experiments with cultures of the 
 typhoid and colon bacilli, the present investigation was confined to 
 the reactions taking place between three strains of B. cholwae ,y///.v, 
 and normal and immune serum' of hogs.. 
 
 The bactericidal properties of the blood serum and other fluids of 
 the body have of recent } T ears attracted a great deal of attention from 
 bacteriologists, and the investigations of the subject have led to very 
 far-reaching results of both theoretical and practical importance. 
 Without attempting to enumerate in full the different properties 
 which blood serum has been found to possess in its various reactions 
 with bacteria and their products, those properties which bear upon 
 the present investigation may be briefh' stated as follows: 
 
 The blood serum of manj^ health} 7 animals when drawn into test- 
 tubes has the power of destroying a larger or smaller number of 
 bacteria when these are introduced into it. Thus large numbers of 
 anthrax bacilli are destroyed by rabbit's blood serum in test-tube 
 experiments. Typhoid, colon, and Asiatic-cholera bacteria are silso 
 destroyed by the blood serum of rabbits. 
 
 The same serum is not equally potent for different bacteria, and 
 the serum from different animals of the same or of different species 
 varies in bactericidal potency for the same organism. The chemical 
 reaction of the serum seems to exert some effect upon this quality of 
 the serum. The more alkaline the serum the more potent is its action 
 apparently, and consequently the venous blood has been found some- 
 times to furnish more potent serum than the arterial blood from the 
 same animal. 9 " 
 
 The injection of animals with the products of growth of bacteria 
 produces different effects upon the properties of the blood serum, de- 
 pending upon the kind of bacteria employed for injection. The injec- 
 tion of the bacteria of the group to which B. cholerx .*///*, B. typhosus, 
 and B. col! communis belong appears sometimes to have no effect upon 
 
 "The figure references refer to bibliography at end of bulletin.
 
 8 HACTKKIOL1 IK 1 l'"\\ 1 K K KI.....M M HUM OF HOO8. 
 
 the bactericidal |>otclicy of tile scrum. At other times it sccin- t' 
 
 increase the potent \. but frequently it has :i very peculiar .lint, for 
 in some cases tin- scrum after the injection (if tin- J'liilual will no lei 
 kill any of tin- bacteria of tin- kind used to inject tin- :iniiii:il. l)iit on 
 the dilutioiiof the serum \\ ith an inditierent fluid, -m-h a- -alt Dilution. 
 it becomes strongly bactericidal for tin- bacteria of this kind. I'.ut 
 this serum beha\cs like the serum from an uninociilated animal with 
 other Imctrria than tho-r with which the animal is inoculated. Thus 
 the M-riini from the blood of a rallit injected with typhoid l>acilli will 
 often fail to kill the typhoid bacilli unless the serum i- givath diluted. 
 but it will kill ju.-t a- many Asiatic-cholera spirilla tin it did before the 
 animal was injected with the typhoid bacilli. 4 The peculiar behavior 
 of the serum from an animal injected with a culture of bacteria, in it- 
 failure to kill the species of bacteria with which the animal is inject, d 
 unless it is diluted, is called the Ncisser- WechslxTg '* phenomenon, 
 after the two investigators who first observed the reaction. 
 
 DKKIXITION OF SERA ISKD IN KXI'KKI M KM -. 
 
 The serum obtained from an animal which has been injected with 
 bacteria is spoken of as immune serum in contradistinction to the serum 
 from an uninociilated animal, which is called normal or fresh serum. 
 Thus si-rum obtained from a hog injected with //. <-Iioh r;e, suls would 
 be known as //. ,-//,,/, /, A-///.S- immune ho^ serum. So wherever immune 
 serum is mentioned in the present paper it is to be understood that it 
 refers to the serum obtained from an animal which has received at 
 last one injection of bacteria, but not necessarily that the serum has 
 either immunizing or germicidal pro|>ert ies. On the other hand, where 
 normal or fresh serum is mentioned, it is to be understood to refer to 
 serum obtained from an uninociilated animal. 
 
 ^-ruiii loses in bactericidal potency on standing after being drawn 
 from the animal: and the higher the temperature to which the serum 
 i- e\j)osed the more rapid the loss of potency. It may remain jx>tent 
 for several days or for even a week or more in the refrigerator. Imt if 
 kept at body temperature it generally lo.-es all bactericidal properties 
 in three or four hours. Heating at .".: or f)'> ('. for ten or tifteen 
 minutes uUo robs the so nun of its bactericidal power, or rather this 
 treatment of immune scrum suspends its bactericidal power, which is 
 restored by the addition of a -mall amount of fresh scrum. This sus- 
 pension of Iwctericidal power by heating at 55 or 56 C. u called 
 inactivating the serum, and wherever inactivated serum is mentioned 
 it is to IM- understood to refer to serum which has Ix-en treated in 
 this \\ay. Inactivated serum to which fresh serum is added, and which 
 has hail its bacteriolytic properties restored in this way, \a termed 
 \ated scrum.
 
 HOW BACTERIA ARE DESTROYED. 
 NATURE OF THE SUBSTANCE CAUSING DESTRUCTION OF BACTERIA. 
 
 In regard to the nature of the substance or substances in the serum 
 which cause the destruction of the bacteria which are introduced, there 
 is some difference of opinion. Some authorities maintain with Buch- 
 ner that there is only one substance, called by him " alexin," which 
 causes bacteriolysis, while others and these appear to be in the major- 
 ity maintain that there are two substances concerned. Those who 
 hold to the former view regard the effect produced by standing or by 
 heating as due to the weakening of the alexin, while those holding 
 the latter view explain this effect by a modification of one of the two 
 substances which they regard as necessary for bacteriolysis. All 
 are not agreed, however, as to the nature of these two substances. 
 Bordet 1>2>3 and the French school generally look upon the substance 
 which is not modified by heat as a sensitizing agent merely, which acts 
 upon the bacteria in such a manner as to make them susceptible to 
 the destructive action of the other body to which the bacteriotytic prop- 
 erties of the serum are directly due. Bordet borrows Buchner's term 
 "alexin " for this active agent, and he gives to the other body the name 
 "substance sensibilisatrice." According to the French school, then, 
 bactericidal serum owes its power to two substances, called, respec- 
 tively, substance sensibilisatrice and alexin. The former resists heating 
 up to 75 C. or even somewhat higher temperatures for an hour or 
 more, while the latter is destroyed by heating at 55 C. or higher in 
 ten or fifteen minutes. Moreover, the substance sensibilisatrice accord- 
 ing to this view is a specific substance in each case. The substance 
 sensibilisatrice for one kind of bacterium sensitizes this one kind only, 
 and while it can not of itself cause bacteriolysis, it nevertheless pro- 
 duces certain changes in the bacteria. On the other hand, according 
 to the view of Ehrlich and his school, this body, the thermostabile 
 body, causes no change in the bacteria themselves, but serves merely 
 as an intermediary, a binding link, serving to connect the bacteria to 
 the active body the alexin of Bordet which according to this view 
 is the active agent in bacteriolysis. To the two hypothetical bodies 
 concerned in the process Ehrlich has given, at different times, differ- 
 ent names. At first he called the thermostabile body "intermediary 
 body," afterwards "immune bod\ r ," and finally "amboceptor." The 
 last two designations are those now generally employed by the Ehrlich 
 school for the body which corresponds, as is evident, to the substance 
 sensibilisatrice of Bordet. To the other body, the alexin of the French 
 school, Ehrlich has given variously the names "addiment" and "com- 
 plement," the last being that now exclusivel}' employed. The torm 
 intermediary body has been employed in a recent publication for the 
 arnboceptors present in the normal serum in order to distinguish these 
 19500 No. 9507 2
 
 10 HA. I 1KI..I.VTI. iMiWKK <>| i:|. ...... sKKl'M "I l!<" 
 
 from iiiiiiiunc amboceptor-. or ambo. -, pi,.! > produced l\ injecting 
 animal- with bacteria or their pro.lu.i-. 
 
 But among those who have accept.-. I mirror less completely the 
 Khrlieh conception of the nature of the Inxlir-. . <>IK . i n.-d in )>: -i, ! -i 
 olysts there is difference of opinion a- t tin- mode of a<ti<.n of 
 the complement. Khrlich himself says in regard to tin- matter 
 'that one will not go amiss if lie a-stime^ \viih IM'eiller that the pro.- 
 ess of bacteriolysis is analogous to digestion, ami attrilmte- i<> the 
 addiment (complement) the character of ad i^e-iive ferment." (Jruhcr/ 
 on the contrary, contends that the complement do.-> not art lik< :( 
 ferment, and that it is erroneous to draw any analogy l-t \\een t Ill- 
 complement and an enzym, since the complement is entirely u-ed 
 up in bacteriolysis, whereas in the process of fermentation, as is well 
 known, the ferment is not used up, hut may In- recovered after tin- 
 action is ended, and used for the fermentation of other material. 
 
 But whatever the exact mode of action may IM>, it is evident from 
 what has just been said that both the Khrlieh and the IJordet -ehooU 
 attribute Imcteriolytic action proper in normal serum to a >ultam-e 
 easily changed by comparatively low temperatures, and called, respec- 
 tively, complement and alexin by the two schools. To the other 
 body concerned in bacteriolysis the amboceptor of Ehrlich and the 
 substance sensibilisatrice of Bordet is assigned by the former the 
 role of a binding link between the complement and the bacterium. 
 while by the latter is assigned to it the property of a sensitizer, or of 
 a mordant as in dyeing. In the one case the bacterial cell i> regarded 
 as not at all injured or otherwise changed by the union with the arnbo- 
 ceptor; in the other case it is the opinion of those holding thi> view 
 that the cell is acted upon and changed by the sensihili.sttrice in such 
 a way that the alexin can penetrate it. 
 
 Bordet* summarizes the difference l>etween his theory and that of 
 Ehrlich as follows: 
 
 According to Khrlieh and Morgenroth the sjHvilic antibody (aensihilisatric.- > ]>la\> 
 tin- !,!. of an actual intermediary (7.wiachenkor|H>r, amlxiceptor), a link of union 
 attaching itaelf on the one haud to the cell, on the other to the alexin. In other 
 words, the aheorption which tin- alexin undergoe** in the presence of the *-n-iti/i-<| 
 cell i.- not due to an affinity nianifcfted hy the cell it-.-li to thix tuilistance. The 
 absorption of the alexin is only indirect; the cell jin- itself to the intermediary 
 gulwtance, which i- itself united chemically by its other |tole to the alexin. 
 
 ( >ur idea of the phenomenon, which we feel we are justified in holding, i.- alto- 
 p-ther different from thin. To us it seems that the Hengibilteatrice which uniu-.- 
 with the cell modifies this in a way which permits it to abxorb the alexin <lir.-.-tl\ . 
 The action of the sensibilisatrice upon the cells is comparable to that of certain fixa- 
 tivi- agents or mordants which confer upon certain substances, (or to the cell- i 
 these substances, as is the case in histological technic) the fiower of absorbing colons 
 which they refuse to absorb before treatment * * * It is to be clearly under- 
 stood, however, that when we speak of mordants in this connection we do not int-nd 
 t<> apply in all details the phenomena of dyeinx to the matter at present under rn- 
 ; \\.- nu-rely mean to draw a comparison which will serve to make our
 
 HOW BACTERIA ARE DESTROYED. 
 
 11 
 
 idea clearer. The hypothesis which we wish to bring out in relief is that in the 
 presence of hemolytic serum, the cell becomes capable of absorbing directly the 
 alexin by means of its own proper elective affinity, and that this power is due to a 
 change caused by the sensibilisatrice. In other words, we do not believe that one is 
 forced to admit, with Ehrlich and Morgenroth, that the sensibilisatrice itself com- 
 bines with the alexin, and that this union is indispensable for the latter substance to 
 attack the cell. 
 
 Bordet furthermore states in the same connection that he agrees 
 with Buchner in regarding the alexin for blood cells and for bacteria 
 as identical that one and the same alexin may attack the most diverse 
 cells; whereas Neisser and others of the Ehrlich school believe that 
 alexins or complements are different in one and the same serum. 
 
 While it is evident from the above that the terms amboceptor and 
 sensibilisatrice are used to designate the same substance, it is scarcely 
 correct to use them interchangeably, since they connote somewhat dif- 
 ferent attributes in the body to which they refer. The same is true 
 of the terms complement and alexin, though to a less degree. 
 
 The following diagrams, obtained from various sources and modified 
 to suit the purpose, will serve to illustrate the process of bacteriolysis 
 according to the views of the Ehrlich 
 school. 
 
 Figure 1 represents in its simplest 
 form the mechanism of bacteriolysis 
 according to the Ehrlich hypothesis, 
 and serves to illustrate the process suffi- 
 ciently for the purposes of the present 
 paper. 
 
 In the diagram the bacteria are rep- 
 resented b}- the parts marked 5, the am- 
 boceptors by those marked , and the 
 complements by those marked k. In 
 No. 1 the bacterium, amboceptor, and 
 complement are represented as just on 
 the point of uniting. No. 2 represents 
 the bacterium and the amboceptor united and the complement on the 
 point of uniting with the unoccupied end of the amboceptor. No. 
 3 represents the process of uniting of bacterium, amboceptor, and 
 complement completed; the bacterium in this case would undergo 
 bacteriolysis. 
 
 It should be borne in mind that according to this theory bacterioly- 
 sis can take place only where the bacterium becomes united to an 
 amboceptor which is itself united with a complement. A bacterium 
 may become united with a free amboceptor i. e., an amboceptor 
 which is not united with a complement but the bacterium in such a 
 case does not undergo bacteriolysis unless a complement subsequently 
 becomes attached to the amboceptor. The complement is incapable of 
 uniting directly with a bacterium; it can do this only through the 
 
 FIG. l. Illustration of the mechanism of 
 bacteriolysis according to the Ehrlich 
 hypothesis.
 
 12 BACTKIUnl.VIlC I'nWKK <>K MU't-H -Mil M "I BOfl 
 
 intervention >f tin- amboceptor. Hut when the complement 
 
 linked t> tin- bacterium by mean- of tin- amhoceptor tin- l>a> i,-rjum 
 
 become- broken ti|) into minute granule- and nit imately di-appear-. 
 
 The twiiid- liy which t lu> ambocept< >r at tache- it-elf to the bacterium 
 on ili'- one hand, and I the complement on the other, an- called hapto 
 phor groups or haptophors (/<). and similarly thi- name i- given to the 
 bond* of union of the bacteria and of the complement. The ambo 
 eeptor thus has two haptophor-. one by mean-, of which it attache- 
 it-elf to the bacterium, the cytophylie ha|)tophor, an<l one by mean- 
 of which it attache-, itself to the complement, the complenientophylic 
 haptophor. The liacteria probably po e each many haptophor- all 
 of the same kind i. e.. hapto|>hors capable of uniting with anil .<! ptm-- 
 of the same kind but for the sakeof simplicity the bacterium is repre- 
 sented in the diagram as having only one haptophor. The complement 
 ha- one hnptophor group and one so-called toxophor group (/). and it 
 is by means of the latter jrroup that the coinplenient a-ts upon the 
 bacterium. The complement may be deprived of this toxophor group, 
 and although it is still capable of uniting with the amlx>ceptor in such 
 a case, it can no longer cause bacteriolysis. This loss of the toxophor 
 group is caused by heating, and it also occurs spontaneously in the 
 -erum on standing. Bacteria subjected to the action of heated serum 
 do not undergo bacteriolysis, but become fixed to the aml>oceptor8, 
 and the amboceptors become united to the haptophor group of the 
 complement which are left unaffected by the heating. It will thus be 
 readily understood why bacteria treated with heated immune serum are 
 subsequently protected from bacterioly-is even when indicated immune 
 -erum or when indicated complement is added to them. The comple- 
 mentophylic haptophor of the amboceptor is in such a ca-e already 
 occupied by the haptophor- of the heated complement, which has 
 in thi- way become deprived of its toxophor group. 
 
 The amboceptors found in ordinary normal -erum are either all 
 alike and in this case they u\\\-\ po---- atlinity for a great many dif- 
 ferent kinds of bacteria or they must differ from one another; and 
 in this case there inu.-t evidently be a great many specific aotbooepton, 
 -ome titled for the hade-Holy .-is of one -pecie- of bacterium, -ome for 
 otheis. This matter -eeni- not yet to have been settled. Hut it is 
 certain that the injection of an animal with certain bacteria or their 
 products causes the formation of a large number of specific amlio- 
 ceptor-: that i- to -ay. of ainlxx'cptor- having affinity only for tin- 
 kind of bacteria with which the animal is injected. Such injection- 
 -eem not to increase the amount of complement. 
 
 Complement is found normally in the serum, that of some animals 
 |x>--e ing more than that of others. The horse- appear- to have a 
 Ia:-L r e amount of complement in the serum. It is not yd -dtled 
 whether the complement i- -peci tic that is, whether the complement 
 for one kind of immune serum can unite with the aml>oceptor- of thi-
 
 HOW BACTERIA ARE DESTROYED. 
 
 13 
 
 FK;. 2. Diversion of complement in undiluted immune 
 serum. 
 
 serum only and not with the immune serum of a different sort or 
 whether complements are general; though they seem for the most 
 part not to be specific. The complement in the serum of horse's blood 
 seems capable of reactivat- 
 ing heated immune serum 
 of various kinds. Still in 
 some cases it would appear 
 as if they were specific. 
 
 With the explanation 
 given above of the nature 
 of amboceptors and com- 
 plements, the phenomena 
 which take place in im- 
 mune serum become more 
 or less satisfactorily ex- 
 plicable. By means of the characteristics ascribed to these bodies it 
 is possible to account for the peculiar behavior of immune serum 
 stated above, consisting in the fact that such serum is frequently 
 more potent when diluted than when it is undiluted. 
 
 Neisser and Wechsberg were the first to observe" this phenomenon, 
 and the theory which they advance to explain it the}* very appropri- 
 ately call the theory of the diversion of complement. As the name 
 implies, they attribute the lack of bacteriotysis in the undiluted 
 immune serum to the turning aside of the complement from the 
 bacteria, or rather from the amboceptors which are attached to the 
 bacteria. They hold that this diversion is brought about by the free 
 amboceptors themselves. In other words, where there are more 
 amboceptors than there are complements present in a serum, a part of 
 these attach themselves to the bacteria and a part to the complements. 
 
 The accompanying diagram (fig. 2), 
 taken from Neisser and Wechsberg, 
 and modified to suit the present descrip- 
 tion, shows two amboceptors a, at- 
 tached to bacteria >, and four ambo- 
 ceptors attached to complements k. 
 Bacteriolysis is not possible in such a 
 condition, because the complements 
 have been diverted from the ambo- 
 ceptors which are attached to the bac- 
 teria. Bacteriolysis can take place 
 only when the complement becomes 
 attached to the bacterium through the 
 medium of the amboceptor. 
 Figure 3 is meant to show the same serum diluted with an equal 
 amount of salt solution. In this case, with the same number of 
 bacteria added, it is evident that one-half of them would be killed, as 
 
 FIG. 3. Partial bacteriolysis and partial 
 diversion of complement in diluted im- 
 mune serum
 
 14 HAITI Kh'I.VIH ToUlK <>K HI IMM >F U<(18. 
 
 i- indicated ly tin- combination lietwecii Iwteria. amboceptdr. :m.l 
 ^implement in No. I. Tin- other half ol tin- bacteria \\<.nl.| e\ i 
 dently escape. the complement being dixcrted by tin 1 free ambo,-epi.r. 
 us shoxxn I iy Nds. _' and H. 
 
 Although these statement- iii regard id Uactei iolx -i- and I In- niechan- 
 i-m probably involved are by no mean- e\haii-iix e. they \\ ill. perhap-. 
 serve tin- purpd-e- df tin- present invcstigatidii and td explain the 
 results hero obtained. It will !> noticed that r\pn -iiiirnt- lia\- i< .-n 
 made with a view t> throuin^ additional li^ht npdii \\\n*.t> pha-< - .f 
 Imctrriolysis already nicnlidiifd. a- they weiv di^rrved in hoo-\ l>lod 
 -ennn, and it Wdidd appear that the re-id t". although diJferin^ in 
 
 -dine in-tance- frolll those of dtlier dh>er\ er-. are neverthele -U-- 
 
 i-eptiMe of interpretation in hannony with more or less linnl\ e-tal- 
 lished hypothec -. 
 
 METHODS USED IN DRAWING BLOOD, AND DESCRIPTION OF 
 CULTURES EMPLOYED. 
 
 The blood wa> drawn from healthy hojjs, or from liojrs injected with 
 cultures of li. cholerw Unix, siibeiitaneously or intravenously. a> stated 
 in each case, at various lengths of time before the drawing. It wa- 
 obtained (1) by cutting off a piece of the tail and allowing the blood to 
 tlow into sterili/ed tubes or tla>k>. (L'| by blei-ding from an artery in 
 the ear, or (tt) by in-erting a canula into the carotid ai'terx <>] jugular 
 vein. In some cases the blood wa- drawn from the carotid and from 
 the jugular at the same operation. After drawing, the blood un- 
 usually placed in the refrigerator to allow the serum to -eparate. but 
 in a few ca-es it wa- u.-ed immediately after drawing. 
 
 All the hogs used in the experiment- were in a perfectly healthy 
 condition to start with. and. as far a- could le a-certained. had ne\ i 
 been -ick. They weighed from S<> to 4<> pounds each. 
 
 The cultures employed were obtained during the course of former 
 exjM'riments. One of them, G. 1'. 4' ( ( ,^. had been repeatedly pa ed 
 through guinea pigs; another, Crawford, had been carried along fora 
 number of years on artificial media without passage through animals; 
 the third, F. '20, had also been carried along for -everal years on arti- 
 ficial media without animal passage. The-e culture- piv.-ented minor 
 point- of difference from one another, but they were all quite typical 
 for II. linl,r tint*. All of them give characteristic growths upon 
 artiticial culture media with the usual fermentation reaction- <>f the 
 u-. and the other features of H. cliolt-rtf #//*, both macroscopic 
 and microscopic, though it is true that the Crawford strain grew more 
 vigorously and gave larger and denser colonies than the other two, 
 ind that the (. 1*. }>'.fJ .-train gave the least vigorous growth on arti- 
 ticial media. Al-o. in the test* whi< h were made to determine the 
 point, the (i. 1*. Jt.:-j -train was much more stronjrly pathogenic foi
 
 BACTERICIDAL POTENCY OF SERUM. 
 
 15 
 
 guinea pigs, rabbits, and hogs than the other two strains. The Craw- 
 ford strain was the least virulent of the three. So the virulence of 
 the three organisms was in inverse ratio to the vigor of growth upon 
 artificial culture media. The culture of B. coli oommwtu was obtained 
 in the course of former experiments from the feces of a normal hog. 
 It showed no pathogenic properties. 
 
 THE BACTERICIDAL POTENCY OF SEBUM FROM THE SAME HOG 
 AT DIFFERENT TIMES. 
 
 The bactericidal potency of the serum from the same hog on differ- 
 ent days was tested by bleeding a hog at intervals of a few days, 
 drawing off the serum after the blood had stood in the refrigerator 
 for twenty-four hours or less, and distributing it into test tubes 1 c. c. 
 into each tube then adding a definite, measured amount of a suspen- 
 sion in salt solution of the bacteria to be tested. At the same time a 
 tube of 1 c. c. of salt solution was inoculated with the same measured 
 amount of bacterial suspension and plates made immediately to deter- 
 mine the number of bacteria added. Plates were made from the serum 
 tubes on the following day. The serum from 4 hogs was used in this 
 experiment, the blood in all four experiments being drawn from the 
 tail. The results will be found in the following tables: 
 
 TABLE I. Bacteriolytic action of normal hog serum from the same animal at different 
 drawings. Blood drawn from the tail. Hog No. 1783. 
 
 Date. 
 
 Time after drawing. 
 
 No. of 
 drawing 
 of blood." 
 
 Culture used to test 
 bacteriolysis. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. c. 
 
 Number of 
 bacteria 
 per 1 c. c. 
 after 1 day. 
 
 1905. 
 April 7 
 
 3 hours 
 
 I 
 
 G. P. 4692 
 
 4,620 
 
 79,800 
 
 7 
 
 do 
 
 I 
 
 B coli 
 
 5 880 
 
 30 800 
 
 9 
 
 do 
 
 II 
 
 G. P. 4692 
 
 30,800 
 
 48,300 
 
 9 
 
 .do 
 
 II 
 
 B. coli 
 
 7,000 
 
 
 
 11 
 
 1 day 
 
 III 
 
 G P 4692 
 
 4 160 
 
 9 800 
 
 11 
 
 do 
 
 III 
 
 B. coli 
 
 225 
 
 53 
 
 14 
 
 . . .do 
 
 IV 
 
 G. P. 4692 
 
 9,800 
 
 4,690 
 
 14 
 
 do 
 
 IV 
 
 B. coli 
 
 9,520 
 
 9,800 
 
 19 
 
 do 
 
 v 
 
 G. P. 4692 
 
 8,655 
 
 142,900 
 
 19 
 
 do ... . 
 
 y 
 
 B. coli 
 
 2,600 
 
 
 
 22 
 
 2 days 
 
 VI 
 
 G. P. 4692. 
 
 4,968 
 
 43,200 
 
 22 
 
 do 
 
 VI 
 
 B. coli 
 
 1,940 
 
 240 
 
 26 
 
 1 day 
 
 VII 
 
 G. P. 4692 
 
 1,800 
 
 1,220 
 
 26 
 
 do 
 
 VII 
 
 B. coli 
 
 2,800 
 
 16, 870 
 
 May 3 
 
 3 hours . . 
 
 VIII 
 
 G P. 4692 
 
 22,628 
 
 17, 510 
 
 3 
 
 do 
 
 VIII 
 
 B. coli 
 
 23,380 
 
 84 
 
 6 
 
 do 
 
 IX 
 
 G. P. 4692 
 
 B 
 
 1,004 
 
 5 
 
 do 
 
 IX 
 
 B. coli 
 
 869 
 
 2,594 
 
 
 
 
 
 
 
 u Roman numerals denote the serial numbers of the drawings of blood. 
 
 As will be seen from Table I, the blood was drawn from hog 1733 
 on nine different days and tested simultaneously upon cultures of G. P. 
 4692 and on the culture of B. coli communis. In four of the draw- 
 ings the serum apparently had no bacteriolytic power for the strain of 
 B. choleras suis employed, while in four of them, although there was 
 also no marked bacteriolysis, there was nevertheless no evidence of
 
 L6 
 
 BACTKKIOLYTK' I'oWKK "I I|.M,| S1-KTM 
 
 EIOO8. 
 
 in\ irivat iiHTi-a-e <>f tin- bacteria introduced. In tin- serum from one 
 of the drawings tin- bacteria were reduced to one-half of the number 
 introduced; this was the greate>t amount of potencx >hox\n iii :HI\ of 
 the drawings. 
 
 With the colon bacillus there \\a^ marked liacteriolysis in one draw- 
 in jf, loss marked in four other drawing. und neither i IK rea.se nor 
 decrease in one of the drawing, while in the other three draxx in;:- 
 there was an increase of the Itacteria introduced. 
 
 It is evident that the serum from this hog Allowed different degrees 
 of potency or alienee of potency upon the different cca-ion- when 
 the bl<x>d was drawn. 
 
 T \III.K II. />'</(/ r/o/i/fiV Hi-linn of normal Innj nfrtiin fnun tin' tainr animal ut iliffrrmt 
 dnnnnyx. ///<*.// ilru >n, from tin- tail. liny .V". 1740. 
 
 Date. 
 
 Tlnif after drawing. 
 
 No. of 
 
 IrauiiiK 
 
 ..n.i.Nni. 
 
 Culture lined to tot 
 Uu-tcrlolyria. 
 
 Number of 
 
 ImrU-ria 
 Introdurcd 
 per 1 <. c. 
 
 Number of 
 tmcteri* 
 per 1 
 after 1 day. 
 
 190T>. 
 
 H ln>iir> . . . 
 
 I 
 
 O. P. 4692. . . 
 
 4 
 
 1.750 
 
 
 do ... 
 
 I 
 
 H. n>li 
 
 975 
 
 8 
 
 g 
 
 ...do.. 
 
 ii 
 
 '. (' 4G9J. 
 
 l .' . 
 
 1 276 
 
 8 
 
 ...do... 
 
 ii 
 
 //. ntti 
 
 .. 771 
 
 70 
 
 9 
 
 ...fin... 
 
 in 
 
 , IV .< 
 
 102 
 
 1,296 
 
 10 
 
 ...do... 
 
 IV 
 
 <i. I'. -KW2 
 
 1.010 
 
 1,562 
 
 10 
 
 .. do... 
 
 IV 
 
 II ciAi . . 
 
 S.110 
 
 9M 
 
 11 
 
 do ... 
 
 V 
 
 Q. P. 4692 
 
 848 
 
 M 
 
 12 
 
 ...do... 
 
 VI 
 
 O. P. 492 
 
 HI 
 
 
 
 12 
 
 do 
 
 VI 
 
 B. coH 
 
 1.700 
 
 1.840 
 
 
 
 
 
 
 
 The blotxl of hog 1740, as is shown in Table II, was drawn on six 
 different days and tested on It. >li and on (i. P. 4>>t'J. It showed 
 marked Imctericidal power for the B. cot! culture in three of the 
 four drawings tested with this organism, but in one there was no 
 decrease in the number of bacteria introduced. With the B. <-/,,>/ f -/';r 
 Kin* culture it will be noticed from the table that the effect varied 
 greatly with the different drawings. While there was no very marked 
 decrease in any case, it is evident in general that the bacteria did not 
 multiply abundantly in the serum in any case. It would seem as if 
 growth had l>een inhibited by the serum, although active lacterioly-i> 
 was lacking except in the serum from one drawing. 
 
 TABLE III. Ikttirrioliitir miion of nttrinnl hoy emm from the mme animal <il Mffertiit 
 Wo<i ilrnirnfrom the tail. Hog \o. 1741- 
 
 I'..t. 
 
 Time after drawing. 
 
 No. of 
 
 .'.: ,'.v ;: i: 
 
 of blood. 
 
 Culture tued to tect 
 bacterioly*!*. 
 
 Number of 
 barteria 
 Introduced 
 pt-r 1 r. r. 
 
 Number of 
 bacteria 
 per 1 c. c. 
 nfd-r Iday. 
 
 1906. 
 May B 
 
 ;; ln>i;r 
 
 I 
 
 ; 
 
 4 926 
 
 82 
 
 8 
 
 
 I 
 
 H. mii . 
 
 1.778 
 
 14 
 
 9 
 
 
 II 
 
 Q i 1 4692 
 
 102 
 
 166 
 
 
 .!,, 
 
 II | 
 
 R nil .... 
 
 1 970 
 
 390 
 
 10 
 
 
 III 
 
 ,, |- |, , 
 
 1 010 
 
 1 9S2 
 
 10 
 
 
 III 
 
 H rnli 
 
 3 110 
 
 40 
 
 11 
 
 .. do 
 
 IV 
 
 . 1 ; 
 
 848 
 
 50 
 
 12 
 
 
 V 
 
 B. rnli 
 
 1,700 
 
 
 
 
 
 
 
 

 
 BACTERICIDAL POTENCY OF SERUM. 
 
 17 
 
 As Table III shows, the blood was drawn from hog 1741 on five 
 different occasions. The serum from four of the drawings was tested 
 upon the culture of B. coli, and it showed marked bacteriolytic prop- 
 erties for this organism in all the tests. The serum from four of the 
 drawings wasjtested upon the G. P. 4692 strain of B. cholerse, suix, but 
 only* two of the drawings showed bacteriolytic properties for this 
 organism. 
 
 TABLE IV. Bacteriolytic action of normal hog serum from the mme animal at different 
 drawings. Blood drawn from the tail. Hog No. 17 4% 
 
 Date. 
 
 Time after drawing. 
 
 No. of 
 drawing 
 of blood. 
 
 Culture used to test 
 bacteriolysis. 
 
 Number of 
 bacteria in- 
 troduced 
 per 1 c.c. 
 
 Number of 
 bacteria 
 per Ice. 
 after 1 day. 
 
 1905. 
 May 8 
 
 3 hours . . . 
 
 I 
 
 G. P. 4692 
 
 4 926 
 
 82 
 
 
 do ... 
 
 I 
 
 Ji coli 
 
 3 788 
 
 686 
 
 10 
 
 ...do... 
 
 II 
 
 G. P. 4692. 
 
 102 
 
 46 
 
 10 
 
 do 
 
 II 
 
 B coli 
 
 1 970 
 
 82 
 
 12 
 
 do 
 
 III 
 
 G. P. 4692 
 
 1 010 
 
 798 
 
 12 
 
 .do 
 
 III 
 
 B coli 
 
 3 110 
 
 30 
 
 11 
 
 .... do 
 
 IV 
 
 G P 4692 
 
 '848 
 
 50 
 
 13 
 
 do 
 
 V 
 
 B coli. 
 
 1 700 
 
 o 
 
 23 
 
 ...do... 
 
 VI 
 
 G. P. 4692 
 
 10,880 
 
 3,376 
 
 23 
 
 do 
 
 VI 
 
 Crawford 
 
 40 500 
 
 2 640 
 
 23 
 
 do 
 
 VI 
 
 F 26 
 
 19 040 
 
 18 280 
 
 24 
 
 ...do... 
 
 VII 
 
 G. P. 4692 
 
 1,682 
 
 
 
 24 
 
 do 
 
 VII 
 
 Crawford 
 
 1 808 
 
 180 
 
 24 
 
 do 
 
 VII 
 
 F 26 
 
 12 040 
 
 1 640 
 
 26 
 
 do 
 
 VIII 
 
 
 320 
 
 o 
 
 26 
 
 . .. do. . 
 
 VIII 
 
 F 26 
 
 80 
 
 1 960 
 
 29 
 
 do 
 
 IX 
 
 F 26 
 
 1 740 
 
 4 480 
 
 29 
 
 ...do... 
 
 IX 
 
 Crawford 
 
 2,320 
 
 
 
 29 
 
 do .... 
 
 IX 
 
 G P 4692 
 
 1 920 
 
 960 
 
 June 5 
 
 s hour 
 
 x 
 
 F 26 
 
 2 840 
 
 26 640 
 
 5 
 
 do... 
 
 X 
 
 Crawford 
 
 3,380 
 
 180 
 
 5 
 
 do 
 
 x 
 
 G P 4692 
 
 1 380 
 
 4 420 
 
 
 
 
 
 
 
 In the experiments with the blood of hog 1742, as shown in Table 
 IV, ten different drawings were made on separate days, and four of 
 them tested on the culture of the colon bacillus, eight on culture G. P. 
 4602, five on culture Crawford, and five on culture F. 26. 
 
 The colon bacillus was greatly diminished in all cases. In one case 
 where 1,700 per 1 c. c. had been introduced into the serum all of them 
 were destroyed, and in another test 3,000 out of 4,000, in round num- 
 bers, were killed. 
 
 The tests with the G. P. 4692 culture showed that the serum was 
 bactericidal in seven tests out of eight. In one of these about 1,700 
 bacilli were introduced and all of them were killed. On the other 
 hand, in the serum from one of the drawings there was an increase of 
 the bacilli introduced. 
 
 The tests with the Crawford culture showed that the serum from all 
 of the drawings tested was actively bactericidal, but in some more 
 than in others. In one of the drawings about 38,000 bacilli per 1 c. c. 
 of serum were destroyed out of the 40,000 introduced. But in another 
 case all were not destroyed although only 1,808 were introduced. 
 19500 .No. 95-07 3
 
 18 BACTKKIoI.YTK POWE1 0* HI. ..... > -IKl\l 01 I ..... -. 
 
 with culture F. _'' showed bacteriolysis in om* drawing; in 
 another drawing there was no increase nor decrease of tin- bacteria 
 introduced. In three drawings there was marked increase of the 
 organisms introduced. 
 
 That the bactericidal potency of the scnini of hogs -should vary :it 
 different times. a- these refill- seem to indicate. should |>crliaps'not 
 be a matter of surprise. Then- arc probably nmny circumstances 
 which influence this property of the serum. A- i- stated eUe \\hrre in 
 this paper, ihe chemical reaction of the serum has Ixvn found ly 
 others to influence the lactericidal power, and doubtle^ there are 
 other as yet oli-cure circuin.stances u hich raise or lower this p,\\er of 
 the sei-uin. The nature of t>aeteriol\ -in- will he found discussed at 
 some length helow in a different conne<-tion. and it would not seem 
 at all improhaMe that there may he more of the-e nt one time than 
 at another present in the blood serum. Indeed, the production of 
 hacteriolysis in serum of the living animal seems to lx> easily Influenced 
 one way or another, and it would not be unreasonable to regard them 
 us \aryinjj from time to time under even slightly changing' conditions 
 of the body. 
 
 Trommsdorf " noticed that human sera derived from normal indi- 
 viduals as well as from those suffering from various diseases \ a ry 
 greatly in bacteriolytic j>ower. Petterson 15 found the same thing 
 with chickens. 
 
 Morgenroth and Sachs 1 " found great variation incytolytic power in 
 serum of various sorts. Thu.s the serum from a horse at one drawing 
 \\ as hemolytic for rabbits' corpuscles but not for those of guinea pigs; 
 three days later the serum from the same horse was strongly hemo- 
 lytic for guinea pigs' corpuscles, but only very slightly for rabbits' 
 corpuscles; twenty-three days later the serum from this hor-e \\ as not 
 hemolytic for guinea pigs' corpuscles, but strongly hemolytic for 
 rabbits" corpuscles. 
 
 It is therefore evident that the cytolytic power of serum is very 
 variable. Not only does the blood from different individuals of the 
 same species differ in this respect, but the serum from the same indi- 
 vidual differs from time to time. This is prolmbly the case with all 
 animals, and. as is apparent in the experiments with hogs, these are 
 no exception to the rule. 
 
 In regard to the above exjM'riiuents. while there is more or less 
 variation between single tests, if one strain only of II. !,,, I, , 
 considered, there seems t<> be a general tendency toward either active 
 l>a. teriolysis or else inhibition of growth.
 
 MODIFICATIONS IN BACTERICIDAL POWER OF SERUM. 
 
 19 
 
 MODIFICATIONS WHICH OCCUR IN THE BACTERICIDAL POWER 
 OF SERUM UPON STANDING. 
 
 As has been previously stated, the blood was placed on ice in all 
 cases where it was not at once used for making tests. In some cases 
 the bactericidal properties of the serum were tested after the serum 
 had stood for various lengths of time. The results of these tests, as 
 will be seen from the accompanying tables, show in general that the 
 bactericidal potency is retained in some cases for as long as nine days. 
 Occasionally, however, the bactericidal power of the serum is dimin- 
 ished by even two days' standing. 
 
 TABLE V. Bacteriotytic action of normal hog serum after standing for various lengths of 
 time in the refrigerator. Blood drawn from the tail, eighth drawing. Hog No. 174%- 
 
 Date. 
 
 Time after drawing. 
 
 Culture used to test 
 bacteriolysis. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. c. 
 
 Number of 
 bacteria 
 per 1 c. c. 
 after 1 day. 
 
 1905. 
 May 20 
 June 2 
 May 26 
 June 2' 
 
 V 
 
 3 hours 
 
 Crawford 
 
 320 
 1,960 
 80 
 3,280 
 
 
 380 
 1,960 
 2,980 
 
 7 davs . 
 
 do . 
 
 3 hours 
 
 F. 26 "... 
 
 7 davs 
 
 ...do... 
 
 i 
 
 The serum from the blood of hog 1742 at the eighth drawing, as is 
 shown in Table V, was tested upon two of the organisms only, 
 namely, Crawford and F. 26. For the Crawford culture the bacteri- 
 cidal power of the serum was retained for seven days, whereas for the 
 F. 26 culture the serum was bactericidal at the start and only inhibi- 
 tory after seven days. 
 
 TABLE Vf. Bacteriolytic action of normal hog serum after standing for various lengtlis of 
 time in the refrigerator. Blood drawn from the tail, ninth drawing. Hog No. 1742. 
 
 Date. 
 
 Time after drawing. 
 
 Culture used to test 
 bacteriolysis. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. c. 
 
 Number of 
 bacteria 
 per 1 c. c. 
 after 1 day. 
 
 1905. 
 May 29 
 June 3 
 May 29 
 June 3 
 May 29 
 June 3 
 
 3 hours 
 
 F. 26... 
 
 1,740 
 1,220 
 2,320 
 1,060 
 1,920 
 700 
 
 4,480 
 1,180 
 
 
 960 
 1,100 
 
 5 days 
 
 do 
 
 3 hours .. 
 
 Crawford 
 
 5 days 
 
 do 
 
 3 hours 
 
 G. P 4692 
 
 5 davs ; 
 
 do 
 
 
 
 At the ninth drawing from hog 1742, as Table VI shows, the serum 
 retained its bactericidal potency for the Crawford culture apparently 
 unabated for five days, whereas for the other two organisms there was 
 apparently more or less inhibition of growth, perhaps, both at the start 
 and after five days, but no very marked bacteriolysis for either of 
 them.
 
 I'll 
 
 i BIOL1 I ic 
 
 "F lfl.>"|. -i 1:1 M 
 
 |, 
 
 I'MII K VII. /.'./.7, -riitli/tn- 'i.Vi/.M ,,f i, HI-UK il liny *'nnn nfl, r 'l'ii,,l,i,. : //,* 
 
 tun, in tin r<_lriii>rnti,r. ///<*.</ ,,i tfi, tail, hull, >lnt 1749. 
 
 lt. 
 
 Jillir 
 
 7 
 N 
 
 9 
 19 
 1 
 
 7 
 1 
 
 19 
 J> 
 1 
 
 I 
 
 9 
 19 
 
 1 mi.' nft.-r .Iriiwin.- 
 
 cultiitv IIM-.I i.. I.-M 
 ttacterloljrvla. 
 
 Nun. 
 : n* 
 Inir.. 
 ,-,-r 1 . ( 
 
 Nilin' 
 Iwi t. 
 iM-r 1 
 
 .lil.T 
 
 I hour... 
 1 -U\ .... 
 
 ! I 
 
 
 
 / . 
 M.760 
 
 M 
 H 
 
 a 
 
 700 
 Ml 
 ' 1 
 
 4.430 
 
 : 
 
 2,000 
 
 
 
 
 - - . . 
 
 
 M 
 
 
 ...do... 
 
 Ml 
 
 
 
 ; H 
 
 11 .ln>- 
 
 
 : M 
 
 ! Imii'r 
 
 
 Crawford 
 
 -. 
 
 1 ,lnv 
 
 
 N 200 
 
 
 ...do .. 
 
 7 440 
 
 
 
 :'. -lax - 
 
 ...do... 
 
 
 
 
 , 
 
 lltln\ 
 
 to 
 
 1 tin 
 
 i hour 
 
 u. I 1 
 
 
 
 do 
 
 ;> 
 
 2 davn 
 
 
 
 '. "l:l\ 
 
 
 ., 
 
 1 'l;tV. 
 
 .1.. 
 
 ' -V. 
 
 11 ilitVK 
 
 ,1 
 
 
 
 
 
 oTh^ i-liiinn-t.-r -itrnitii- tlmt tlu-r<- w-rt' too many roloniiii (.. iinint. 
 
 >iTiun from tho l)lood at the truth drawing from hor ITli'. :i- 
 Table VII sliows. prcsci vrd it> hactcricidiil powt-r to a marked degree 
 for tlu Crawford culture uj) to and including the >rcond day of stand- 
 ing. It appeal^ to liave IMTM >oiuc\vhat more potmt on tho fourth 
 than on the third day of ^tandiiiir. Imt tho dinVrenee in potency of the 
 ^eruin on the-e t\\<> days i> hardly >uliicient. perhaps, to IK- of any 
 great signiricance. For the other two strains there was at most inhi- 
 bition shown in some of the tots. lut no decided baeterioly-U either 
 before or after standing, except perhaps in the case of the F. L''> -train 
 after t\.<> day-. But in the case of this organism the serum appeared 
 to have been more |M>tent after -landing for three da\ - than it \\a- 
 previou- to thi> time. 
 
 TMII.K VIII. -- Itiiilrrinhftir ,i,-lii, i, <,j normal IK><J trrutn aflrr ulnitilhuj t'r utriinin length* 
 "f tini' in tin- rifri'firntor. liliMxl ilrnnn fruin tin tail. I'-n nlli ilrnn imf. /// \. 
 
 I'M 
 
 Tlm nflrr drawing. 
 
 ('ilium- ii^.-<l in t.-t 
 bactcrlolyd*. 
 
 Sui In-rol 
 
 Hilr."ltn . -I 
 pt-r 1 r < 
 
 Niini>N-rof 
 bacteria 
 
 p<-r I 
 af l.-r 1 day. 
 
 1905. 
 
 Jinn- U 
 
 | hour . ... 
 
 1 | 
 
 2.SMO 
 
 . . 
 
 16 
 
 
 
 1,000 
 
 1 INI 
 
 19 
 
 
 - 
 
 
 4. MO 
 
 
 22 
 
 in .1.1 \ - 
 
 do 
 
 ., .. 
 
 
 12 
 
 i hour 
 
 ('rnwlord. .. 
 
 ,. 
 
 400 
 
 16 
 
 
 
 I Ml 
 
 2*0 
 
 19 
 
 
 
 
 1.440 
 
 32C 
 
 
 
 
 .V> 400 
 
 . 
 
 12 
 
 
 ' I' Ifl92 .. 
 
 
 800 
 
 19 
 
 
 do. 
 
 1 71" 
 
 
 22 
 
 
 do 
 
 , .. 
 
 
 
 
 
 
 
 
 iiarmctcr iii(nlRe> that then- were too many >l<>m<- lo count
 
 COMPARISON OF BACTERICIDAL POTENCY OF SERUM. 
 
 21 
 
 The serum from the blood of hog 1742 at the eleventh drawing, as 
 shown in Table VIII, had marked bactericidal power for all three 
 strains at the start, and this was retained for as long as seven days for 
 the Crawford culture, whereas there was inhibition only for F. 26 
 after four days, and after this there was not even inhibition for this 
 organism. The effect upon the G. P. 4692 culture was similar to that 
 upon the F. 26 strain. 
 
 TABLE IX. Bacteriolytic action of normal hog serum after standing for various lengths 
 of time in the refrigerator. Blood drawn from the jugular rein, twelfth drawing. Hog 
 No. 1742. 
 
 Date. 
 
 Time after drawing. 
 
 Culture used to test 
 bacteriolysis. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c.c. 
 
 Number of 
 bacteria 
 per 1 c.c. 
 after 1 day. 
 
 1905. 
 June 22 
 
 i hour 
 
 Crawford 
 
 4 320 
 
 
 23 
 
 1 (lav 
 
 . do 
 
 4,060 
 
 
 22 
 
 i hour 
 
 F. 26 
 
 3,380 
 
 
 23 
 
 1 dav 
 
 do 
 
 640 
 
 00 
 
 22 
 
 i hour . . 
 
 G. P. 4692. 
 
 1,240 
 
 
 23 
 
 1 day 
 
 do 
 
 340 
 
 00 
 
 
 
 
 
 
 a This character signifies that there were too many colonies to count. 
 
 The serum from the blood of hog 1742 at the twelfth drawing (Table 
 IX) was taken from the jugular vein, and it will be observed from 
 the table that the serum showed no bacteriolytic power either at the 
 start or on standing. In order to test whether the lack of bactericidal 
 power was due in this case to the fact that the blood was drawn from 
 the vein, experiments, which are hereinafter given (page 22), were 
 made to compare the bactericidal power of arterial blood serum with 
 that of venous blood serum. 
 
 COMPARISON OF THE BACTERICIDAL POTENCY OF THE 
 SERUM FOR DIFFERENT STRAINS OF BACTERIA. 
 
 SAME 
 
 If in the foregoing tables comparison is made between the results 
 obtained with the same drawing of blood upon the various organisms, 
 it will be seen that quite decided differences appear. 
 
 Selecting the most striking contrasts, in one case there was a decided 
 increase of the B. choleras, suis, culture G. P. 4692, while in the same 
 blood 7,000 B. coli per 1 c. c. were all destroyed. It is true that in 
 this case a great many more of B. choleras suis were introduced at the 
 start than of B. coli, and this may have influenced the result some- 
 what. But in another case where approximately the same number of 
 organisms of the two kinds were introduced at the start there were 
 veiy few of the B. choleras, suis, if any, destroyed, whereas nearly all 
 of the B. coli were destroyed. In still another case there was a very 
 great increase of B. choleras suis, while all of the B. coli were de- 
 stroyed. In one case there was no notable increase of the B. choleras 
 suis. while there was a decrease of the B. coli.
 
 22 
 
 HACTKKH'I N I h POWKB OF HI..H,|. s|KI\| Ql BOOS. 
 
 It is e\ ident, therefore. that tin- bactericidal pouer of hog >erum is 
 different for tins strain of />'. ,!,,,/, n .-//. and for the II. ,,/;, \\\ go 
 far as this rim In- determined l>\ tin- method- :it pre-.-nt in use. 
 
 THE POTENCY OF SERUM FROM ARTERIAL BLOOD COMPARED 
 WITH THAT FROM VENOUS BLOOD. 
 
 As hereinlM'fon- staled. thr following experiment! were undertaken 
 
 because of iln- iv-ull- .ilit.-iin.-d \\ith tin- -.-rum fnun tin- \en,.ii- M ..... 1 
 in tin experiment in which it appeared that there was neither inhibi- 
 tory nor bactericidal power in the serum. In the experiments pre- 
 vious to this one the blood was drawn from the tail, and was 
 con-et|uently mainly arterial. It spurted from a severe. 1 artery when 
 the tail was cut. In the experiments at present under consideration 
 the blood was drawn from the carotid artery or from the jugular vein 
 either at the same operation or on different occasion-. 
 
 TABLK X. Itacteriolytic action of arterial nrrmn i-<n}M\rl irlth tlml of i-faou* *mtm, 
 Ixilli x,-nt I'roin tit,' K'lint- inirninl luxj (\<>. 17.1.1) nt the Mine operation. T'nll, .//-./ 
 nf liltMnl. Titt* inmle at intt'ri'al a 
 
 Time- after drawing. 
 
 Culture u..l in ti-t 
 tiactcriulysls. 
 
 NtlllllMTI'f 
 
 ItwctcriH 
 intnxtiioMl 
 perlc.t-. 
 
 NlllIllNTl 
 
 |HT 1 
 
 1 .1,,J. 
 
 AM. -rial 
 M-niin. 
 
 >( bacteria 
 f. c. after 
 
 \'. 
 
 Hma 
 
 1 ilav 
 
 F. 26... 
 
 \,m 
 
 m 
 IM 
 
 1,480 
 420 
 . II- 
 9. IWO 
 900 
 
 60 
 1A.320 
 2,200 
 40 
 200 
 140 
 
 lino 
 
 !] 
 
 100 
 
 
 ,,. 
 
 
 
 80 
 120 
 40 
 6.820 
 
 , , 
 
 140 
 
 J <\H\* 
 
 <\<>. 
 
 
 do. . . 
 
 
 1 (lav 
 
 Crawfonl . . . 
 
 J iln\ 
 
 ...do... 
 
 
 
 
 1 .lav 
 J duv 
 
 
 0. F.46W 
 ...do... 
 
 9 djn 
 
 do 
 
 
 
 The serum from hog 17H3 at the tenth drawing, as shown in Table 
 X, was taken at the same operation from the carotid artery and from 
 the jugular vein. In the t<--N with the Crawford culture the serum 
 from the arterial, as well as that from the venous blood, seem both to 
 have |>o-^r>M-d considerable jx>tenry, and to have ] I it in about 
 
 equal degree. They furthermore appear to have retained their 
 potency, much or all of it. for nine days. For (i. P. 4'.'.'i > culture 
 neither serum appears to have had strong bactericidal pouer in any 
 They both -eem to have had some inhibitory jxjwer for thi^ 
 organism both at the start and after nine days. For culture F. 26 
 there appear- to have leen no difference between the two kind- 
 serum after having stood for one day, but the serum from the venou- 
 bloo.l would seem to have retained it- potency upon -landing more 
 tenacioii-lv than that from the arterial blood.
 
 ARTERIAL AND VENOUS BLOOD SERUM COMPARED. 
 
 23 
 
 TABLE XI. Baeteriolytic action of arterial strum compared with that of renous serum, 
 both Kern from the same normal hog (No. 1733) at the game operation. Eleventh draw- 
 ing of blood. Texts made at intervals after drawing. 
 
 Time after drawing. 
 
 Culture used to test 
 bacteriolysis. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. K. 
 
 Number of bacteria 
 per 1 c. c. after 
 1 day. 
 
 Arterial 
 serum. 
 
 Venous 
 serum. 
 
 1 dav 
 
 F. 26... 
 
 5,420 
 6,520 
 
 10,480 
 20,160 
 280 
 10,960 
 160 
 400 
 20,640 
 7,280 
 2, 520 
 2,960 
 21,240 
 
 00 
 
 220 
 27,680 
 8,960 
 2,600 
 10,860 
 360 
 580 
 60 
 3,300 
 3,340 
 1,360 
 2,320 
 
 2 days 
 
 do 
 
 r> davs 
 
 ...do... 
 
 8,580 
 240 
 8,160 
 8,660 
 26,240 
 1,920 
 2,160 
 2,720 
 a oo 
 2,240 
 
 8 davs 
 
 do 
 
 1 day 
 
 Crawford 
 
 2 days 
 
 ...do... 
 
 5 davs 
 
 do 
 
 8 days 
 
 . ..do 
 
 Idav 
 
 G. P. 4692 
 
 2 days 
 
 do 
 
 5 days 
 
 do 
 
 8 davs 
 
 do 
 
 
 
 "This character signifies that there were too many colonies to count. 
 
 The blood obtained at the eleventh drawing from hog 1733, as shown 
 in Table XI, was drawn from the artery and from the vein at the 
 same operation. There seems to have been a difference in the potency 
 between the two kinds of serum for all three organisms. For the 
 Crawford culture the serum from the arterial blood seems to have 
 been quite strongly bactericidal at the start, but the serum from the 
 venous blood was apparently not so. The tests made after the serum 
 had stood for two days would indicate that both kinds of serum were 
 potent. The serum from the venous blood in this case seems to have 
 gained in potency upon standing, a result previously noted in the 
 case of culture F. 26 with a different serum, but it seems necessary to 
 conclude that an error of some kind must have crept in where such 
 results are obtained, since they are at variance not only with the 
 a priori probabilities of the case but also with the usual experience. 
 On the whole, the results in this experiment seem to indicate for the 
 Crawford culture a greater potency, or at least a retention of a greater 
 amount of potency, on the part of the venous than of the arterial blood. 
 
 For culture F. 26 in this experiment the arterial serum appeared less 
 potent at the start and after eight days than the venous serum, but 
 after standing five days it was more potent than the venous serum. 
 Here again the serum seems to have gained potency on standing. 
 
 For G. P. 4692 the stronger retention of potency of the venous 
 serum as compared with the arterial is apparent in the tests made five 
 days and eight days after drawing. No difference between the two 
 sorts of serum is noticeable in the tests made with this organism one 
 day and two days after drawing. 
 
 The fact that blood through which a stream of CO 2 was conducted 
 becomes more alkaline led Hamburger 9 to make tests of the compara- 
 tive bactericidal potency of serum from ordinary blood and that from
 
 24 
 
 i RIOL) 'I. r>\\ IK OP MI. -I 1:1 M "I 
 
 Mood llin.nyli \\ In. h ( ( li:i.l In-.-n pa-xi-d. M- \\.-ll a- !' the difference 
 
 in tlii- rexp-ci Itetueen arterial and venou- Uood. Mr found. in fa.-i. 
 i h;ii serum from \enou> |I|IMH| and from l>l....:l through which CO, had 
 (Missed is more potent than UterbU-blood serum. 
 
 EFFECTS OF HEAT UPON THE BACTERICIDAL POWER OF HOO 
 
 SERUM. 
 
 A large number of (.>!- \\.-re made parallel with those above recorded 
 to determine the effect of heat upon the gerwicidal power of hog 
 serum, mid also to determine whether there i- anv difference lct\\ < -n 
 the potency of the heated -crum for the different strains of It. cholerx 
 SHIM upon which it was tested. 
 
 The method employed in making the te-K consisted in immer-inir 
 the tubes containing the serum up to the plugs in a water Imth which 
 \\a^ kept by means of a thermoregulator exactly at the temperature to 
 be tested. The results are shown in the following tables, from whieh 
 it will be seen that the serum was subjected to temperatures of 51, 
 52, 53, and 54 C. for ten minutes and for thirty minutes at different 
 times. Tests were also made at temperatures al>ove 54 C.. but these 
 are not tabulated for the reason that they gave results similar to those 
 at 54. 
 
 TABI.R XII. Effect* <>f hi-nt n/xm the barlirinhilli- jtowtr of normal hog terum. Blood 
 drawn from ll toil mul /.</./ in tin refrigerator for varion* length* of time. Temper- 
 ature ftnjtloiffil, :,i C. 
 
 Date. 
 
 Titm- nftT 
 
 ilruwiiii:. 
 
 N". i.f hiv 
 and drawiiiK 
 of bl.xxl. 
 
 Culture iix-<l 
 
 til ! 
 
 tiactfriolysi*. 
 
 Number of 
 
 tiarlcrin 
 intr.Mlurtil 
 IKT 1 . . 
 
 Arterial blood. 
 
 l^'iiirth of 
 expxiirc to 
 ht-nt. 
 
 Numberr 
 
 [*Tl <..-. n 
 
 N..t 
 h.-at.-il. 
 
 fbnct-ri 
 ftiT lilny. 
 
 M.-nl.-.l. 
 
 1*K 
 April 7 
 
 June 12 
 
 :t li-uir*... 
 .1.. 
 i liniir 
 |.lnv .... 
 T.IMV- .... 
 I."l\- .. 
 
 J li<>iir 
 I >\n\~ .... 
 
 10 <1 >... 
 
 ; liniir 
 
 T .lm 
 
 17881... 
 
 <!<> 
 ITU XI 
 .lo 
 do 
 ... .do 
 do 
 do 
 do 
 
 do 
 
 .. I- MB... 
 
 F. a; 
 do 
 
 d.. :.. 
 
 ( 'raw ford 
 
 4.GX 
 
 I'. H-'M) 
 
 l.UUU 
 4.040 
 
 1,860 
 
 t,m 
 
 1 IKI 
 
 .. i.., 
 
 16. 1'JO 
 1.740 
 8. WO 
 
 TC.flOO 
 30. KX> 
 
 I.IK) 
 
 M,MO 
 
 OB 
 
 400 
 
 :NI 
 
 M 
 
 90 
 
 800 
 1.2HO 
 
 OD 
 
 ,, nog 
 ,, no 
 
 1.100 
 
 <l x 
 
 ao 
 440 
 1. Ill) 
 4,200 
 
 11.200 
 I.K40 
 2K.MO 
 
 10 minute*. 
 
 1)0. 
 
 90 minutes. 
 Do. 
 
 Do 
 
 i... 
 Da 
 Do. 
 Do. 
 Do. 
 
 Da 
 Da 
 
 
 
 
 
 -. i- u;s 
 .1.. 
 .... .1.. 
 
 Thi huni. i. r -ii:niii.- that there were U many <-il..ni-> io . ..nut 
 
 It would appear from the results recorded in Table XII that heat- 
 ing for ten minutes at 51 C. produced no etfert in the two tests which 
 were made in this manner, one on the G. P. 4692 strain of B. chol- >: 
 .>"/>. and the other on the colon culture. The same temperature for 
 thirty minutes robbed the serum of much or all of its power in 8 of 
 the 11 tests.
 
 EFFECTS OF HEAT UPON BACTERICIDAL POWER. 25 
 
 TABLE XIII. Effects of heat upon the bacteriolytic power of normal hog serum. Blood 
 drawn from the tail. Temperature employed, 52 C. 
 
 Date. 
 
 Time 
 after 
 draw- 
 ing. 
 
 -9 
 
 No. of 
 hog and 
 drawing 
 of blood. 
 
 Culture 
 used to test 
 bacterio- 
 lysis. 
 
 Num- 
 ber of 
 bac- 
 teria 
 intro- 
 duced 
 per 
 1 c. c. 
 
 Number of bacteria per 1 c. c. 
 after 1 day. 
 
 Length of 
 exposure 
 to heat. 
 
 Arterial blood. 
 
 Venous blood. 
 
 Not 
 heated. 
 
 Heated. 
 
 Not 
 heated. 
 
 Heated. 
 
 1905. 
 June 28 
 
 July 12 
 
 1 day . . . 
 2 days . . 
 
 1733 X.. 
 do .... 
 
 F. 26 
 ...do... 
 
 3,880 
 4,920 
 380 
 940 
 1,480 
 420 
 2,440 
 9,880 
 300 
 5, 420 
 6,520 
 2, 160 
 2,720 
 
 60 
 16, 320 
 2,200 
 40 
 200 
 140 
 9,760 
 8,160 
 100 
 10, 480 
 20, 160 
 2,620 
 2,960 
 
 1,540 
 15, 200 
 1,720 
 3,960 
 460 
 300 
 1,920 
 9,840 
 100 
 
 320 
 600 
 400 
 80 
 120 
 40 
 5,920 
 6,560 
 140 
 220 
 27, C80 
 3,300 
 3,340 
 
 10,720 
 3,360 
 
 a 30 
 25, 080 
 
 00 
 
 X 
 
 3,080 
 10,000 
 
 30 minutes. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 
 Do. 
 Do. 
 Do. 
 
 9 days 
 
 do 
 
 do 
 
 1 day 
 
 do . 
 
 Crawford 
 
 2 days . . 
 
 do.... 
 
 ...do... 
 
 9 days . . 
 
 ...do... 
 
 do 
 
 1 day 
 
 .do 
 
 G. P. 4692... 
 
 2 davs 
 
 do.... 
 
 ...do... 
 
 
 do 
 
 . do 
 
 1 day . . . 
 2 days . . 
 1 day 
 
 1733 XI ... 
 do.... 
 do.... 
 
 F. 26 
 do 
 G. P. 4692... 
 
 5,680 
 41,720 
 2, 240 
 
 (?) 
 
 14,880 
 2.440 
 2,180 
 
 2 days ' 
 
 do 
 
 ...do 
 
 
 
 
 "This character signifies that there were too many colonies to count. 
 
 It will be seen from the above table that the effect of an exposure 
 of thirty minutes showed a marked influence in some cases while in 
 others it was apparently without effect. In these experiments the 
 blood was drawn at the same operation from the carotid artery and 
 from the jugular vein, and it would appear that the serum from the 
 venous blood was somewhat more sensitive to the action of the heat 
 than the serum from the arterial blood, if any conclusions are per- 
 missible from the somewhat limited number of observations. In the 
 arterial there was in only one case a marked difference between the 
 potency of the heated and of the unheated serum out of the 12 cases 
 in which it was tried. In the serum from the venous blood, on the 
 other hand, there was a marked difference between the heated and the 
 unheated serum in the 11 te^ts which were made. On the whole the 
 effect of heating at 52 C. would appear to be uncertain; sometimes 
 heating seems to have a marked effect upon the bactericidal power of 
 the serum; at others it seems to lack this effect. 
 
 19500 No. 9507 4
 
 BA< rKKlOLYTIC I'oXVKK <>K HI. -MMM "! HOGS. 
 
 T \IIIK XIV. Kipi-ct* a f hull III>H tin Inn-It riot (///< fxmrr f iu>rnuil )nj *nnn. 
 ilrnirii I'niiit lltr tail. 1'i-iii/n-riitnri- nni>l'ir<l, .',.1 ('. 
 
 /;/,./ 
 
 
 
 Time ftT 
 drawing. 
 
 NO. Of In < " lid 
 
 drn liik- ( 
 
 blood! 
 
 Cotton 
 
 llwll 
 burl. 
 
 U-i- 
 
 Nunilx-r 
 of l>ai-(i-Ma 
 
 Intro 
 
 Sum 
 hartrrU 
 
 nfli-r 
 
 Sol 
 
 \' "' 
 
 XT 1 C. r. 
 
 1 da> 
 
 l.i-IIL-t ' 
 
 n- to 
 
 190ft. 
 
 \|T I 1 .' 
 
 
 i : . . v 
 
 U. P. 4092... 
 
 - , . , 
 
 i , ... 
 
 ; . .- 
 
 10 nn i 
 
 19 
 
 J davs 
 
 
 
 ll.roli 
 
 ', r IMW 
 
 i ' - 
 
 ; . 
 
 ..: -i n 
 1 ... 
 
 
 -"' 
 
 <|,i 
 
 
 H riili 
 
 I 940 
 
 i 
 
 
 
 26 
 
 
 
 11 
 
 do 
 
 
 1.800 
 
 
 
 Do 
 
 Mnv 
 
 ;i hittip* . 
 
 1 1 
 
 
 ::. i. - 
 
 17 M 
 
 
 
 
 
 
 do 
 
 H.eoli 
 <i T. 4fi92 
 
 i m 
 
 - 
 
 I 004 
 
 9.920 
 
 IS 720 
 
 1 
 
 Do 
 
 
 i 
 
 
 
 17401 
 
 H. ro/i 
 1-.92 
 
 i 
 
 975 
 
 .'. 04 
 1,750 
 
 ,.,. 
 
 Do. 
 
 n 
 
 10 
 
 
 
 1740 II 
 
 '1746111!!! 
 
 171" IV 
 
 <;. i'. ! 
 
 H.oJi 
 
 i, I'. ! 
 
 i MB 
 
 \M 
 
 1.010 
 3.110 
 
 70 
 1 ] | 
 i M 
 994 
 
 1,900 
 
 . ' 
 1 1 . 
 
 Do. 
 
 Do. 
 Do. 
 
 |l<> 
 
 11 
 12 
 12 
 
 8 
 
 
 
 do 
 do 
 
 
 1740 VI 
 
 
 1711 1 
 
 <;. r ii 
 
 ^. P. 4692 
 
 
 Ml 
 
 1,700 
 
 450 
 
 1,840 
 
 .' -l 
 
 15,900 
 4 1 II 
 
 Do 
 Do 
 Do. 
 
 1 
 
 10 
 
 
 
 
 ...do.. 
 
 do 
 
 . 1T ",',!:::::: 
 
 17M III 
 
 
 
 I'.'.'J . . 
 li. -1, 
 G. 4692 
 
 3,778 
 102 
 1,970 
 1,010 
 
 II 
 
 390 
 |,M 
 
 1 ! I 
 910 
 1.2X0 
 
 i :> 
 
 Do. 
 
 Do 
 
 10 
 
 . . .do . . . 
 
 do 
 
 
 3,110 
 
 40 
 
 7" 
 
 Do 
 
 11 
 12 
 
 
 
 ...do... 
 
 1741 IV 
 1711 V 
 
 ;.. 4692... 
 Hi i 
 
 Ml 
 1,700 
 
 MO 
 
 
 3.286 
 1.1M1 
 
 Do 
 Do, 
 
 8 
 
 do 
 
 17 1'. 1 I . . 
 
 ; i (092 
 
 4 925 
 
 
 
 Do. 
 
 8 
 
 do 
 
 do 
 
 I r 
 
 
 
 
 
 1" 
 
 
 1712 II 
 
 . 1'. 
 
 
 M 
 
 
 Do 
 
 1" 
 
 do 
 
 
 j rrrf. 
 
 1 970 
 
 
 7:t4 
 
 
 U 
 U 
 
 11 
 
 do 
 do 
 
 rum. 
 
 do 
 171.' IV 
 
 i 1'. ?J2 
 
 i. r i 
 
 1,010 
 
 :l. no 
 
 798 
 30 
 50 
 
 Mil 
 
 1.758 
 
 Do. 
 
 Do 
 
 u 
 
 Julv 12 
 
 i VlHV ""III! 
 
 1742V 
 173:1X1 ' .. . 
 
 It. f,tli 
 
 r H 
 
 1,700 
 
 5 420 
 
 
 
 10.480 
 
 1,181 
 
 Do. 
 
 1 " minute*. 
 
 u 
 
 12 
 13 
 
 u 
 
 13 
 
 -'day- 
 1 day 
 J diiv 
 1 dy 
 
 do 
 do 
 do 
 do 
 do 
 
 do 
 Crawford 
 
 .... do 
 
 (J. p. 1- 
 
 2,100 
 
 no 
 
 20 160 
 
 M 
 
 400 
 2,960 
 
 H -" 
 
 2! 440 
 2.180 
 
 Da 
 
 Do, 
 Do 
 Do. 
 
 12 
 
 1 dav ... 
 
 17: XI'-.. 
 
 I M 
 
 5. 120 
 
 
 
 
 13 
 12 
 13 
 
 '.'lav* 
 1 da\ 
 
 Ida? .'.'. 
 
 2 ilny- 
 
 do 
 do 
 do 
 ...do 
 do 
 
 
 
 r raw lord 
 .... do 
 (!. P. 469 1 .? 
 
 
 6.520 
 
 10. MO 
 
 .. . M 
 
 41.720 
 2R.160 
 
 (T) 
 
 Do 
 Do 
 Do 
 
 "This i-luirartcr *i>riiit:rs (lint tln-n- \\-rr hm iimny t-nlonio in rouiil. 
 * Arteiial I>I<>1 M-riiin. 
 
 ' Venous M.H| MTtlll). 
 
 A- -lin\vii Ity the results recorded in Tahlr XH". lie:itin<: at 
 had tlie ftlect in most casos of jrreutly diminishing the liitctericidal 
 power of the -enmi. though this is not to lie noted in all <-:i-e-. e\-.-n 
 \\here the xeriim \\:i- exposed for thirty minute^.
 
 EFFECTS OF HEAT UPON BACTERICIDAL POWER. 
 
 27 
 
 TABLE XV. Effects of heat upon the bac'eriolytic power of normal liog serum, 
 drawn from the tail. Temperature employed, 54 C. 
 
 Blood 
 
 Date. 
 
 Time after 
 drawing. 
 
 No. of hog and 
 drawing of 
 blood. 
 
 Culture used 
 to test 
 bacteriolysis. 
 
 Number of 
 bacteria in- 
 troduced 
 per 1 c. c. 
 
 Number of bacteria 
 perlc. c. after 1 day. 
 
 Length of 
 exposure to 
 heat. 
 
 Not 
 heated. 
 
 Heated. 
 
 May 26 
 June 2 
 May 26 
 June 2 
 May 29 
 June 8 
 May 2'J 
 June 3 
 May 29 
 June 3 
 5 
 6 
 7 
 8 
 9 
 19 
 5 
 6 
 7 
 8 
 9 
 19 
 5 
 6 
 7 
 8 
 9 
 19 
 May 23 
 23 
 23 
 24 
 24 
 24 
 
 3 hours 
 
 1742 VIII.. 
 do 
 ...do... 
 
 Crawford . . . 
 do 
 F. 26 
 
 320 
 1,960 
 80 
 3, 280 
 1,740 
 1,220 
 2, 320 
 IOM 
 1,920 
 700 
 2,K40 
 2,240 
 3,440 
 2,200 
 1.360 
 4,040 
 8,380 
 5,200 
 7,440 
 3,500 
 2,640 
 1,440 
 1,380 
 3.C40 
 180 
 360 
 l.SSO 
 1,740 
 10, 8SO 
 40,500 
 19,040 
 1,682 
 1,808 
 12,040 
 
 
 380 
 1,960 
 2,980 
 4,480 
 1,180 
 
 
 960 
 1,100 
 26, 640 
 51,760 
 900 
 1,340 
 18, 280 
 a oo 
 180 
 120 
 760 
 3,160 
 1,380 
 oo 
 4,420 
 6, 320 
 3, 420 
 2,000 
 
 00 
 
 00 
 
 3.376 
 2,640 
 18,280 
 
 180 
 1,640 
 
 1,160 
 2,380 
 5,280 
 9, 360 
 13.600 
 1,180 
 '., 520 
 1,880 
 5,040 
 1,260 
 119,700 
 41,840 
 6,440 
 7,280 
 20,480 
 oo , 
 28,000 
 8, 320 
 13,000 
 4S,000 
 
 00 
 00 
 
 4,820 
 27, 760 
 
 00 
 
 17,960 
 
 00 
 
 00 
 
 13,180 
 58,800 
 
 00 
 
 2, 720 
 7,600 
 26, 320 
 
 10 minutes. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 30 minutes. 
 Do. 
 Do. 
 Do. 
 Do. 
 Do. 
 
 7 days 
 3 hours 
 
 7 davs 
 
 . do 
 
 do . .. 
 
 3 hours 
 
 1742 TX 
 
 do 
 
 5 days 
 
 do . 
 
 do 
 
 3 hours 
 
 do 
 
 Crawford 
 do 
 
 5 days 
 
 ...do 
 
 3 hou rs 
 
 do 
 
 G. P. 4692. 
 
 f> davs 
 
 do 
 
 do 
 
 J hour 
 
 1742 X . 
 
 F. 26 
 
 1 dav 
 
 do 
 
 do 
 
 2 days 
 
 do 
 
 ...do... 
 
 3 days 
 
 do 
 
 do... 
 
 4 days 
 14 davs 
 
 do.. 
 ...do ... 
 
 do 
 do 
 
 i hour 
 
 do. 
 
 Crawford 
 do 
 
 1 dav do 
 
 2 days .do 
 
 ...do ... 
 
 3 davs 
 
 .. .do . 
 
 .. do 
 
 4 days 
 
 . do.. 
 
 do 
 
 14 days do 
 
 do 
 
 hour do 
 1 dav do 
 
 G. P. 4692 
 do 
 
 2 days do 
 
 3 davs . ' do . . . 
 
 do 
 
 .do . . 
 
 4 days 
 14 days 
 
 do 
 
 ...do ... 
 
 do 
 ...do... 
 
 . .do 1742 VI 
 
 do 
 
 do do 
 
 do ' dn 
 
 Crawford 
 F. 26 
 
 . ...do. 
 
 1742 VII 
 
 G. P. 4692 
 
 do 
 ....do... 
 
 do 
 do 
 
 Crawford 
 F. 26 
 
 
 
 
 a This character signifies that there were too many colonies to count. 
 
 As is evident from the preceding table the effect of exposure for ten 
 minutes at 54 C., as well as exposure for thirty minutes at the same 
 temperature, is practically in all cases to weaken or suspend the 
 bacteriolytic power of the serum. The few exceptions to be seen in 
 the table are without significance in view of the many cases in which 
 the serum lost in power. 
 
 In summing up the results of heat upon the bacteriolytic power of 
 normal hog serum it would seem evident that temperatures below 54 
 C. are uncertain in action for the lengths of time employed, but that 
 54 C. even for ten minutes serves quite uniformly to suspend or at 
 least to weaken the bacteriolytic power of the blood serum. A num- 
 ber of tests were made at 55 C. and 56 C., but as these did not show 
 any results that were not to be expected from those at 54 C. it would 
 seem superfluous to give them in detail. 
 
 EFFECTS OF INJECTING CULTURES OF B. CHOLEILffi SUIS UPON 
 THE BACTERIOLYTIC POWER OF HOG SERUM FOR THIS OR- 
 GANISM. 
 
 Very conflicting results have been obtained by various investiga- 
 tors in regard to the effects produced upon the bacteriolytic power of 
 blood serum by the injection of animals with cultures of bacteria. 
 Some have failed to note any effect of such injections, either to in-
 
 BAOTJCBIOLTTIC 
 
 -M:IM 
 
 i ..... -. 
 
 civa-e ..r .Iiinini-h till' haeteriolytic potency; other- ha\e found that 
 
 lli> potcncN was diminished, others again that it wa- increased. I'n 
 
 der the supposition that this discrepancy in iv-ult- was due pn-hap- 
 
 to tin- te-ts having been made at different interval- of time after in 
 jeetion. and that the serum taken from th- -aim- animal at dillen nt 
 period- might exhibit dihVrence- in potenc\. <.r that it might at 
 tain times he devoid of power, te-t- \\eiv made of -erum taken at 
 
 various intervals after OM or more injection-. The re-uli- < ( f these 
 
 experiment- are -nmmari/ed in the following table-. 
 
 TMII.K \\'l. H'l'trrioli/tii- ftoinr of xt-runt fruiii tin <iin: // ( .\<>. 17.', 1 1 I,. !,,,-> nn<l 
 ilt,r iiijti-ti'ni intli II. rlin/,-r,i xnin, ,-nlt' 
 
 Ciiltur. H-..-.! in i.-i 
 IwirliTiiilyM-. 
 
 Time lil.MMl \Mi-ilniwn r.-lali\. (.. inj.-i-in.n. 
 
 NiiniU-r ..f 
 lia.-tcriH in 
 
 triMlllcell 
 
 IHT 1 
 
 NniiilH-r <if 
 
 iMleterill |T 
 1 < e. llfl.T 
 
 1 d.l>. 
 
 Cnif..r.l 
 
 
 
 
 
 jilt.-r lir-t in jit -ti< >M 
 
 
 
 1". 
 
 
 K. ji; 
 
 ! iln\ > ai'i.T s.'coti.l injection 
 loilnv- ai'i.T third injortiim 
 B.'forc injii-tinii 
 
 6.200 
 
 
 
 
 IL. 
 
 :i '1 n - aft.T lir>t inji-rti.ni 
 
 
 11 800 
 
 
 '. il -i\ - afl.T MTiiinl i ii j 11 -lii Hi 
 
 : MM 
 
 '2 &W 
 
 l. 
 
 in i!av- after Hi inl inject inn 
 
 fi,200 
 
 | j . 
 
 
 
 h,. 
 
 ltd on- inject inn 
 :! .In \ x after tirvt injection 
 
 1,060 
 
 tu P.. 
 
 in 
 
 12 WO 
 
 ]>,, 
 
 '.' i|n\ ~ alter NOOOO inject inn 
 
 MO 
 
 1 nJO 
 
 Di. 
 
 10 i\n\- after third injection 
 
 4,100 
 
 16,700 
 
 
 
 
 
 The results uiven in Table XVI were obtained with the serum from 
 a hojr which was injected nt various intervals suhcutaneou-lv \\itha 
 In-ef-broth suspension from an arar culture of the (i. 1*. ''.'_' -tmin. 
 
 The lir-t injection was made with 5 c. c. of a su-pen-ion e(pii\alent 
 in den.-ity to a twenty-four hour typhoid culture diluted about one- 
 half. The second injection was made with 10 c. c. of a sii-jH-n-ion of 
 about the same strength. The third injection was made with a -u- 
 pen-ion con-isting of the entire growth in twenty-four hours of an 
 a gar culture. Before each injection the blood was drawn from the tail 
 of the animal and placed on ice for three days. After thi- the -erum 
 \\a- <li awn otf from the blood and tested on the three -trains in all 
 cases except the serum. from the blood taken at the tir-t drawing, which 
 \\a- tested only upon the culture with which the animal was subse- 
 quently injected culture (J. I*. 4M2. 
 
 As there were no test- made with culture Crawford before the 
 animal was injected, it is impossible to be SUIT whether it \\a- bac- 
 tericidal for thi- -train at this time or not, but judging from its act ion 
 on(i. T. H'.!'J before injection it would seem proluihle that it would 
 have been bactericidal or. at least inhibitory for culture Crawford, 
 since the latter strain was quite uniformly more sen-it iv- to the 
 germicidal action of serum in other experiments. The -erum after 
 the injections possessed quit^ strong l>acteriolytic properties for cul- 
 ture Crawford.
 
 INJECTIONS OF CULTURES OF B. CHOLERA 9UI8. 
 
 29 
 
 It is also possible only to surmise from the results with G. P. 4692 
 that the serum probably had at least inhibitory power for F. 26 before 
 the animal was injected. It seems to have retained its inhibitory 
 power after the first injections for this organism, but to have lost much 
 in potency after the third injection. With culture G. P. 4692 the 
 results seem to be more or less in accord with those obtained with F. 
 26, though it is true that three days after the first injection the serum 
 exhibited very strong bactericidal power for G. P. 4692 stronger 
 than for F. 26. Still the results can not be compared too closely in 
 these two cases, since the number of bacteria introduced differed 
 widely. 
 
 TABLE XVII. Bacteriolytic power of serum from the same hog ( No. 1755} before and after 
 injection with B. cholerse suis, culture F. 26. 
 
 Culture used to test 
 bacteriolysis. 
 
 Time blood was drawn relative to injection. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. c. 
 
 Number of 
 bacteria per 
 1 c. c. after 
 1 day. 
 
 Crawford 
 
 Before injection .". 
 
 
 
 Do 
 
 3 days after first injection 
 
 2,180 
 
 40 
 
 Do 
 
 9 days after second injection. ...... 
 
 240 
 
 
 
 Do 
 
 10 davs after third injection 
 
 5,200 
 
 
 
 F.26 
 
 Do 
 
 Before injection 
 3 days after first injection 
 
 800 
 16,700 
 
 47,400 
 380 
 
 Do 
 
 9 days after second injection 
 
 7,600 
 
 11,880 
 
 Do 
 
 10 days after third injection 
 
 8,200 
 
 660 
 
 G. P. 4692 . . . 
 
 Before injection 
 
 
 
 Do 
 
 3 days after first injection 
 
 38,400 
 
 9,800 
 
 Do 
 
 9 davs after second injection 
 
 6,340 
 
 3,800 
 
 Do 
 
 10 days after third injection 
 
 4,100 
 
 11,200 
 
 
 
 
 
 The results of the experiments with the serum of hog 1755 are 
 recorded in Table XVII. In this experiment the injections were made 
 and the tests applied precisely as with hog 1754, except that culture 
 F. 26 was used for injecting the animal instead of culture G. P. 4692. 
 The concentration and amounts of culture were the same in both experi- 
 ments, and the methods of procedure otherwise were the same. The 
 serum from the hog before injection was tested upon the F. 26 strain 
 only; after injection it was tested with all three. 
 
 For the Crawford culture, as already stated, there was no test of the 
 serum made before the injection of the animal. The tests made with 
 this organism after each of the th'ree injections showed that the serum 
 possessed considerable potency, and there seems no evidence that the 
 injections were followed by diminution of potency for this organism. 
 
 For F. 26 culture the serum seems to have had but little potency to 
 start with before injection, but it seems rather to have gained than 
 lost in potency for this organism after injection. 
 
 For G. P. 4692 culture the results of the three tests are somewhat 
 difficult of comparison, perhaps, but the serum after the third injection 
 would appear to have been somewhat less potent than that after the 
 second, and, also, after the first, if the difference in the initial doses 
 in these two cases may be disregarded as a factor in determining the 
 results.
 
 80 
 
 HAiTKRInLYTH 1 I'oWKR 'K HI.ooH 
 
 <! I! 
 
 Tvlll.K XVIII. /.''. ' , r ,,f i-niiii from tin- IMIIIII- lioij ( .\'<t. 
 
 utter f/i/Vr/iiiii ii , tit />'. f/u*/rr:r i>ni/>, riill "/. 
 
 ' 
 
 Ciilmr.- tiM>d U> IWt 
 
 baetoriolyrii. 
 
 Time blood WM drawn n i.m\.- in mj.-.-n,,ii. 
 
 Nutiitx-r <>( 
 linrtrrin 
 tinr--' 
 per 1 
 
 \nnii 
 
 il |MT 
 
 ulii-r 
 
 I'niw lunl . . 
 
 llcliiro injection 
 
 TOO 
 
 
 
 lo 
 
 Do 
 
 :illiT tirM llijrrlloli 
 
 Mill r -i mini iiiji-< tii.n 
 
 I'" 
 I'l" 
 
 HO 
 
 o 
 
 Do 
 
 III ililN" lllIiT tlllnl lllJiTtlnll 
 
 
 . 
 
 
 Ih-forv inji-rl inn 
 
 
 
 
 
 after lirl mji-< linn 
 
 M "">' 
 
 
 
 Do 
 
 .ifd-r M-.-IHI.I Injection 
 In iln\ uftcr tliinl Injection 
 
 ; m 
 8,200 
 
 
 ( , | \(Q> . . . 
 
 Kcfnre injection 
 
 
 
 Do 
 
 3 I|HVH niter llrt injection 
 
 iij jjj 
 
 &.MO 
 
 
 tt <1a>' BfliT M'cnllil injection 
 
 . Ml 
 
 M 
 
 Do 
 
 10 dv ufti-r thinl injection 
 
 4,100 
 
 - ... 
 
 
 
 
 
 [n the experiment with tin- srnun of the Mood of ho^ 17.'.<;. the 
 n-.nlt> <>f which :irc ^ivrn in Tahlr XVIII. the method of procedure 
 u:i- the same ;i- with t he >rruiM of hoyfs 1 7. VI and 1755, just de-i-rilied. 
 the only difference IMMIIJJ in the strain >t' oiyani>in employed. In the 
 ca>e of hog 175G the Crawford Culture WM u>ed. and the .-ernin \\a- 
 tested upon this organism only before injection and on all three 
 -train> after the injections. 
 
 With the Crawford culture the serum seems to have been unaf- 
 fected ly the injections of the hog. It seems, at any rate, not to Inivc 
 lt in potency, and from the result after the second injection, if any 
 conclusion is justifiable, it seems possibly to have gained. 
 
 With F. _'; it >eem- to have lo>t in potency, if the result obtained 
 after the tirst injection is compared with those after the other two. 
 
 With (i. 1*. 4t>'.'2 culture the serum seems to have lost in potency 
 after the third injection, but it is uncertain whether it gained or l"-t 
 after the lir>t two. 
 
 TABLE XIX. llnrlrriiih/tit' f.ovrr of neritm from thf mint hoq ( A". 1798) brfarr <nul 
 after mju-tinn u-ith H. chdltTH unit, culture F. 6. 
 
 Culture nvil 
 bacterioljmta. 
 
 Time blood wn drawn relative to injection. 
 
 NiiinlM-r of 
 bacteria 
 introduced 
 per 1 
 
 Number of 
 iMdi-ria |K-r 
 1 . . c. after 
 Iday. 
 
 f ^ 
 
 IM reinjeetion 
 
 , 1 
 
 l.MO 
 
 Do .. 
 
 in iln\" after tlrM injection 
 
 . s 
 
 7 -JUO 
 
 Do 
 
 9 dayx after <-coii'l injection 
 
 , ,-,, 
 
 . .. 
 
 Do 
 
 1 1 iliivu after ihlnl inject inn 
 
 : 
 
 ., .., 
 
 
 
 
 
 In the experiment with the >erum of hog 1798, Table XIX. the 
 animal wa^ given three intravenous injections of suspensions in beef 
 broth from agar cultures of the F. '2K strain. The tirst one was made 
 with a very dilute -u>pension. weaker than would correspond with a 
 twenty-four hour In-ef-broth typhoid culture, and the other two injec- 
 tion-, were made with -imilar. >ii>|>cn>Mons. 
 
 It would appear from the results given in the table that the serum 
 w.i- not actively bactericidal before the injection of the animal, but 
 it seems to have had some inhibitory power. Ten days after the tirat
 
 INJECTIONS OF CULTURES OF B. CHOLERA SUIS. 
 
 31 
 
 injection there seems to have been no striking change. The serum 
 was still inhibitory, but was neither markedly bactericidal nor can it 
 be said to have been devoid of potency. But nine days after the 
 second injection, and fourteen days after the third injection, the serum 
 seems' to have lost greatly in the inhibitory power which it possessed, 
 it would appear that in this case the injections had the effect of ulti- 
 mately diminishing the power of the serum. 
 
 TABLE XX. Bacteriolytic power of serum from the same hog (No. 1809) before and after 
 injection with B. choleric suis, culture G. P. 4692. 
 
 Culture used to test 
 bacteriolysis. 
 
 Time blood was drawn relative to injection. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. c. 
 
 Number of 
 bacteria per 
 1 c. c. after 
 1 day. 
 
 F. 26... 
 
 Before injection 
 
 5,920 
 
 5,900 
 
 Do 
 
 14 davs after injection ... 
 
 7,300 
 
 1,800 
 
 Crawford . 
 
 Before injection . . 
 
 5,700 
 
 
 
 Do 
 
 14 davs after injection 
 
 13,000 
 
 56,600 
 
 G. P. 4692 
 
 Before injection 
 
 1,140 
 
 10.9CO 
 
 Do 
 
 14 days after injection . 
 
 7,300 
 
 7,900 
 
 
 
 
 
 In the experiment given in Table XX the serum of hog 1809 was 
 tested on the three different strains of B. choleras suis before the animal 
 was injected and on the same cultures fourteen days after the hog had 
 received subcutaneously about 5 c. c. of a dilute suspension from an 
 agar culture of culture G. P. 4692. In the tests made with the serum 
 upon the F. 26 strain there seems to have been a slight diminution of 
 potency after the inoculation, but the difference in the numbers of 
 bacteria surviving bacteriolysis in the serum from the hog before 
 injection and those surviving after injection is not large enough to 
 permit of very definite conclusions. The same may be said in regard 
 to the organism with which the hog was injected culture G. P. 4692 
 but the results in this case are perhaps somewhat more significant. 
 It would seem justifiable, however, to conclude that there was no 
 marked increase nor decrease of bacteriolytic power for F. 26 culture 
 or for G. P. 4692 produced by the injection. The result with Craw- 
 ford seems to show plainly that the serum was less potent for this 
 organism after injection than it was before. 
 
 TABLE XXI. Bacteriolytic power of serum from the same hog (No. 1836) before and 
 after injection ivith B. cholera suis, culture F. 26. 
 
 Culture used to test 
 bacteriolysis. 
 
 Time blood was drawn relative to injection. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. c. 
 
 Number of 
 bacteria per 
 1 c. c. after 
 1 day. 
 
 Crawford 
 
 Before injection 
 
 32 200 
 
 a 3 840 
 
 Do 
 
 1 hour after injection 
 
 32, 200 
 
 a 5, 340 
 
 Do 
 
 3 davs after injection 
 
 1 560 
 
 
 F. 26 
 
 Before injection .. . . 
 
 30,400 
 
 a 247 800 
 
 Do 
 
 1 hour after injection 
 
 30,400 
 
 " x 
 
 Do .. 
 
 3 davs after injection 
 
 5 000 
 
 
 G. P. 4692 
 
 Before injection 
 
 35 000 
 
 a 47 4CO 
 
 Do 
 
 1 hour after injection 
 
 35,000 
 
 a 36, 600 
 
 Do 
 
 3 days after injection . 
 
 6 800 
 
 
 
 
 
 
 a Serum diluted in the proportion of 1.4. 
 
 6 This character signifies that there were too many colonies to count.
 
 HACTI i:i"i.i ii< i'"\\ i i; 01 BLOOD -i-:i:rjf OF li> 
 
 In tln experiment with the >erum from tin- l>l>o<l of hog 1830, given 
 in Table XXI, the animal \\a- injected int ravenously with a -u-; 
 -ion from an a gar culture of -train !'. L'I', of about the density of a 
 :M-hour l>eef-broth culture of the typhoid bacillus. Before injection 
 the hog was hied to obtain -enim for testing the power of the 
 normal serum. The amount of blood obtained before injection and 
 that obtained one hour after injection wa> in-utlicient for the test to 
 be made with the- quantity of serum usually employed, so the -enim 
 was diluted in both of tlie-e ea-es with three pan- of physiological 
 salt solution to one part of serum. There were no tests made with 
 the undiluted -rum. 
 
 With the Crawford culture the serum seems to have been quite 
 potent before injection, and also one hour after injection, but three 
 days after injection it appears in have been entirely devoid of bacter- 
 iolytie power for this organism. 
 
 With F. _' -the strain used to inject the animal the serum had 
 only weak potency before injection, and if there was any effect pro- 
 duced by the injection it was in the direction of weakening the potency 
 of the serum for this organism. This apparent diminution of potency 
 i> to be noted even in one hour after the injection, and is still more 
 pronounced after three days. 
 
 With the (i. P. 45t-J strain the serum was apparently inhibitory at 
 the start before injection and one hour afterwards, but three days 
 after injection it seems to have lost even the power to inhibit the 
 growth of the organism. 
 
 The results of this experiment seem to show that in this case the 
 effect of injecting the hog was to suspend the bactericidal power of 
 the serum for the homologous" organism a- well a- for the other 
 -trains upon which it was tested. 
 
 TABLE XXII. Baekrtobftfc powtr <>f serum from the Mine hog (,V. 18,59) before and 
 after injection icith B. cholerx suit, culture (i, P. 4692. 
 
 Culture used to test 
 bacteriolysis. 
 
 Time blood was drawn relative to injection. 
 
 NiimlM-r "f 
 bacteria in- 
 troduced 
 per 1 c. c. 
 
 Number of 
 bacteria per 
 1 c. c. after 
 Iday. 
 
 
 Before HIM rt ion 
 
 3 760 
 
 80 
 
 Do... 
 
 *J hour* after injection ................. . . 
 
 .. TI'.I 
 
 ' i.,. 
 
 Do 
 
 1 day alter injection ...................... .... 
 
 i... 
 
 M i"' 
 
 Do 
 
 F. 26. . . 
 
 - niter Injection 
 
 600 
 
 \ .'.. 
 
 I-.,.,, 
 .i 
 
 Do 
 
 'i hours alter injection . 
 
 1 020 
 
 HO 
 
 DO :.:::...:: 
 
 Do 
 
 1 day alt-r injection 
 2 da v<< after injection 
 
 MO 
 
 840 
 
 ... ... 
 
 .' . 
 
 O. P. 46M... 
 
 
 2,840 
 
 . -v.i 
 
 Do 
 
 '_' Hour* niter injection 
 
 | |.i 
 
 9.200 
 
 Dor... 
 
 1 lav niter inject ion 
 
 200 
 
 :.j .... 
 
 Do 
 
 2 day* alter injection . ... 
 
 200 
 
 1 i"' 
 
 
 
 
 
 Homnln((ou i the term now universally employe*) in denote the organism with which the 
 animal in any given case Is Injected, aa In the present cmae.
 
 INJECTIONS OF CULTURES OF B. CHOLERA SUIS. 
 
 33 
 
 In the experiment given in Table XXII the animal hog 1859 was 
 injected intravenously with 5 c. c. of a suspension of culture G. P. 
 4692. The suspension was made in beef broth from a 24-hour agar 
 culture as usual, and was equivalent in density to a 24-hour beef-broth 
 culture of the typhoid culture diluted with about equal amounts of 
 broth. Tests were made of the potency of the serum from the blood 
 of the animal before injection and after injection with all three strains 
 of B. cholerse suis. Owing to the fact that the animal was killed in a 
 moribund condition two days after injection, it was impossible to make 
 tests with the serum from the blood later than the second day after 
 injection. 
 
 It would seem hardly necessary to discuss the results with the differ- 
 ent organisms in detail, since they all appear to point to a lessening 
 of the potency one and two days after injection. Before injection the 
 serum from the blood seems to have been bactericidal for the Crawford 
 strain only; still for the other strains it was at least inhibitory, whereas 
 after injection it had lost much, if not all, even of its inhibitory power 
 on the first and second day after injection, in the tests made two 
 hours after injection the results appear somewhat discrepant; the 
 serum seems to have lost potency for Crawford, and gained for F. 26; 
 but, after all, the figures are perhaps not very significant in these 
 cases. 
 
 The results in this experiment may have been complicated by the 
 fact that the animal from which the blood was drawn was suffering 
 from acute infection with B. choleras suis at the time the blood was 
 obtained. It may not be correct to regard this serum as immune in 
 the sense in which this term is usually understood that is to say, 
 serum from the blood of an animal given injection of bactei 'a and re- 
 covered. Nevertheless, the results are perhaps not without interest, 
 since they show certainly a very marked difference between the serum 
 from the blood of the hog before injection and that from the blood 
 after injection. 
 
 TABLE XXIII. Bocteriolytic power of serum from the same hog (No. 1860) before and 
 after injection with B. cholerse suis, culture F. 26. 
 
 Culture used to test 
 bacteriolysis. 
 
 Time blood was drawn relative to injection. 
 
 Number of 
 bacteria in- 
 troduced per 
 1 c. c. 
 
 Number of 
 bacteria per 
 1 c. c. after 1 
 day. 
 
 Crawford 
 Do 
 Do 
 
 Before injection 
 1 day after injection 
 2 days after injection 
 
 1,460 
 1,240 
 140 
 
 
 20 
 
 a go 
 
 Do 
 Do 
 
 3 days after injection 
 4 davs after injection 
 
 5,400 
 1,620 
 
 00 
 
 o 
 
 F. 26 ... 
 
 Before injection 
 
 2,100 
 
 80 
 
 Do 
 Do 
 
 1 day after injection 
 2 days after injection 
 
 700 
 1 100 
 
 880 
 
 Do 
 
 3 days after injection 
 
 1 500 
 
 ao 
 
 Do 
 
 4 davs after injection 
 
 2,260 
 
 5,580 
 
 Q. P. 4692 
 Do 
 
 Before injection 
 
 1 day after injection . 
 
 10,200 
 4 100 
 
 17,400 
 60 
 
 Do 
 Do 
 Do 
 
 2 days after injection 
 3 days after injection 
 4 davs after injection . . 
 
 1,500 
 9,900 
 1 380 
 
 00 
 
 30 
 
 1,020 
 
 
 
 
 
 a This character signifies that there were too many colonies to count.
 
 84 HA<TKKI"|.VH I-\\I-.K <>K m.nm -i ui'M m- IKKS. 
 
 In the experiment recorded in Table \\lll. (lie animal IIOM- 1 
 \\lis injected fust \vilh about "J r. r. of a dilute sUs|M'l|sion of MM :ej:il 
 
 iiltuiv in beef 'broth, but most of tin- material was injected meie|\ 
 under the >kiii. as tin- attempt to strike a vein \va> not successful. 
 Two lay> after the first injection a second injection of about I'..". < 
 of H similar .suspension from the same M r: ij,, ,,f bacterium. in this 
 all of the suspension, was int roduced into the vein. Two days after 
 the second injection a third injection was made similar in all r>--\ 
 to the second. The sii-peiisions for all three of the injections \ 
 heated at '>~> < '. for thirty minutes. The blood was drawn from 
 
 the animal In-fore injection and on the liiM. second, third, and fourth 
 daja after the last injection. The results seem to le quite uniform for 
 the .serum on the second and third days after the injections; on both 
 days the serum showed apparently entire loss of bactericidal jnwer for 
 all three organisms. On tl>e fourth day after the last injection the 
 
 Beruin had apparently gained in potency, particularly for the Crawford 
 stniin. The results obtained with the serum one day after the last 
 injection are not very striking, but they perhaps permit of the conclu- 
 sion that the serum had not lost in potency at least to any marked 
 extent. 
 
 AS previously stated, the results of the injection of animals with 
 cultures or with the products of rrowth of bacteria have been most 
 conflict intr in various hands in >,, far as the effect of >uch injections 
 upon the bacteriolytic properties of the blood serum from the animal 
 in test-tube experiments is concerned. 7 
 
 A numtier of investigators have found that the injection of an ani- 
 mal with cultures or with the products of growth of certain bacteria 
 increases the bactericidal power of the blood serum for the homologous 
 organism. Others have found that there is no effect upon the bacteri- 
 cidal potency of the serum produced by such injections, while still 
 others have found diminution or complete l<s of j>ower for the 
 homologous organism to follow the injections. Among others Neisser 
 and Wechsberg 11 have found that the so-called immune >erum i. e., 
 the serum from an animal which has received injections of certain 
 Iwcteria no longer possesses bactericidal power for the homologous 
 organism when the serum is undiluted. The results with the diluted 
 serum will be discussed later. Huxton* has found that the serum 
 from the blood of a normal rabbit is capable of destroying very lar-jv 
 numbers of the typhoid and paratyphoid bacilli, but that after the 
 rabbit has been injected with these organism.- it no longer kills any of 
 the homologous strain. 
 
 But while there is undoubtedly conflict in regard to the matter, the 
 preponderance of evidence certainly seems to show that the >erum i< 
 frequently, if not always, weakened in bactericidal power or com- 
 pletely robbed of this power for the homologous organism by th -
 
 EFFECTS OF. DILUTION UPON BAOTERIOLYTIC POWER. 35 
 
 injections, and the results recorded above seem in many instances to 
 bear out this result in the case of hogs injected with B. choleras, *'//'.. 
 But it would also seem apparent that there is a tendency in some ca-f- 
 to a restoration of the lost potency on further injection of the animal. 
 
 These conclusions seem justifiable at least from the experiments \\ ith 
 the F. 26 and the G. P. 4692 strains. The results with the Crawford 
 strain differ somewhat in this respect from those with the other two 
 strains, and this may be due to the fact, already mentioned, that this 
 organism is of only feeble pathogenic potency. It seemed to be much 
 more easily killed by the serum generally than the other two strains, 
 and it probably also has less power than these to cause the reaction 
 in the serum which is necessary for the production of immune serum 
 as this is understood. The serum of hogs injected with this strain 
 would consequently perhaps be expected to behave more like the 
 serum from an uninoculated animal. In some cases the serum of the 
 blood of hogs injected with the F. 26 or with the G. P. 4692 strain 
 seemed to have retained bactericidal power for the Crawford strain 
 after having lost it for the other two. 
 
 The variations which have been already alluded to as occurring in 
 the serum of animals from time to time, and which are due to causes 
 as yet obscure, must be borne in mind in drawing conclusions from 
 the results of experiments with the serum from animals injected with 
 cultures. Of course it is impossible to determine in the present case 
 the extent to which this spontaneous variation, as it is at present con- 
 venient to call it, may have affected the results. 
 
 In view of the many conflicting results with the serum of the blood 
 both of normal and of injected animals, in so far as the bactericidal 
 properties are concerned t/ it is evident that there are some obscure fac- 
 tors in the process which the methods at present in use are incapa- 
 ble of determining. If the blood serum from one and the same animal 
 is found to be bactericidal at one time and not at another, where the 
 animal is kept under apparently the same conditions, and if, further- 
 more, the injection of cultures is found by certain observers to have 
 no effect upon the bactericidal potency of the serum, by others to 
 increase the potency, and by others again to lessen the potency, there 
 must be gome very subtle factors which enter into the problem, else 
 they would scarcely be overlooked by so many investigators. 
 
 EFFECTS OF DILUTION UPON THE BACTERIOLYTIC POWER OF 
 NORMAL AND IMMUNE SERUM. 
 
 DILUTION OF NORMAL SERUM. 
 
 The effects of dilution upon the bactericidal power of hog serum were 
 tested in the manner usually employed in such cases. The method for 
 obtaining the blood was to cut oft a piece of the tail and allow the 
 blood to run into sterile test tubes or flasks, which were put into the
 
 i RIOL1 ii' r\vn: 
 
 HI..M.M -i 1:1 M 
 
 muss. 
 
 refrigerator immediatelx . Tin- >ermn wa- <li-;i\\n <>!l a- -i><>n a- i: had 
 -eparated. and 1 c.c. of i( \\ a ^ put into each of three tube... Yarioii- 
 dilution-.. a> mentioned in tin- taMe>. were al>o prepared with ph\ )0 
 logical salt solution, and |>ortion> of these dilution-* \\eiv di-t riluited 
 into te>t tube*, three tiil>r- from each dilution, each containing 1 
 In most casrs o. 1 or t'.o;, ,-.<-. of neutral. \ -pinni/cd U'ef broth was 
 added to inMiiv>ullicient nutrient material for tin- bacteria which \\en- 
 subsequently introduced. Tlii- \\a- done in conformity with the 
 method ordinarily employed in such experiment-. Kach of the tul>e> 
 was finally inoculated with a carefully niea-mvd amount, usually 
 C.C., of a -u-penxi.n in phy-iolou-i-al salt solution from a recent agar 
 culture. One -eiie-. each was inoculated with one of the three -train- 
 of 11. </!>/, r.i v//v. Inaddition to the tulx's of undiluted and of diluted 
 xenim. three lulu's of physiologic*] salt solution with the addition of 
 0.05 c.c. of leef broth were also prepared in each ea-e. and inoculated 
 at the same time as the serum tule-* with an amount of culture e^ual 
 to that used for the .-erum tuljes. These tubes constituted the cheek-.. 
 Immediately after the inoculation of the tubes, agar plates were made 
 from each of the tubes of salt solution with carefully measured 
 amounts, and after twenty-four hours at room temperature agar plates 
 were made from all the tubes of a series from lnth the undiluted 
 and diluted serum, and from the salt solution. In nearly all cases 
 0.05 c. c. was used for each plate. The colonies were counted after 
 twenty-four or forty -eight hours in the incubator at body tiwnperature. 
 
 TAKI.K X X I V. Bactcriolytie power of normal hog *tnun on tlilnlion. 
 
 No. of 
 hog. 
 
 Culture used to test 
 Lxu-terioljrrifc 
 
 Number of 
 tartfria 
 Introduced 
 per 1 c. c. 
 
 Number of bacteria in serum after 24 
 hour*. 
 
 Number 
 
 .. i..,, 
 teria in 
 
 V.llltlOll. 
 
 (MOoted. 
 
 lMllltr.1 
 
 1 to 50. 
 
 Dilut,-,! 
 
 i to KM 
 
 i.iiut.ii 
 1106,000. 
 
 L7M 
 
 1W8 
 1809 
 
 M 
 
 :- 
 1MO 
 
 Crawford 
 
 840 
 2,620 
 
 840 
 S.400 
 
 6,700 
 
 :.._<> 
 1,140 
 
 32.200 
 30,400 
 
 ..,,.., 
 
 : : 
 1,020 
 
 1,460 
 2.100 
 10.200 
 
 
 
 1.9HO 
 1,600 
 
 2,040 
 2,900 
 
 5,900 
 10,900 
 
 f>3,MO 
 
 f> -:. HOO 
 
 ' IT. Hi 
 
 2,900 
 3, WO 
 
 
 M 
 17.4<io 
 
 26.400 
 1.400 
 2.700 
 
 1 I 
 
 620 
 
 ;,.. ,. 
 
 2,880 
 
 81.200 
 1,748 
 
 74,200 
 
 - ' 
 1.9W 
 
 00 
 
 : . M 
 
 no 
 
 2,2X0 
 
 . 1' 
 
 900 
 
 
 
 60,800 
 
 
 p 26 
 
 
 . 1' 4692 
 
 
 Cniwfiinl 
 
 
 
 
 
 '. 1' 4692 
 Crawford 
 
 
 at 
 
 73,000 
 
 
 00 
 
 OB 
 OB 
 0* 
 
 !-:' 
 D ..' 
 
 F. 26 
 
 O. P. 4692 
 
 Crawford . . . 
 
 Dilnt. .1 
 1 > 10. 
 
 Iilnt.-.l 
 1 to 100. 
 
 DOvb ! 
 
 It.. 1.000. 
 
 
 
 m.fioo 
 
 37.200 
 1.160 
 : ,000 
 
 , 
 
 00 
 
 30, 4*0 
 
 ll.4il 
 
 808.000 
 
 ; . 
 
 1H7.600 
 
 ;. ,,-. 
 
 ..; (00 
 
 61.400 
 
 
 
 OB 
 
 OB 
 OD 
 B 
 
 i;-. Ml 
 -. . 
 445.200 
 
 F.26 
 
 O. P. 4492 
 
 Crawford 
 
 F.26 
 
 O. P. 4092 
 
 Crawford . . . 
 
 F. 28 
 
 O. P. 4M2 
 
 
 oThia <-h*rnrter kigniflea that thvre were too many colonien to count. 
 
 * IMluted 1 to 4.
 
 EFFECTS OF DILUTION UPON BACTERIOLYTIC POWER. 37 
 
 The results given in Table XXIV show that the serum taken from a 
 hog before injection i. e., normal serum as a rule, loses in potency 
 upon dilution; that is to say, a given amount of the diluted serum 
 kills actually fewer bacteria than the same amount of the undiluted 
 serum. This was to be expected a priori and from the results of 
 others with other organisms and with other animals. But the point 
 which seems specially worthy of note, and which will be discussed in 
 a different connection in this paper, is that the diminution of potency 
 on dilution is not in proportion to the degree of dilution. Not only is 
 this the case, but in at least 4 out of the 18 experiments given in 
 the table there was apparently an actual increase of power on dilution. 
 Those familiar with the subject will at once see the bearing of these 
 results upon the difference between the bacteriolytic properties of 
 normal serum upon the one hand and of immune serum on the other. 
 As it is attempted to show below, the difference seems, after all, to be 
 one of degree only, and not a difference of kind. 
 
 In interpreting the above results it must of course be borne in mind 
 that the number of bacteria found in the serum after twenty-four hours 
 probably does not represent merely the bacteria which are not killed 
 by the serum, but it represents in addition the number resulting from 
 the multiplication of these after bacteriolysis has ceased. Thus, in the 
 case of the serum from hog 1859, if 1 c. c. killed approximately 4,000 
 of the Crawford bacilli, 0.1 c. c. of the serum should evidently have 
 killed 400 and have left 3,600 to multiply, unless in addition to the 
 bactericidal power the serum also possessed inhibitory power for the 
 bacteria which survived bacteriolysis. If the serum had exhausted its 
 bacteriolytic power in ten or twelve hours and exerted no further 
 influence after this time, there should have been in the 1 to 10 dilution 
 after twenty-four hours a multiplication of the 3,600 bacilli left, 
 resulting in some 4,000,000 or 5,000,000 even at the rate of only one 
 generation an hour, as a simple calculation shows. But even if only 
 one bacillus were left after twelve hours, there would result over 
 4,000 in twent\*-four hours, provided there were no hindrance to 
 multiplication at the rate assumed above. It would seem, therefore, 
 that either relatively more bacilli were destroyed by the diluted serum 
 than by the undiluted, or that, aside from the bacteriolytic action, the 
 diluted serum exerted an inhibitory effect in the experiment quoted; 
 for at the end of twenty-four hours there were only about 1,000 bacilli 
 in the 1 to 10 dilution instead of the 4,000 calculated as the minimum 
 number possible under the supposition stated. 
 
 The dilution of 1 to 1,000 in the same experiment should evidently 
 have killed only 40 bacilli if it acted with the same relative intensity as 
 the undiluted, i. e., if it possessed just one one-thousandth of the potency 
 of the undiluted serum. Supposing that all bacteriolytic action ceased 
 after fourteen hours, the 3,960 bacilli left alive increasing at the rate 
 of one generation an hour would be represented by over 4,000,000
 
 HAITI- :i:i"i vi 1- POWEfl "i iti.".|. -KUM ..i HOO8. 
 
 bacilli ill ten Inuu^, when -a- as a matter of fact tin-re \\eiv --nly about 
 :;-JJMN in this .liluti.'H aft, -r t\\ent\ four hour-. 
 
 Tin- time at \\hich bactei io|\ -is i- exhausted in serum at room t.-in- 
 p.-ratures \\as not detei mined, hut tests made at tin- end of three or 
 four hour- showed that up to thi- tiuif there was little or no appreci- 
 able effect Upon the number of bacteria introduced, -o that thcaltove 
 
 a uiiiption that tin- proce-- i- exhausted in twelve or fourteen hours 
 limy or may not be correct. Hut whether it i- or i- not. the relative 
 etlcct of the various dilution-, would be shown ly assuming the \< 
 6BI to come to an end simultaneously and thi- i^ prohahly the c-ae 
 at any time within the twenty four hours, the oliject of the ealcula- 
 tioi. IMMIIJT merely to show that no delinite rule of proportion < oiild be 
 oliM-rved, and that the diluted serum in all ea-e> d,-xt roved relatively 
 more Kaeteria than the undiluted -.enim. While dilution of normal 
 -erum eau-e^ a marked diminution in the haeterieidal power, it doe* 
 not >eem to dimini-h it to the extent that would be exj)ected 11 priori. 
 In other words, a dilution of 1 to 1< doe- not >eem to de-troy just 
 one-t*nth the number of bacilli that the undiluted -fimn destroy-, but 
 a much larger number than this, or at least if it does not destroy a 
 larger number it prevents a> relatively rivat an inerea-e as that 
 whi-h takes place in the undiluted serum. 
 
 Still another method of compari>on which apjx'ars permi->il'le. and 
 which leads to the same conclusion, is to calculate the number of 
 bacteria which should result in a dilution of 1 to KM) if the same rela- 
 tive increa-e took place as in the 1 to 1<> dilution; or to compare the 
 relative increase in any of the dilutions with that in the undiluted 
 serum in the experiment ^i\en above. For such a comparison it may 
 lie assumed that the organisms which were not destroyed in fourteen 
 hour- multiplied in the undiluted and in the dilated serum at the same 
 rate, or at least that if the serum retarded multiplication this effect wa> 
 more marked in the more concentrated serum than in the more dilute. 
 It would s. Tin evident, then, that the relatively smaller number of 
 bacteria in the diluted -eriiin can lie accounted for only on the >up- 
 po-ition that the diluted serum retains more bacteriolytic power than 
 would be exacted from the decree of potency shown by the un- 
 diluted scrum, according to the simple rule of proportion. This point 
 -eeins no t to have }x>en alluded to in the literature, but it would seem 
 nevertheless not without significance perhaps, since it tends ap- 
 parentlmto show that the difference U-tween normal serum on the one 
 hand ana immune serum on the other is nierelv a diflerence of degree 
 and not a difference of kind. The matter of the actual identity in the 
 behavior of normal sera and of immune sera on dilution, in spite of 
 the apparent difference shown by them, will be discussed more fully 
 in u different connection below . where the attempt is made to -how 
 that the law of complement diversion applies to normal serum as well 
 as to immune scrum.
 
 EFFECTS OF DILUTION UPON BACTERIOLYTIC POWER. 39 
 
 DILUTION OF IMMUNE SERUM. 
 
 The results obtained by tests made to determine the effect of 
 dilution upon the bacteriolytic power of immune hog serum that is to 
 say, of serum from hogs injected with cultures in the main agree 
 with those observed in all recent investigations in this direction 
 with the serum of other animals injected with t3 T phoid, paratyphoid, 
 Asiatic cholera, and other bacteria. The behavior of the serum under 
 such conditions is very peculiar ; it seems to be in conflict with all 
 laws of proportion which are applicable to other chemical reagents, 
 particularly in regard to germicidal substances in general. With ger- 
 micidal substances in general, as it is hardly necessary to state, the 
 more concentrated the germicide the more powerful the destructive 
 power. A strong solution of carbolic acid is a more powerful germi- 
 cide than a weaker solution. But not so with immune blood serum. 
 Paradoxical as it ma} 7 seem, the bacteriolytic potency of the immune 
 serum increases upon dilution. The undiluted immune serum may be 
 weak or wanting in bactericidal properties, and yet become more and 
 more potent upon dilution. In other words, 1 c. c. of undiluted 
 immune serum may be found to kill few, if anj T , bacteria ; it may be 
 found even to furnish a good nutrient medium in which the bacteria 
 grow ; whereas 1 c. c. of a dilution consisting of 1 part of the same 
 serum to 10 or 100 or 1,000 parts of dilute salt solution may be found 
 to kill many hundred bacteria introduced into it. It goes without 
 saying, of course, that there is a limit to this increase of potency upon 
 dilution. Where the dilution goes beyond a certain point the potency 
 becomes weakened. There is for each immune serum an optimum 
 dilution for bacteriolysis, above and below which there is less and 
 less potency. Thus, as will be seen in some of the experiments 
 described below, the greatest potency in one case was in a dilution of 
 1 part of serum to 100 parts of salt solution. In the case of a dif- 
 ferent serum the optimum dilution for bacteriolysis was 1 part of 
 serum to 1,000 parts of salt solution. 
 
 Buxton very appropriately speaks of the phenomenon just described 
 as the formation of bacteriolytic zones, of which three may be recog- 
 nized in every immune serum a pro-killing zone, a killing zone, and 
 a post-killing zone. The action of the undiluted immune serum lies 
 in the pro-killing zone, that of the optimum dilution lies in the killing 
 zone; that of dilutions in which bacteriolysis becomes again weakened 
 lies in the post-killing zone. Of course it is to be understood that these 
 zones are not sharply marked off from one another, but that they grad- 
 ually merge into each other. The pro-killing zone, for example, may 
 extend over dilutions of several strengths, and the same is true of the 
 killing and of the post-killing zones. Thus in some of the experiments 
 given below there appeared to be no marked difference between the 
 bactericidal power of the undiluted serum and the same serum diluted
 
 40 
 
 BAOTERIOLYTIO I'-'WKK <K KMIOD SKKl'M OK HOGS. 
 
 in the proportion of 1 to lo<>r 1 to lo<>; tin- pro-killing /one in these 
 QMe* would I... - :l id |,, ,.\ti ml t<> the dilution- o| 1 t.> 1" or 1 to I '". 
 The /one- a IT therefore spoken of a- longer or shorter, accordin 
 whether they apply t<> many or to few dilution-. 
 
 The jMViiliar behavior of immune serum ju-l de-ei -il.ed \\a- iii--t 
 ol-erved l>\ Nei--er and Weehsberg," and is called tin- Neisser- 
 \Yechsl>erg phenomenon. While it is true that tin- phenomenon was 
 ol.-er\ed (j ui to frequently in immune hog serum, a- will be -ho\\n 
 later. it was not as n<>ti<val>l<> in sonic ca-e- a- it wa- in other-. :md 
 moreover the three /ones varied in length, not only after the injee 
 tion of ditTerent strain^ of II. </////,// .v///.v in ditl'eient hog, but also 
 after the injection of the >ame strain in ditlernit hoj^s. 
 
 It ha> already been shown that normal serum of hogs gains rela- 
 tively in potency on dilution, and. as has just Keen Mated, immune 
 -eimn al-o puns in potency. The two behave alike, and at mo-t then- 
 is only a difference of degree between the amount of gain in either 
 a-e. But even a difference of degree between tiie two sorts of serum 
 i- not always noticed in the present experiments, for the normal serum 
 acted in some cases just like immune serum. It wa- not only relatively 
 more potent on dilution, but it killed actually more bacteria per 1 
 c. c. of serum in certain dilutions than in dilutions containing more of 
 the serum. 
 
 TABI.K XXV. /.'//. <t ./' <lihitin >i/>n immune hoy nmtin UK <<;///.// //'/// / 
 (liliilinii (//;</ //</ iinniinl vrniiii. Hmj ;.- / /;,/// ,-nlhii. <,. /'. 
 
 Culture 
 
 ll4-<) Illtl-t 
 
 Imi-tiTin- 
 ly>iv 
 
 Tinn- blood wan ilniun 
 relative In injeetiini. 
 
 Niimlier 
 
 of bacteria 
 
 inlnxlueed 
 per 1 e. e. 
 
 NiiintNTof luieteria in MTIIIII niter ?! 
 
 Illlllpi. 
 
 Number 
 
 terin in 
 elieckmll 
 
 Dilution. 
 
 * 
 
 00 
 
 :. 1 
 L.0001 
 
 UC 
 
 2,700? 
 
 ' 
 
 
 200? 
 
 I n.lililte.1. 
 
 Hilllte.1 
 1 to IIP. 
 
 iMhK <1 
 
 Diluted 
 1101,000. 
 
 (twwfiinl .. 
 F. ' 
 <i.l'.40n.. 
 
 l^'iUri 1 iniei'iiiin 
 
 3,700 
 3.760 
 600 
 600 
 1,080 
 
 i.oao 
 
 M 
 2.S40 
 
 200 
 
 no 
 
 80 
 1,400 
 
 ,.,, L,, 
 
 l-.HKI 
 
 2,900 
 80 
 
 3..HKI 
 
 :i... li NI 
 8,800 
 
 M BOO 
 12.400 
 
 1,100 
 
 200 
 4.600 
 700 
 i ,000 
 
 11. IM, 
 
 27. HOD 
 l.NOO 
 
 -'7. li i 
 22.400 
 
 :i 
 
 ll'.t.XW 
 4.900 
 HO 
 4.MO 
 1K7.600 
 
 l-., 
 9.440 
 16. ON) 
 
 19.200 
 400 
 
 OCO 
 
 
 520 
 12,400 
 / 
 i 
 6,500 
 900 
 
 00 
 00 
 
 00 
 
 1.600 
 
 .' lioiirx lifter iiijfi-tiini 
 1 day after inje<-ti(.n. . 
 ' (lavs after injeetioli. 
 Hi'fure injeetiiin 
 
 '2 hours after injtftiini 
 1 iliy after inji-^tioii .. 
 J ilays after Injcetinn. 
 Befi>ri' injection 
 
 '2 IKIIIIN niter injeetioli 
 1 day after injceiion.. 
 2 days after Injection. 
 
 a Thin ehnrnetcr xiKnlne* that then- were too many colonies to munt. 
 
 An example of the difference often to be seen between the bac- 
 tericidal power of the senim from a hog hefore and aft<>r injection 
 with culture- of 11. ,-l,,l, // .v///X insofar as the .'tied of dilution is con- 
 cerned, is given in Table XXV. The animal in this case was injected 
 with a culture of the (J. I'. 4t'.:-j -train, and the blood was drawn 
 before and at several interval.- after injection. Before the injection 
 the serum from the blood of this hog showed inhibitory, but no l>ac- 
 tericidal power for the organism with which tin' animal was injected.
 
 EFFECTS OF DILUTION UPON BACTERIOLYTIC POWER. 
 
 41 
 
 In this case there were 2,840 of these organisms introduced per 1 c. c. 
 into the undiluted serum, and after twenty-four hours there were 
 3,800 per 1 c. c. present. In the dilution of 1 to 10 the same number 
 of organisms introduced about doubled in number in twenty-four 
 hours; in the dilution of 1 to 100 the s%me number of organisms 
 increased about fivefold; in the 1 to 1,000 dilution there was a count- 
 less increase. In the undiluted serum from the blood of the same hog 
 two days after inoculation there was a sixty-two fold increase of the 
 200 bacilli per 1 c. c. which were introduced, while in the 1 to 10 dilu- 
 tion with the same number of bacilli introduced there were only 140 
 alive after twenty-four hours, in the 1 to 100 dilution only 400, and 
 in the 1 to 1,000 there were 1,600. In other words, the normal serum 
 diminished in potency on dilution while the immune serum increased 
 in potency on dilution. It should be noticed, however, that the dimi- 
 nution of potency of the normal serum is an actual and not a relative 
 decrease on dilution. In this case, as in others, the normal serum 
 killed more bacteria, relatively, when it was diluted than it did when 
 undiluted. 
 
 TABLE XXVI. Effect of dilution upon immune hog serum as compared with the effect 
 of dilution upon the normal serum. Hog 1798, injected urith culture F. 26. 
 
 Culture 
 used to test 
 bacterio- 
 lysis. 
 
 Time blood was drawn 
 relative to injection. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. c. 
 
 Number of bacteria in serum after 24 
 hours. 
 
 Number 
 of bac- 
 teria in 
 check salt 
 solution. 
 
 Undiluted. 
 
 Diluted 
 Ito50. 
 
 Diluted 
 1 to 500. 
 
 Diluted 
 1 to 5,000. 
 
 F. 26 
 
 Before injection 
 
 2,620 
 8,400 
 
 5,920 
 2,400 
 840 
 5,700 
 13,000 
 2,660 
 1,140 
 7,300 
 
 1,980 
 7,200 
 
 38,000 
 36,600 
 
 160 
 359,600 
 1,680 
 1,600 
 5,100 
 
 1,400 
 106, 400 
 
 38,400 
 686,800 
 26,400 
 400 
 6 12, 000 
 2,700 
 2,000 
 69,100 
 
 1,740 
 103,600 
 
 84,000 
 681,200 
 81,200 
 27,600 
 620,400 
 32,800 
 5,500 
 67,500 
 
 2,280 
 6,900 
 
 rt 00 
 
 6112,000 
 140 
 
 00 
 
 6175,000 
 5,200 
 26,600 
 boo 
 
 
 Crawford . . 
 G. P. 4C92.. 
 
 10 davs after first injec- 
 tion. 
 9 days after second in- 
 jection. 
 14 days after third in- 
 jection. 
 10 days after first injec- 
 tion. 
 9 days after second in- 
 jection. 
 14 days after third in- 
 jection. 
 10 days after first injec- 
 tion. 
 9 days after second in- 
 jection. 
 14 days after third in- 
 jection. 
 
 
 cc 
 
 X 
 
 00 
 CO 
 
 73,000 
 
 CC 
 
 This character signifies that there were too many colonies to count. 
 6Dilutions used were 1 to 10, I to 100, 1 to 1,000. 
 
 That there is a difference in the behavior of the blood serum of dif- 
 ferent hogs injected with the same strain of B. ckolerse suis is shown 
 by comparing the results given in Table XXVI, hog 1798, with those 
 shown in Table XXVII, hog 1860. Both of the animals were injected 
 with culture F. 26. Before injection the blood of hog 1798 showed 
 considerably more bacteriolytic power on dilution for the organism 
 with which the animal was afterwards injected than the blood serum 
 of hogs did in other cases, though, as has been seen, normal hog
 
 l-J 
 
 I: \ I ) Kl-'I.N IK I'.'U IK "| ULoop -1 Kl M oK II- 
 
 scrum frequently retains :i c.msider.ihle dcgr ,f potency upon dilu- 
 tion. This serum ap|>ears to have possessed about an equal d< 
 potency in all tin- dilutions which were tried, and to have Keen al>.ut 
 as potent wlu'ii diluted :is it was when undiluted. Ten day- nl'tei in 
 jection its behavior was v^ry peculiar, for it was apparently 
 potent a- the nuriual serum when il \\a- undiluted and in the I to 
 'dilution, luit it had ls| potency in t he dilut ions of 1 to .".o ;md 
 1 to 500. Nine days after a second injection it had apparently lost in 
 bactericidal potency, both when diluted and undiluted. Fourteen 
 days after a third injection it seems to have gained in power in all 
 strengths. If this is eoinj)ared with the behavior of the serum from 
 the blood of hog IM'.H, Table XXVII, a hog also injected with the F. 
 J'. -train, it will be noticed that there is a marked difference. 
 
 TAHI.E XX VII. Efferi of dilution </*// immune hog nerum at compared with the effect of 
 
 ililiiliii iifHDi thr normal ternm. ll<><) JSi',n, injfrl,</ intnn nniixlii n four MUXCtrive 
 days ii'ilh culture F. 2(S, nlmni .' <-. <. lirntxl ir tliirt;/ minnti* nt '>8-^5S C. 
 
 culture 
 uned to teat 
 
 Time l>loo<l ua- ilra \vn 
 
 Number ol 
 bacteria 
 
 NunilMT of bacteria in wnim after 24 
 boon. 
 
 Number 
 
 .,; ', .. 
 fi.rlu In 
 
 bacterio- 
 
 1VM. 
 
 relative to inj.-etion. 
 
 Introduced 
 
 |ier 1 '. c. 
 
 Dndltatad, 
 
 l>ilntc<l 
 1 to 10. 
 
 Dilated 
 
 1 to 100. 
 
 Dilute,] 
 
 1 to 1,000. 
 
 icrin in 
 ahaekai 
 Milutlon. 
 
 ('niwforil 
 
 Hefore injection 
 
 1,460 
 
 o 
 
 80 
 
 12,500 
 
 118 400 
 
 1M Till 
 
 
 1 day after injection. . 
 
 1/JI" 
 
 20 
 
 
 
 ft. 200 
 
 
 2 ilaysafter injection. 
 
 140 
 
 a 30 
 
 , 
 
 19, INK) 
 
 , . , 
 
 460 
 
 
 :t ilaysafter infection. 
 
 I i 
 
 x 
 
 b 
 
 
 6 30 
 
 00 
 
 
 1 clnys after injection. 
 
 1,00 
 
 
 
 
 
 I M 
 
 14,400 
 
 1 Ml 
 
 F.26 
 
 Before injection 
 
 J.I (Ml 
 
 M 
 
 ;- 
 
 504. C0 
 
 V . 
 
 
 
 1 ilay after injection.. 
 
 in 
 
 880 
 
 
 70,000 
 
 
 10.000 
 
 
 .' <la>> after injection. 
 
 1,100 
 
 OD I / 
 
 200.200 
 
 
 A4.400 
 
 
 :t 'lays after Injection. 
 I ilays after injirtion. 
 
 1,000 
 
 i ' , 
 
 L.OO 
 
 / ft 30 
 
 2,860 6.900 
 
 IS. 600 
 
 <i. 1'. 4692.. 
 
 Hefore injection 
 
 10,200 
 
 17. lii 11 IIKI 
 
 '1. HO 
 
 MB m 
 
 a ,.., 
 
 
 1 ilay after injection.. 
 
 1,100 
 
 <X) H, MOO 
 
 10,600 
 
 ., v ,.. 
 
 .- 
 
 
 J ilaysafler injection. 
 
 1,600 
 
 OB I / 
 
 , l. ;,., 
 
 i i ,, 
 
 4.400 
 
 
 :< 'laysaftcr injection. 
 
 9.900 
 
 a | Ax 
 
 
 69 
 
 ' 
 
 
 4 ilny* after injection. 
 
 :,o.>o 
 
 2,100 
 
 2,4*) 
 
 10.800 
 
 < 
 
 oThU character MKniiie* tliat there were too many colonies to count. 
 6 Dilution- 11-.. 1 W.TC 1 to :*i, I to. '-in, 1 to 5,000. 
 
 In the experiment with the blood serum of hog I860, Table XXVII, 
 the animal was injected on four successive days with <|tiite den-e MM- 
 pen-ions, considerably denser than a twenty-four-hour culture >f 
 typhoid. The >uspensi(, n^ \\ere heated for thirty minutes ; ( t ;,s ;,: ( '. 
 Iwfore injection, and the injections were made intravenously. On 
 each day before injection, and previous to any injection, blood was 
 drawn from the tail of the animal, and after the separation of the 
 serum this \\as used on the three strains of //. , }n>l< r;> .-///*. Doubtless 
 the fact that the test- were made while the animal was being immu- 
 ni/ed had some effect upon the results obtained, and for this reason 
 they are different in some measure from those obtained in other 
 experiments in which the serum was tested only after the animal had 
 
 been immunized. 
 
 The undiluted serum from this ho<_r before injection was quite 
 strongly bactericidal for the Crawford and the F. _'', -trains, but it
 
 EFFECTS OF DILUTION UPON BACTERIOLYTIC POWER. 
 
 43 
 
 WHS at most mereh 7 inhibitory for the G. P. 4692 strain. After the 
 second and third injections the serum seems to have lost all potency 
 for all three organisms, at least when undiluted. But after the fourth 
 injection the serum seems to have regained its potency both when 
 undiluted and in certain of the dilutions. 
 
 TABLE XXVIII. Effect of dilution upon immune hog serum as compared with the effect 
 of dilution upon the normal serum. Hog 1885, injected with Crawford culture. 
 
 Culture 
 
 used to test 
 bacterio- 
 lysis. 
 
 Time blood was drawn 
 relative to injection. 
 
 Number of 
 
 bacteria 
 
 introduced 
 per 1 c. c. 
 
 Number of bacteria in serum after 24 
 hours. 
 
 Number 
 of bac- 
 teria in 
 check salt 
 solution. 
 
 Undiluted. 
 
 Diluted 
 ItolO. 
 
 Diluted 
 1 to 100. 
 
 Diluted 
 1 to 1,000. 
 
 Crawford . . 
 F. 26. 
 
 1 hour after injection 
 1 day after injection. . 
 1 hour after injection 
 1 day after injection . . 
 1 hour after injection 
 1 day after injection. . 
 
 5,860 
 3,200 
 16,600 
 6,900 
 9,600 
 4,600 
 
 18,200 
 71,100 
 oo 
 
 31,000 
 3,600 
 
 
 5,200 
 110, 400 
 40,400 
 7,200 
 9t>,000 
 
 33.600 
 11,000 
 
 OO 
 
 28,000 
 10,700 
 
 a oo 
 
 CO 
 00 
 
 00 
 00 
 00 
 
 00 
 
 oo 
 102,200 
 
 oo 
 
 00 
 
 1,280? 
 
 G. P. 4692.. 
 
 a This character signifies that there were too many colonies to count. 
 
 In the experiment of which Table XXVIII gives the summary the 
 hog was injected intravenously with a dilute suspension of the Craw- 
 ford culture. Two injections of the organism were given on two con- 
 secutive days, and blood was drawn from the animal one hour and 
 twenty-four hours after the last injection. No blood was drawn 
 before injection in this case; 
 
 There seems to have been no difference between the behavior of the 
 blood on dilution and that of ordinary normal blood on dilution. 
 Omitting one or two unimportant discrepancies, the serum lost more 
 and more in potency upon further and further dilution. 
 
 TABLE XXIX. Effect of dilution upon Immune hog serum as compared with the effect of 
 dilution upon the normal serun,. Hog 1809, injected with culture G. P. 4692. 
 
 Culture 
 
 used to test 
 bacterio- 
 lysis. 
 
 Time blood was drawn 
 relative to injection. 
 
 Number of 
 bacteria 
 introduced 
 per 1 c. c. 
 
 Number of bacteria in serum after 24 
 hours. 
 
 Number 
 of bac- 
 teria in 
 checksalt 
 
 solution. 
 
 Undiluted. 
 
 Diluted 
 Ito60. 
 
 Diluted 
 1 to 500. 
 
 Diluted 
 1 to 5,000. 
 
 F. 26 
 
 Before injection 
 
 5,920 
 2,400 
 ' 5,700 
 13,000 
 1,140 
 7,300 
 
 5,900 
 1,800 
 
 56, 600 
 10,900 
 7,900 
 
 109,200 
 l> 1<>8, 000 
 520 
 ft-1,000 
 2,880 
 &9.200 
 
 <l 00 
 
 ''224,000 
 
 00 
 
 f'6,l>00 
 123,200 
 b 25, 900 
 
 oo 
 b oe 
 oo 
 '< v> 
 60,800 
 b oo 
 
 oo 
 OO 
 
 oo 
 oo 
 73,000 
 
 OO 
 
 Crawford . . 
 G. P. 4692 .. 
 
 14 days after injection. 
 Before injection 
 14 days after injection. 
 Before injection 
 14 days after injection. 
 
 a This character signifies that there were too many colonies to count, 
 b Diluted in the proportion of 1 to 10, 1 to 100, 1 to 1,000. 
 
 In the experiment summarized in Table XXIX the hog was injected 
 subcutaneously with 5 c. c. of a suspension of G. P. 4692, but there 
 seems to have been little if any effect produced upon the bacteriolytic 
 power of the serum. It is true that with the Crawford strain there is 
 some evidence of loss of potency in the undiluted serum, and of 
 increased potency upon dilution in the serum after the injection of the 
 animal, but the effects on the whole are not striking.
 
 44 
 
 SKKI'M 
 
 ,i 
 
 TMUI \\\ /-on mi in unt hog itfrn in > 
 
 ul ililittinn 11/1,11 lh> nnrmtll Hfritm. //</ IMfll. ni/V.7.--/ //. ' 'run -fnl > 
 
 Culture 
 
 twclerlo 
 
 K | 
 
 'I 1. I.H.I .i.,lniw n relative 
 In injection. 
 
 Number 
 
 Intro 
 
 |.er 1 c c 
 
 Nmnherof Uicleriii IIIMTIIIII i, 
 hour- 
 
 tin In 
 -alt 
 
 1 M.ll 
 
 luted. 
 
 IMI.ilMl 
 Ito.V). 
 
 i io 000 
 
 lMlule.1 
 
 Hi. la\ suit, r tir-t injection.. 
 a d*v after M-comi injection 
 1 1 ilnv- after thinl Injection. 
 Hi la\- after tint Injection. . 
 .' il:i\ ai'icr ccoui injection 
 11 .lay* after thlnl injection. 
 
 10 day* after ilrst injection. 
 9 tUvK nfier M-cotnl injection 
 14 day* after thinl inj.-ctlon. 
 
 8.400 
 MO 
 
 ton 
 
 1.1 in 
 
 7 BO 
 
 ,.., 
 
 ' 60 
 
 (1 
 1-11 
 
 ' -J" 
 
 3. .-WO 
 7!00 
 
 |9 MOO 
 
 oo MM 
 
 640 
 
 19,000 
 
 :i 000 
 
 not 
 
 of 
 
 : 000 
 
 "I'.l -1l 
 
 0, 1 1 
 
 fc8,100 
 
 172.000 
 
 / 
 
 I ,-. 
 
 . n 
 
 7^600 
 '21.000 
 
 
 ..nl .. 
 
 ., ] |I.T; 
 
 o 
 
 
 
 
 78.000 
 
 
 
 "This cluirnctcr ^ik'iiilic- tliat there \\rn- I.... many coloni. 
 'Hilule.1 1 to 1(1. I to 1IKI. I to 1,000. 
 
 The hog (1801) in (he experiment abown in Table XXX wa- injeeted 
 
 on three different occasions with the Crawford organism. No te-t- 
 were made of the serum before the inoculation of the animal, but there 
 -ems little or no evidence of the reaction usually seen in the serum of 
 animals injected with bacteria in the tests which were made after each 
 injection. The serum in most of the test- showed !<>-- of bacteriolytic 
 power on dilution, behaving in this respect like normal serum, ll i- 
 true that with the homologous -train Crawford -the -enini ten <lay> 
 after the first injection and fourteen day- after the third injection 
 appeared to be somewhat more strongly bacteriolytic in the dilution 
 of ltx>5,<HX)and ltol,(X><>. respectively, than in the le diluted -erum: 
 but on the whole the serum .seemed to act more like normal than like 
 immune serum. 
 
 TABI.K XXX I. KtJ'i-i-t <>f >Hlntii,n HJXHI innmine hog xerinn nx n.ii,j,,ij,<l mil tin ,f 
 Man tin- normal writ in. Jim/ Jfi.iH, injn-t,<l mil, ni/liii, /'. S6. 
 
 Culiun- 
 
 UXtKl ' 
 
 (it-tiTiii- 
 
 Crawfonl . . 
 K. 26 
 
 Tinii- l.l. .-! waxdrawn 
 rt-lntivi- to injection. 
 
 Number of 
 
 luicieriii 
 
 hatrodooed 
 
 |HT 1 c. c. 
 
 NiiinlMTof liHctcrin in M-riiin after Jl 
 
 bam. 
 
 NiiiiiU-r 
 of 1-jirtc- 
 ri in 
 
 cheek silt 
 
 Dilution. 
 
 
 
 
 at 
 
 / 
 
 ... 
 
 Dtodttmad. 
 
 . 1840 
 <i. r t.S40 
 
 JI7.MO 
 
 a an 
 
 "47.400 
 
 , | ... 
 
 OB 
 
 lilllte.l 
 1 to Id. 
 
 I>illlte,l 
 
 l>i iiinl 
 
 1 to 1. (111. 
 
 Before injii-tfon 
 
 oo m 
 
 i M 
 00 (00 
 ... (00 
 ft. 000 
 
 a, ooo 
 
 , ... 
 
 ao 
 
 240 
 
 117,600 
 
 
 0| ... 
 
 - I'- 
 ll MM) 
 
 ; . 000 
 
 OB 
 1 .1 Ml 
 
 r 
 
 00 
 OB 
 
 30K.OOO 
 
 1". i 
 
 7 m 
 
 OC 
 
 '" 
 OB 
 
 
 ' 
 
 1 hour iifli-r iiiji-<-tloii .. 
 Sdayx iifti-r injcctiini . 
 
 Mfforr illjrctioll 
 
 ' I' MW2.. 
 
 1 hour nfii-r injection .. 
 - after injection. . . 
 Before Injection 
 1 hour after injection . . 
 Sdaynaftcrinji tion. . . 
 
 IMlilteil in the |.ro|Nirtion of 1 | art of MTIIIII to :: of |-l.\ !..:. cu nl -nit solution. 
 *>Thi chiinicier 'iuiiitli^ Hint there were !> iiiiiny colunjc* to count. 
 
 In the experiment with the serum fr<m the blond of hor \W*. Table 
 XXXI. the animal was ujven intrax enoii>ly ." c.c. of a dilute beef-broth 
 >u|>fn>ion of an ajjar culture of F. _'' -train. < )ne da\ after the first
 
 SUMMARY OF DILUTION EXPERIMENTS. 45 
 
 injection the animal was given a second injection of the same amount 
 of the same strain. The blood was drawn before any injection was 
 made, one hour after the first injection, and three days after the second 
 injection. 
 
 The results oDtained with the serum from the blood of this hog, at 
 least in so far as the strains G. P. 4692 and Crawford are concerned, 
 are in striking contrast with the results obtained with the serum from 
 the blood of hog 1801 (Table XXX). In the case of hog 1836 the ani- 
 mal was injected with a more virulent strain than was the case with 
 hog 1836, and it is possible that this may have had some influence 
 upon the results in the two cases. But aside from this the two sera 
 must have had different potency originally, for even the normal serum 
 of the blood of hog 1836, before the animal was injected, will be seen 
 to have shown increase of potency upon dilution; the dilution of 1 to 10 
 was more potent than the dilution of 1 to 4. There was no test made of 
 the undiluted normal serum for the reason that not enough of the blood 
 was obtained to use for the purpose. This increase of potency on 
 dilution of the normal serum of hog 1836 is to be seen particularly in 
 the tests with the Crawford strain, in which the dilution of 1 to 4 
 showed 3,840 bacilli per 1 c. c. whereas the dilution of 1 to 10 showed 
 only 320 bacilli after the same exposure one day in each case with 
 the same number of organisms 32,200 per 1 c. c. introduced. This 
 serum in other words seems to have behaved somewhat like immune 
 serum to start with, and this behavior seems to have been intensified 
 by the injections, as will be seen by examining the table. 
 
 SUMMARY OF THE DILUTION EXPERIMENTS. 
 
 On summing up the results of the experiments made with normal 
 and immune hog serum on dilution, it will be noticed that the normal 
 serum seems to differ greatty in its behavior in different cases; that 
 while it sometimes shows marked diminution of potency upon dilution 
 it sometimes shows increased potency on dilution. The immune hog 
 serum also shows variation in behavior. 
 
 But the results which would seem to be of especial interest are those 
 in which the immune serum showed strong bacteriolytic power when 
 undiluted, and lost in power on dilution in certain proportions, but 
 gained potency upon further dilution. In these cases there appeared 
 to be a combination of the behavior of normal and of immune serum, 
 as this is usually stated to occur. 
 
 As already stated above, the characteristic behavior of immune 
 serum is said to consist of the formation of bacteriolytic zones. A 
 typical immune serum reaction consists of a pro-killing zone, a killing 
 zone, and a post-killing zone on dilution. In the pro-killing zone, 
 represented by the undiluted immune serum, there is no bacteriolysis. 
 In the killing zone, represented by one or more dilutions, there is
 
 I'. n\< IKUIOI.Y n< I-IIXVH: >K in. ...... NEBUM 01 HOO8. 
 
 bacterioh -i-. In tin- p<>-t killing zone, represented by further dilu- 
 tion than tin- killing /one. there is again lack of bacteriolysis. Bui in 
 tin- re-ult- at pre-ent under con-iderat ion tin- serum showed a killing 
 /one to begin within the undiluted serum, thus imitating to tin- extent 
 the normal -erum. 
 
 The peculiar behavior ju-t alluded to was to he >een in sexn.il 
 instances given in the preceding tallies. Perhaps the mo-i -trikm-_r 
 example- are furni-hed by the lehavior of the immune >era from I 
 171'San.l l^iix ,.,,.,. Tablr \\IYi. I,, the-.- -era with the Crawford 
 culture there wa- strong bacteriolysis shown in both ca-e- undiluted. 
 The Ixos \\a.- still trough potent in the 1 to :> dilution, but the 17'.^ 
 serum was much less potei-t in this dilution than when undiluted. 
 lioth sera seem to have lo-t all potency when diluted in the proj>ortioii 
 of I to 500. But the jx)int to be specially noted is that they both 
 -.mi to have Lfained ^-really in the dilution of 1 to .".JUKI; particularly 
 is this the case with the 17!* scrum. 
 
 For the present it is merely nece ary to cull attention to the-e 
 re>ult-: they will l>e taken up and discussed in another connection fur- 
 ther along in the present pajx-r. 
 
 GENERAL DISCUSSION. 
 
 Judging from the results of the above experiments as a whole, it 
 seems apparent that the blood serum of hojr- ha^ but feeble bactericiilal 
 potency for K. cl>!< r;> A-///.V. at least for the more strongly pathogenic 
 strains. This would naturally lead to the presumption that the known 
 susceptibility of hogs to infection by intravenous injection was due to 
 this cause, bat it doe- not -eem a- yet >ettled that the sii-cept ibilitv 
 of an animal in general is in direct proportion to the want of bacteri- 
 cidal power of its blood. In fact, in some instances, as is well known. 
 quite the contrary has been shown to be the ea-e. Nuttall, Huchner. 
 and many others have shown that rabbit serum in te-t tubes will kill 
 large numbers of anthrax bacilli, and yet a comparatively small num- 
 ber of these organisms introduced beneath the skin of a rabbit i- . r 
 tainly fatal to the animal, (iuinea-pig serum, as recently >hown by 
 Buxton,' may be expected to kill approximately I,OOU.<MMI typhoid 
 bacilli per 1 c. c., and yet the injection of a relatively small amount 
 of this organism intraperitooeally ifl certainly fatal for guinea pig-. 
 In spite of this, however, a- \VoltF' : " ha- pointed out. it is probably all 
 a matter of dovigr. and bacterioly-i- i- an ellicient means of defense 
 where the number of bacteria concerned is not large. On the con- 
 trary, where the number of bacteria exceeds certain limits, bacteri 
 o|\-is i- not only not a mean- of defen-e. but it actually lil>erate- the 
 di -ease-producing -ub-tance> the endotoxin- from the Ixxiies of the 
 bacteria.
 
 GENERAL DISCUSSION. 47 
 
 Pfeiffer" was the first to give this name, endotoxins, to the poison- 
 ous substances which are present in the bodies of the bacteria of 
 certain species, and it is now quite generally the opinion that it is the 
 liberation of these that brings about disease in infection with these 
 bacteria. So long as bacteria of this kind remain intact, they are 
 thought by those holding this view to be incapable of producing dis- 
 ease. Mechanical injury and other harmful effects formerly attributed 
 to the action of bacteria are now thought by many not to exist. 
 
 The effects of inoculations of hogs with cultures of B. choleras suis, 
 and the conclusions to be drawn from these experiments are on the 
 whole in harmony with those derived from experiments with various 
 bacteria injected into rabbits, guinea pigs, and other animals. Thus 
 Loeffler and Abel 12 found that undiluted immune blood serum from 
 dogs failed to protect guinea pigs from injections of virulent colon 
 bacilli. Neisser and Wechsberg 14 obtained analogous results with 
 immune rabbit serum in test tubes, the undiluted immune serum failing 
 to destroy the bacteria of the kind used to produce the immune serum. 
 Buxton,* as already quoted, found that the undiluted serum of rabbits 
 injected with cultures of typhoid or paratyphoid bacilli behaved in a 
 similar manner. It was to be expected, therefore, that the serum of 
 hogs injected with cultures of B. cJiolerae suis would show loss of 
 bacteriolytic power in undiluted serum. The behavior of the diluted 
 serum will be considered below. 
 
 In the foregoing experiments with the serum from the blood of 
 hogs injected with cultures of B. cholerse suis, it appears that 
 the efiect of the h'rst injection was sometimes, as stated, to deprive 
 the undiluted serum of its power to kill B. cholerae suis. It is true 
 that this behavior was frequently lacking. But if the results after 
 several injections are examined, it will be found that the undiluted 
 serum frequently regains its power. This is noticeable in a number 
 of cases if not in all, and it perhaps accounts for the experience of so 
 many observers that animals frequently die from injections of com- 
 paratively small doses after having been treated with carefully graded 
 amounts of culture. Thus Wolff 20 found that strong, healthy rabbits, 
 instead of becoming more and more accustomed to injections of 
 cultures, withstood these less and less readily, even without increasing 
 the dose; that after remaining apparently unaffected by previous 
 injections, they often die suddenly on the inoculation being repeated 
 with the same sized dose. This supersensitiveness on repeated in- 
 jections has often been observed in various animals and with various 
 bacteria. It has also been met with in the course of the present 
 experiments with hogs. In fact, it does not seem so difficult to grade 
 the first dose so as to avoid killing the animal as It is to strike upon 
 the proper subsequent doses. The return of bacteriolytic properties 
 in the serum following further injection of animals may account for
 
 48 BA< TKKH-I.VI K POWXB 'i BLOOD 9BBUN 01 H"< 
 
 (hi- result, lli- lir-t injection- depriving the -ermn of the l.a.-tn i.|\ ti. 
 DOWer Mkd thttt preventing the liberation <f endotoxin and it- <-on 
 M'.|IICMCC-. and the subsequent injection- producing :i ivturii of 
 bacteriolylic |HWT and causing the liberation of the cndotoxin. 
 
 It would seem porhap-. difficult to reconcile the fai-i (hat tin- -mini 
 becomes more |)oteut for tin- protection of animals other than the 
 individual which produces it. and that \ rt fnun tin; very fart of it- 
 bacteriolytic i>ower it should U> injurious to the animal furni-hing it. 
 It would appear paradoxical to lind that -mini obtained from an an! 
 nial jMissessed the property of protecting other animal-, and, at the 
 same time, not only failing to protect tin- animal furni-liin^ it. hut 
 actually increasing the >u>ceptihility of this animal. Here again 
 WoltF offers the explanation that it is a matter of dosage. If the 
 numher of Imctoria injected into the animal furnishing the >ermn, or 
 into the animal which has been ^iven a protective dose of the -erurn. 
 i- not so large that on In'ing disintegrated the amount of toxin is >uf- 
 ficiently large to cause disea-e. the animal escapes. There are appar- 
 ent difficulties in the way of this explanation, but it is, perhaps, 
 nevertheless not without weight. 
 
 In this connection the results obtained by Elischer and Kentxler* 
 on comparing the bactericidal power of unheated immune serum 
 i. e., immune serum containing its own proper complement with the 
 same serum heated and reactivated by the addition of fresh rabbit 
 serum would seem to apply. The latter was found to be more potent 
 than the former, and this would seem in a way to offer an explanation 
 of the protective power of immune serum for an animal into which it 
 is injected on the one hand and of the failure of such protective prop- 
 erties of the same serum for the animal from which it was obtained 
 on the other. Elischer and Kentzler interpret their results as indi- 
 cating that immune serum contains an insufficient amount of com- 
 plement for the full development of its bactericidal ]>ro]M>rties. So 
 in other ca>e-. the amount of complement in immune serum may be 
 insufficient to enable the serum to protect the animal from which it 
 was obtained, while it would find sufficient complement in the serum 
 of a fresh animal. To state this view briefly, the excess of aml>ocep- 
 tors in immune serum finds the requisite amount of complement in 
 the normal serum of uninfected animals. 
 
 When the behavior of hog serum on dilution i> considered, the 
 explanation afforded by the theory of complement diversion seems 
 applicable, not only for the behavoirof immune >erum in which there 
 was shown often to be increased bactericidal |>otency on dilution, but 
 also in the case of normal serum, as it will be attempted to show. 
 
 It will be recalled from the statement^ in regard to the theory of 
 complement diversion given on page 1:5 that in accordance with the
 
 GENERAL DISCUSSION. 49 
 
 views of the Ehrlich school generally it is held by Neisser and 
 Wechsberg, the originators of the theory, that bacteriolysis is due 
 to the action of a substance called complement, which is present in 
 normal and in immune serum, but that this substance can act on the 
 bacteria only when it becomes united to these through the medium of 
 certain bodies called amboceptors. The complements are held to be 
 incapable of uniting directly with the bacteria. Now, according to 
 the theory of complement diverson, if there are more amboceptors 
 than complements present in any given serum, all the complements 
 which are present unite with a corresponding number of amboceptors, 
 and if any bacteria are introduced into the serum they unite with the 
 free amboceptors in preference to uniting with those having comple- 
 ments attached. In this way the complements become diverted from 
 the bacteria by the excess of amboceptors. It will also be recalled 
 that while there are amboceptors in normal serum, these become 
 increased when the animal from which the serum is obtained is 
 injected with bacteria. The amboceptors formed by these injections 
 are specific; that is to say, an animal injected with B, choleras suis 
 develops amboceptors in the serum which are capable of uniting with 
 B. choleras suis only. Reference to figure 4 (page 51) will serve 
 to elucidate whatever may be obscure in the above explanation of the 
 theory of complement diversion. 
 
 Bearing in mind that normal serum contains a relatively large 
 amount of complement and a relatively small number of ambocep- 
 tors, it is evident that by introducing normal serum into immune serum 
 it is possible to increase the amount of complement without adding 
 materially to the number amboceptors. In fact, by making a series 
 of dilutions of immune serum and adding to each lot in the series a 
 definite amount of normal serum, it is possible to obtain a set of dilu- 
 tions containing various amounts of amboceptors and approximately 
 the same amount of complement. Evidently if the immune serum is 
 merely diluted and no normal serum is added, the complements 
 present will be diluted in the same proportion as the amboceptors. 
 
 If the amount of complement is kept constant in the way above 
 described, by the addition of a constant amount of normal serum, it is 
 evident that in some of the dilutions of the immune serum the ambo- 
 ceptors will have enough or more than enough complements to com- 
 bine with the amboceptors, and that in such cases the conditions 
 necessary for bacteriolysis will be fulfilled the amboceptors with the 
 complements attached will unite with the bacteria. In other words, 
 the undiluted immune serum, having an excess of amboceptors over 
 complements, fails to show bacteriolysis, while the diluted immune 
 serum with complements added, having no excess of amboceptors 
 over complements, causes bacteriolysis.
 
 f'H HA I I Kh'l.'i I l< I'.'WIIJ <>|. MI..i| -I i;i \| .., BOOB. 
 
 Buxton' ha- raised the objection to tin- theory that it can account 
 for those cases only in which the an:, unt of complement is kept . ,,u 
 slant in the dilutions of immune serum, while the amboceptoi - alone 
 
 re diminished. HA makes the point that in experiments such as arc 
 recorded in the present pa;>er, in which amlioceptoj-s and comple- 
 ments arc diluted to an equal extent, the theory of complement (Inci- 
 sion is inapplicable; and >ince the pr<t-e-- i- probably the -anie in loth 
 cases, both where the complement is kept to a on-taut amount by 
 heating the serum and adding a definite quantity of normal scrum on 
 the one hand and where the serum is not heated and ha- no normal 
 serum added on the other hand, the theory a- a whole i- not tenable. 
 Valid as this objection appears from Buxton's presentation, it appear- 
 on further consideration a- scarcely to be insuperable, for the theory 
 seems to afford a satisfactory explanation in every case \\liere the 
 ambocepton are in excess of the complements, and, granting A cer- 
 tain proportion between these in the undiluted serum, it i- not n- 
 sary for the complement to be present in an equal amount in the undi- 
 luted serum and in the dilutions for the occurrence of the diversion 
 of the complement to take place. All that the theory would seem to 
 demand is that the undiluted serum contain an insufficient amount of 
 complement to satisfy the amboceptois which are free and those which 
 are attached to the bacteria. Bacteriolysis will take place just in pro- 
 ]>ortion to the paucity of the free amboceptors, for, according to the 
 theory, those bacteria which are not coupled to free amboceptors 
 are liable to bacteriolysis by becoming attached to the amboceptor- 
 complement combinations. 
 
 In figure 4 it is attempted to show that comp.ement diversion may 
 well account for those cases in which the complement is diluted in 
 tin 1 same proportion as the amboceptors. This point could be more 
 strikingly shown if a larger number of amboceptors and complements 
 could be taken, and a much greater relative difference could l>e made 
 in the diagram between the number of amlxxjeptors and of comple- 
 ments, but for obvious reasons it is impracticable to do this. Thus, if 
 it were feasible to show 100 amboceptors and 100,000 amboceptor- 
 complement combinations in 1 c. c. of undiluted serum, it could be 
 demonstrated that 100 bacteria introduced would all esea|>e bacterio- 
 lysis in this serum, only 10 bacteria would escape in 1 c. c. of a dilu- 
 tion of 1 to 10 of the same serum, only 1 bacterium would escape in 1 
 c. c. of a dilution of 1 to 100, and none would escape in 1 c. c. of a 
 dilution of 1 to 1,000. In other words, theoretically in serum in which 
 the above conditions prevailed, taking 1 c. c. in every case and intr<> 
 ducing 100 bacteria, there would be no bacteriolysis in the undiluted 
 serum, but complete bacteriolysis in the same serum in the 1 to 1,000 
 dilution.
 
 GENERAL DISCUSSION. 
 
 51 
 
 In figure 4 the amboceptors are represented by the parts marked 
 #, the complements by the parts marked #, and the bacteria by the 
 parts marked b. No. 1 is intended to represent the undiluted serum, 
 and all except two of the amboceptors are shown as having diverted 
 a corresponding number of complements, while the two amboceptors, 
 which are not united to complements, and which represent the excess 
 of these bodies over complements, are united to a corresponding num- 
 ber of bacteria. Evidently in such a case there would be no bacte- 
 riolysis, since all the complements have been diverted from the 
 bacteria. In No. 2 half the amount of the serum, as in the first case 
 
 FIG. 4. Diversion of complement in Immune serum, not heated, and \rithout the addition of 
 
 foreign complement. 
 
 that is to say, 1 c. c. of a 1 to 1 dilution is represented, with the 
 same number of bacteria added as in the undiluted serum represented 
 by No. 1. Under the conditions represented by No. 2 it is evident 
 that one-half of the bacteria would be killed. In No. 3 is represented 
 1 c. c. of a dilution in which 0.25 c. c. of the original serum is contained. 
 In this case all the bacteria would be killed, since there are no longer 
 any free. amboceptors to unite with the bacteria, or, at any rate, there 
 would not be enough of the amboceptors to divert the complement in 
 all of the four portions into which it is assumed the serum is divided. 
 If the amboceptors are to be regarded as chemical substances in solu- 
 tion, then there would not be enough in &ny one of the four portions 
 to cause diversion of the complement. If, on the other hand, the
 
 .'_' BA<TKK1I.YTI< I-..W1K <>K HI IM'M "I MIN! 
 
 amlo,-ept,.r- are l>>lie- in tin- >hirt -MX,., thru it i* e\ idmt that two 
 of the four portion.- would show liict-i i>|\ -i- ,.f <>ne half f tin- la- 
 teria, while the other two would -li>\\ ...mplrte ta<-teriol\ -i-. a inn 
 ing uniform distribution of (lie amboceptors. In rithrr cast- the 
 diluted scrum would be more powerfully bactericidal than the undi- 
 luted serum. It will be observed that the complement is not k< pt 
 at u constant amount in the dilutions represented by the diagram-, 
 but that it is diluted simultaneously and to a like degree with the 
 amlK>ceptors. 
 
 I-'rom what has been said above it would appear that diven&n of 
 complement seems a rea>oimble explanation of the phenomena whieh 
 are observed in unheated un reactivated serum, and it therefore >emis 
 to overcome Buxton's objection to the application of the theory to the 
 phenomena observed under such condition-. 
 
 It is aside from the purpose of the present article to try to meet in 
 general objections to the theory of complementdiversion, or todi-cu-- 
 this theory apart from whatever bearing it may have upon the pre-ent 
 experiments; but incidentally the objection raised by Buxtonof bring- 
 ing in accord with the theory of complement diversion the fact that 
 Asiatic-cholera spirilla are killed about as readily by typhoid and para- 
 typhoid immune serum as by normal serum may perhaps be overcome 
 by consideration of the fact that the injection of an animal with any 
 one kind of Imcterium probably has no effect upon the aml>oceptor- 
 complement combinations for any other kinds of bacteria which are 
 present in the serum of the animal undergoing inoculation. The effect 
 is probably very sharply specific, even going so far in some cases as to 
 affect different strains of the same organism, as is shown by the results 
 of some of the experiments in the present paper. It seems not at all 
 inconceivable, at least, that an animal injected with any Driven kind of 
 bacterium might well be deprived of the power of killing the kind 
 of bacterium with which it was injected, owing to the excessive pro- 
 duction of specific amboceptors, without interfering with the ambo- 
 ceptor-complement combinations for any other bacteria which may be 
 originally present. 
 
 In view of the very specific reaction which takes place on injecting 
 an animal with bacterial cultures, there would really seem to be no 
 good reason why immune serum should act differently from normal 
 serum for any but the homologous organism. The amboceptors which 
 are newly formed in consequence of the injections could not reasonably 
 be supposed to have the power of depriving the amboceptors originally 
 present of the complements to which they are attached. If it is true, as 
 assumed, that in normal serum there are am boceptor-coniplement com- 
 binations which will destroy D. cholera? #//*, and in the same serum 
 aml)oceptor-complement combinations which destroy //. typhoawi, the 
 injection of the animal with cultures of B. cholerxauis would evidently
 
 GENERAL DISCUSSION, 53 
 
 increase the amboceptors for this organism, and these newly formed 
 amboceptors would have the power of uniting with any free comple- 
 ment, but not with any complement already united to the B. typhoxux 
 amboceptors. So that while the newly formed amboceptors would 
 and probably do find free complements with which to unite, the} 7 could 
 hardly be expected to tear loose the bonds between the original ambo- 
 ceptors and complements. 
 
 As has been pointed out by Neisser and Wechsberg in their experi- 
 ments, the increase of bacteriolysis usually observed on the dilution of 
 immune serum does not appear to be explicable by the conception of 
 Bordet that the immune body the amboceptor is merely a sensitizing 
 agent. For if the amboceptor were in reality merely a "substance 
 sensibilisatrice " it is not likely that immune serum would be more 
 active when dilute than when concentrated. At least it would be only 
 reasonable to presume that a reagent possessing the properties ascribed 
 to the sensibilisatrice by Bordet and his followers would lose rather 
 than gain in power upon dilution, and that a given amount of the 
 undiluted sensibilisatrice would be more potent than the same amount 
 of the diluted substance, as is the case with other reagents. On the 
 other hand, Ehrlich's conception of the amboceptor affords a satis- 
 factory explanation of the behavior of immune serum upon dilution, as 
 has been shown in the above discussion of the theory of complement 
 diversion. 
 
 The results obtained in the present investigation in which the bog 
 serum appeared to increase in potency upon dilution seem best ex- 
 plained by the theory of complement diversion. But in these experi- 
 ments, as in all others of like nature, it would seem possible that the 
 process of bacteriolysis or the failure of bacteriolysis may at least be 
 complicated by the formation of antibacteriolysins on the injection of 
 the hogs or by the presence of antilysins normally present in the 
 serum. Opinions a re. divided upon the question as to whether there 
 are such antilysins present in normal or in immune serum. But if 
 there should be such antibodies present, it is reasonable to suppose 
 that these would be weakened in power on diluting the serum, and 
 that consequently such serum would be more potent when diluted than 
 when undiluted, unless the lysins and antilysins should be diluted in 
 like proportions. 
 
 There seems to be no difference of opinion in regard to the develop- 
 ment of qualities antagonistic to bacteriolysis in serum under certain 
 circumstances, but the matter in dispute is whether this antagonistic 
 property is due to special antilysins or whether it is due to the pres- 
 ence of nonspecific, normal amboceptors or of superabundance of 
 immune amboceptors which absorb the complement, and in this way 
 prevent bacteriolysis. 
 Pfeifl'er and Friedberger, 16 who were the first to detect this antago-
 
 54 HMTKKI>M I I' P.'UIK <>K IU.""I> -I KIM Of H<<8. 
 
 ni-tic action on the part ( normal scrum. hold that under certain cir- 
 cumstances specific antilxxlies tire formed, which coinliiin> with the 
 complement. In other word-, they regard the antih -in- a-anticom 
 plements. Their 0zperime&tl < % <nsi>t of triturating normal serum 
 with bacteria ami centrifugali/.ing. I'lider -uch cir.-um-tance- the 
 -iipernatani serum acquires the proj>erty of neutnili/ing the bacieri- 
 lytic jxwer of immune serum. Thus normal scrum, which in it-e|f 
 exerts noantihactcriolytic power, becomes EBtiDBCterfolytic for cholera 
 immune scrum when it is triturated with cholera bacilli and centrifu- 
 galixed or filtered through a bacteria-proof filter. 
 
 Gay" attributes antilytic pn>i>erlics developed in this way to the 
 formation of specific precipitines. He finds that only when then- i^ a 
 specific precipitation is there any antilytic property shown by henio 
 lytic sera, and since hemolysis and bacteriolysis are probably identical 
 processes, the same statement would apply to the latter a- well as to 
 the former. 
 
 Gay's view has been called into question by Sachs, 11 who attribute- 
 the antilytic properties of serum to normal, nonspecific amlnjceptors 
 which he regards as acting as neutralizes of the complement. The 
 only difference l>etween the explanation offered by Pfeiffer and Fried- 
 bergeron the one hand and Sachs on the other is that while the former 
 look upon the antilysins formed in their experiments as newly formed 
 bodies derived in part from the bacteria, Sachs contends that the 
 antagonistic bodies are present in the serum from the start. He 
 states, at least, that this is the only point in which he take- i uc with 
 Pfeiffer and Fricdlwrger on the matter of the formation of antily>in- 
 for hemolytic scrum. 
 
 Lipstein,"on comparing strongly agglutinating immune sera, found 
 that while l>oth the specimens examined possessed equal agglutinating 
 power only one of them showed the phenomenon of complement diver- 
 sion that is to say, antibaetcriolytie properties. He was also unable 
 to discover any constituent of the normal serum which showed diver- 
 sion of complement, but on the contrary he found this phenomenon 
 to take place in immune serum only. In other words, the antagonistic 
 action of serum to Iwcteriolysis he attributes solely to the diversion of 
 complement by the specific, immune amlxx-eptors. 
 
 In spite of this conflict of authority, it would seem at least that the 
 Neisser-Wechsberg phenomenon permits of easiest explanation by the 
 theory of complement diversion. For if the failure of the undiluted 
 immune serum to produce Iwicteriolysis were due to the presence of 
 antilysins, it would U less potent upon diluting the serum, it is true, 
 and consequently exert less power u|>on the Imcteriolysins when diluted 
 than when undiluted, but the relative effect would remain the same. 
 It is nevertheless prolmble that if Imcteriolysins are present in immune 
 -enini they complicate the process.
 
 GENERAL DIWCUSSION. 55 
 
 While the theory of complement diversion seems to have been used 
 by all investigators to explain the bacteriolytic phenomena of immune 
 sera only, it would not seem inapplicable to those which take place in 
 normal serum as well. The theory seems at least to explain the 
 behavior of the normal serum of hogs upon dilution in the present 
 investigation. Even in those cases where the normal serum showed 
 diminution of potency on dilution, the diminution was not in direct 
 proportion to the dilution, as has been previously noted; but on the 
 contrary the diluted normal serum acts more powerfully, relatively, 
 than the undiluted normal serum. This relatively greater potency of 
 the diluted normal serum (>tr the undiluted may be accounted for 
 by the theory of complement diversion, if it is assumed that there is 
 an excess of amboceptors over complements in the normal serum of 
 hogs to start with, as certain considerations will show. 
 
 Difficulties in the way of the acceptance of the explanation just 
 offered are of course very obvious. In the first place, normal serum 
 generally has been found to contain an excess of complement over 
 amboceptors; in fact, in adding normal serum to heated immune 
 serum, this is done for the purpose of increasing the amount of com- 
 plement without adding to the amboceptors. In heating immune 
 serum it is the complement which is rendered for the time inactive, as 
 it is called, and the addition of fresh serum reactivates the immune 
 serum by means of the new complement contained. The addition of 
 fresh serum to the heated t}^phoid immune serum of a rabbit reacti- 
 vates the immune serum. On account of the large amount, or of the 
 great number, of complements contained in the serum from the blood 
 of a normal horse this can be used to reactivate several different kinds 
 of immune sera. Then again, as stated above, in the process of im- 
 munization, the newly formed amboceptors probably find free comple- 
 ments with which to unite in the serum of the animal undergoing 
 injection. 
 
 While the force of these and of possibly other objections can not be 
 denied, it would seem evident from the experiments herein reported 
 that the only difference between the behavior of immune serum on 
 the one hand and of normal serum on the other, upon dilution, was a 
 difference of degree and not of kind. If this is true, the theory of 
 complement diversion must be applicable to both or to neither. 
 
 Evidently there are only three possibilities in regard to the relative 
 number of amboceptors and of complements in an} 7 given serum either 
 there is just the proper proportion of amboceptors to complements or 
 there is an excess of the one or of the other of these. If the ambo- 
 ceptors and complements are present in just the proper proportion, 
 then there would be a simple relation between the undiluted and the 
 diluted serum. The serum would become relatively and actually less 
 and less potent in exact ratio to the extent of the dilution. If a given
 
 .">', BAt n Ki"i.vn< I-MWKR OF -BI.< ..... IKRDV "i- HOO8. 
 
 number of bacteria were killed by 11 certain amount of tin- undiluted 
 - ruin, a certain f motion of this numtier would be destroyed hy the 
 same fraction of the serum. If tin- complement i" in BXOMt, evidently 
 the same rule would hold: tin- free compleinents would neither help 
 nor hinder the process. Hut if there were mi excess of ainboceptoifi, 
 a part of the bacteria corresponding to the amount of tlii- excess 
 would escape bacteriolysis, according to the theory of complen 
 diversion. On dilution, however, there would le relatively more l.ac- 
 teria killed where there are free aml>oceptor present in the undilutrd 
 serum, as already explained in the description of the Nei--er \Ve<-|isl>erg 
 
 theory. Aft 
 
 * . ^^ 
 
 Since none of the experiments in the pre-ent investigation have IK in 
 
 made to determine whether the serum from normal hog's blood con- 
 tains free auilxx-eptors, and no experiments of the kind with hog's 
 blood seem to have been so far reported, the matter must for the 
 present l>e left undecided. But the as>imiption of such ti condition in 
 the normal serum would seem necessary to account for the results 
 obtained. 
 
 A specific example taken from one of the experiments already 
 described may serve to show the application of the theory of complement 
 diversion to the phenomena observed on diluting normal hog sera in so 
 far as the effect of dilution upon the bacterioly tic potency i- concerned. 
 Into the undiluted serum from the blood of hog 1851>, and into each 
 of the various dilutions, there were introduced before injection of the 
 animal 3,760 bacilli of the Crawford strain per 1 c. c. of the serum. 
 The number of bacilli surviving after twent3'-four hours in the undi- 
 luted serum was 80 per 1 c. c. There are two conditions which may 
 be assumed to exist in the serum, either of which would account for 
 the escape of the 80 bacilli, disregarding for the present the fact -Ue- 
 where discussed that there is a multiplication of the bacteria surviving 
 Inicteriolysis and assuming that the 80 Imcteria in the present c*M 
 represent merely those bacteria which escaped bacterioly>is. Hither 
 there were not enough amlweptor-coinpleiiients for all the bacteria 
 introduced in other words, then- were >o short of these or, if there 
 were enough or more than enough aml>oceptor-coniplements. t lien- 
 were also 80 free amlMn-eptors. Hither of these two conditions would 
 account for the escape of the 80 bacteria in the experiment. A simple 
 calculation from the result obtained on diluting the serum will show 
 which of these conditions is most likely to have prevailed. If there 
 had been a paucity of amboceptor-complenients if there had ln-en. 
 in other words, just 3,680 of these instead of over H.7'.n. which was 
 the number of bacteria added then a dilution of 1 part of the >ei um 
 to 9 parts of salt solution should have killed only :'.',^ bacilli per 1 
 \\hen these were introduced; whereas, as a matter of fact, this dilu- 
 tion killed :i,60o \trr 1 c. c. There would appear, therefore, no ground
 
 CONDITIONS AFFECTING BACTERIOLYSIS. 57 
 
 for assuming the failure of bacteriolysis in the immune serum to have 
 been due to a paucity of amboceptor-complements in the undiluted 
 serum. 
 
 Suppose, now, that the other condition mentioned above existed in 
 the serum, namely, that there was an excess of amboceptors over and 
 above the amboceptor-complements, and that there were more ambo- 
 ceptor-complements than the bacteria introduced. A simple calcula- 
 tion will show that such a supposition will readily account for the 
 results obtained in the diluted serum as compared with the results in 
 the undiluted. If there were in the undiluted serum 26,000 ambocep- 
 tor-complements and 80 free amboceptors per 1 c. c. then on dilution of 
 1 to 10 there would evidently be 2,600 amboceptor-complements and 8 
 free amboceptors per 1 c. c. of the diluted serum. Consequently 8 
 bacteria would escape bacteriolysis on account of being attached to the 
 free amboceptors, but 2,600 would be destroyed. 
 
 The above calculations are, of course, not intended to indicate pre- 
 cisely the processes involved, for these are probably complicated by 
 the multiplication of the surviving bacteria, as mentioned above. 
 Still, even if such a complication is admitted, the probabilities in favor 
 of the condition last named become even greater, as a moment's con- 
 sideration will show. 
 
 A further complication may also arise from the possibility, however 
 remote, that the amboceptors and complements are not uniform!}" dis- 
 tributed in the serum that while they may perhaps usually be so 
 distributed, they may be found more abundantly in one part of the 
 serum than in another. 
 
 SUMMARY OF THE CONDITIONS AFFECTING BACTERIOLYSIS. 
 
 The conditions under which bacteriolysis would take place, and 
 those under which no bacteriolysis would take place in any given 
 serum, may be summarized as follows: 
 
 1. Complete bacteriolysis could take place only where there were 
 no free amboceptors and where there were at the same time a number 
 of amboceptor-complements equal to or greater than the number of 
 bacteria introduced. 
 
 On dilution in a serum of this kind there would be a loss of bac- 
 teriolytic power in proportion to the degree of dilution if the ambo- 
 ceptor-complements were exactly equal in number to the bacteria 
 introduced. If there were more amboceptor-complements originally 
 in the undiluted serum than the bacteria introduced, then on dilution 
 there would be relatively more bacteria destroyed. If the excess of 
 amboceptor-complements is large enough, there could of course be 
 enough present in the diluted serum to kill as many bacteria as were 
 killed by the serum before dilution.
 
 '.^ BA TKKInM I I. POWJEB 'K IU."'I -MUM "I- II". 
 
 J. Partial kwetoriolysift would follow when there were fewer ainbo 
 ceptor- pn -cut in tin- -eruin than the bacteria int rod need ami when 
 at the -ame I line there were any Rmbooeptor-OOOlpleineilt0 pre-ent. 
 
 The extent of bacterioly-i- upon dilution would depend upon the 
 number of amlxweptor-complemcnt- present originally. 
 
 H. No hacterio|y-i- could take place if the free amboceptor- wen- 
 e(|iial in number to the bacteria introduced, or if they were in cxce-.- 
 of this number, either in the undiluted or the diluted -erum. 
 
 Tin: im.i;i . ,i DMPI.KMKNT itivi:i;.-ioN. 
 
 In spite of the serious difficulties alluded to it \\ould neverthele-- 
 appear that the theon of coiiipleiuent diversion give- an insight into 
 the proce-x of bacteriolysis OOCnring in normal serum in ><> far a- thi- 
 i- allected by dilution in tho-e ca-e- at lea-t \\here the experiiu. ni - 
 >how that the serum U actually or relal ively more potent upon dilution 
 than when undiluted irrespective of the nature of the serum, whether 
 thix ix normal or immune. But this would leave the result- obtained 
 in certain other experiments still unexplained. It will be recalled 
 that in certain experiments the M-riim was found to be bactericidal 
 when undiluted, that it lost in bactericidal potency on dilution, but 
 that it irained in potency upon further dilution. It is the.e result- 
 which would he left unexplained by the theory of complement diver- 
 sion as thus far dUcuxsed. One example of this behavior amon^ other- 
 is to t>e seen in Table XXVI. in the serum of hog 1708, ten day- 
 after the inoculation of the animal with the F. :.''. strain of 11. rJ,t,1eraB 
 . I none of the tests there were introduced into the undiluted -ermn 
 and into the various dilutions in round numbers .v>o Crawford bacilli. 
 The result after twenty-four hour.- wa- that all the bacilli \\eiv 
 de-t roved in the undiluted serum, while in the dilutions of 1 to ."><' and 
 1 to .".uo there \\a- an increa-e of about thirty-three fold and one 
 hundredfold, respectively. Hut in the dilution of 1 to 5,(X>" there 
 were only II" bacilli renmining. In other word-, the undiluted 
 serum showed strongly bactericidal power, and the dilution of 
 1 to :..i'oo ;d-o showed considerable potency, while the dilution- 
 bet ween the-e two showed at Ilio-t but feeble power. If this re-lilt is 
 
 analyxed. it will be seen that the -erum appeared to act like normal 
 serum at the -tart when undiluted and in the lir-t dilution-, but that 
 on further dilution it appeared to act like immune serum. This siig- 
 ~ that po ibly the normal ambyeeptor-comph-nient- per-i-t ing in 
 tin- -erum may have been more potent than the immune amboceptor- 
 formed in consequence of the injection, that the tir.-t eflect of dilution 
 wa- to reduce the potency of the normal arnU.ceptor complement-, and 
 that further dilution enabled the immune amlxjceptors to produce the 
 phenomenon of complement diver-ion. This would le ijuite in accord 
 
 with certain view- e\p|V ed elsewhere in this |Ml|>er to the ellect that
 
 THEORY OF COMPLEMENT DIVERSION. 59 
 
 the injections of cultures or their products while they cause the for- 
 mation of new amboceptors leave the normal amboceptor- complements 
 unaffected. 
 
 In the above discussion it is assumed that the two bodies concerned 
 the amboceptor and the complement of Ehrlich, the sensibilisatrice 
 and the alexin of Bordet are capable of uniting and do actually 
 unite independently of the presence of bacteria or of other cells. But 
 Bordet 3 has recently published a series of investigations tending to 
 show that the experiments of Ehrlich and Sachs 5 , which constitute the 
 chief evidence in favor of this view, are capable of quite a different 
 interpi-etation from this, and that this interpretation is in fact not justi- 
 fiable from the results of the experiments which consisted in the 
 demonstration of the fact, not denied by Bordet, that ox serum will 
 produce cytolysis only when the serum has in it amboceptors and com- 
 plements simultaneously. It is not possible, as in some other cases, to 
 produce cytolysis by sensitizing cells with ox amboceptors that is, 
 with heated ox serum and, after washing these sensitized cells, add- 
 ing complement that is, fresh serum. Cytolysis with ox serum 
 takes place only when the heated ox serum and some unheated fresh 
 serum (horse serum was the kind used in the experiments) are 
 employed at the same time. This is interpreted by Ehrlich and Sachs 
 as showing that while the free amboceptors present in the ox serum 
 will not unite with the cells they will and do so unite when the}' are 
 previously attached to complements. But Bordet's results appear to 
 show quite plainly that in this case the horse serum which was used 
 as complement produces cytolysis quite independently of the ox serum, 
 and that while cytolysis takes place more promptly when heated ox 
 serum ox amboceptors are added, the ox serum is not necessaiy. 
 Bordet therefore regards the experiments as showing- that the heated 
 ox serum acted merely as an accelerator of cytolysis. 
 
 Bordet summarizes his conclusions as follows: 
 
 We see but one rational explanation of the peculiar action of ox serum that there 
 exists in the serum a peculiar substance capable of resisting heat of 56 C. and 
 which remains unaltered for many months in this heated serum. The substance 
 is probably of an albuminous or colloid character, and does not adhere to the 
 normal corpuscles, but is precipitated upon the corpuscles which are previously 
 charged with sensibilisatrice and alexin. We believe that it is a veritable process 
 of glueing of absorption depending upon molecular adhesion. In con- 
 
 formity with the statements of Ehrlich and Sachs, experiments show that the 
 corpuscles of guinea pigs become hemolyzed in a mixture of fresh horse serum and 
 of ox serum, the latter having been heated to 56 C., while they resist hemoly- 
 sis if they are first subjected to the action of the heated ox serum and have the horse 
 serum added subsequently. But the interpretation offered by Ehrlich and Sachs, 
 according to which the sensibilisatrice furnished by the ox serum does not unite 
 with the corpuscles unless it (the sensibilisatrice) is previously connected with alexin 
 derived from the horse serum is n(h correct. In the first place, the sensibilisatrice, 
 which plays a preponderating and most essential role, is not contained in the ox
 
 i'l I HACTr Kl.'I.VHc 1'oUKR OF BI I' -I-.WM K H<" 
 
 -.nun ut all, hut i- furni-hed by tin- h.ir-e scrum. ( 'on-eo.ncntly theft) 
 
 > U-have lik- all <f their conveners, in tin- nonse that they do not require the 
 pn-scii.-e ! tin- alexin In-fore they are capable of uniting \\ ith tin- corpuscles. 
 
 Finally, this interpretation leaves completely in the dark thf MTV -^-.'ial |>ccnliari- 
 
 : the cu.ses of heinolysis in (jiutiti.m. 
 
 The (teculiarity of ox serum consist* in the prewnce of a certain element which 
 - ">ti ('.. ami also reeistw tanding, and is of the nature of a >lliil, doubt lew 
 albuminoid, and which, furthermore, in al^irlKsl by curpuwlff which have IKT..III,- 
 rharxi^l witli HHibilisatric- and aK-xin, but which remain^ fn-- in tin- presence of 
 normal OOCjMMlflt OT Oi corpusrlrs imTt'ly sennit i/c. I i , cnrpn>cli-.- tn-atvd \\ith 
 heated Heriun alunc. Tin* absorption of thi>rolloii| by corpuscle which have btfii 
 tn-att-d with lx>th wnsihilisatrire and alcxin ha the effect of energetically aggluti- 
 natiim them and of rendering them more siiM-eptible to liemoly.-i.- except un<l. 
 tain circumstance*. * The absorption of the sensiti/ed and alcxinized cor- 
 puscles is very likely due to molecular adhesion, the preliminary treatment h:. 
 niiMlilieil the corpuscles in so far a* their adhesive properties are concerned. I n-ler 
 them* conditions the absorption may take place inde|>en<lcntly of the upecieu of 
 animal from which the corpuscles are obtained; it may even take place with the 
 corpuscles of the name animal which furnishes the colloid, an in the cane of ox wrum. 
 
 N"\\, if the contention of Bonlct a> ><-t forth al>ov is correct, and 
 if tin- t\v<> lolir> conrrriH-d in cytolysis do not unite, then of cour-e 
 the tlu-ory of complement diversion must fall, since it requires as the 
 lir-t condition the union of amboceptors with complements. Never- 
 theless, there appears to be as yet no other explanation of the phe- 
 nomena observed on diluting immune serum, and. a^ an attempt ha* 
 IXMMI made to show in the present paper, there seems to be no other 
 explanation of the behavior of normal serum on dilution. 
 
 CONCLUSIONS. 
 
 In summarizing the results of the present investigations it is seen 
 1. That the bactericidal potency of the serum from the same hog 
 
 varies from time to time. 
 
 _'. That the serum from one and the same drawing differs in poteiu-y 
 
 for different strains of /?. chnl,>rn> xuix. 
 
 3. That while the effect of standing is to weaken the potency, this 
 tl. , t is more marked in some sj)ecimens of serum than in other- 
 under the same condition-. 
 
 4. That the bactericidal power of the serum from the venous blood 
 of hoefs is not ahvay* more potent than that from the arterial blood. 
 
 5. That heating the serum at about 54 C. for thirty minute*, inacti- 
 vates the serum. 
 
 6. That the Neisser-\Veehsl>erg phenomenon is sometimes seen in 
 and sometimes missed from the Mood of hogs injected with cultures of 
 //. cAokrM MM. 
 
 7. That the theory of complement diversion may not only account 
 for the behavior of immune serum. l>ut also for that of normal >erum 
 on dilution, or, at least, that the theory, if applicable in the one case, 
 is equally so in the other.
 
 BIBLIOGRAPHY. 
 
 (1) BORDET, JULES. 
 
 Agglutination et dissolution des globules rouges par le serum. Deuxieme 
 memoire. Ann. de 1'inst. Past., t. 13, no. 4, p. 273-297. Paris, Apr. 25, 
 1899. 
 
 (2) BORDET, JULES. 
 
 Sur le mode d'action de scrums cytolytiques et sur 1'unite de Palexine dans un 
 meme serum. Ann. de 1'inst. Past., t. 15, no. 5, 303-318. Paris, May 25, 
 1901. 
 
 (3) BORDET, JULES, and GAY, FREDERICK P. 
 
 Sur les relations des sensibilisatrices avec Palexine. Ann. de 1'inst. Past., 
 t. 20, no. 6, p. 467-498. Paris, June 25, 1906. 
 
 (4) BUXTON, B. H. 
 
 Bacteriolytic power of immune serum and the theory of complement diver- 
 sion. Jrn. of med. research, v. 13, no. 5 (whole no. 90, n. s. v. 8), p. 431- 
 485. Bost, Aug., 1905. 
 
 (5) EHRLICH, P[AUL], and SACHS, H[ANS]. 
 
 Ueber den mechanismus der amboceptorenwirkung. Berl. klin. woch., 
 jahrg. 39, no. 21, p. 492-496. Berl., May 26, 1902. 
 
 (6) ELISCHER, JULIUS v., and KENTZLER, JULIUS. 
 
 Ueber die baktericide eigenschaft des typhusserums. Berl. klin. woch., 
 jahrg. 42, no. 29, p. 897-900. Berl., July 17, 1905. 
 
 (7) FRIEDBERGER, E[UGEN]. 
 
 Die baktericiden sera. Kolle und Wassermann's Handbuch der pathogenen 
 mikroorganismen, bd. 4, teil 1, Jena, Gustav Fischer, 1904. See p. 497. 
 
 (8) See citation 7, p. 521. 
 
 (9) See citation 7, p. 561. 
 
 (10) GAY, FREDERICK P. 
 
 , The fixation of alexines by specific serum precipitates. Cent. f. bakt., abt. 1, 
 Orig., bd. 39, hft. 5, p. 603-610. Jena, Oct. 10, 1905. 
 
 (11) LlPSTEIN, A. 
 
 Die komplement ablenkung bei baktericiden reagenzglasversuchen und ihre 
 ursache. Cent. f. bakt., abt. 1, Orig., bd. 31, no. 10, p. 460-468. Jena, 
 Apr. 16, 1902. 
 
 (12) LOEFFLER, F[RIEDRICH], and ABEL, R[CDOLF]. 
 
 Ueber die spezifischen eigenschaften der schutzkorper im blute typhus- und 
 coli-immuner tiere. Cent. f. bakt., abt. 1, bd. 19 no. 2|3, p. 51-70. Jena, 
 Jan. 23, 1896. 
 
 (13) MORGENROTH, J., and SACHS, H[ANS], 
 
 Ueber die completirbarkeit der amboceptoren. Berl. klin. woch., jahrg. 
 39, no. 27, p. 631-633. Berl., July 7, 1902. 
 
 (14) NEISSER, MAX, and WECHSBERG, FRIEDRICK. 
 
 Ueber die wirkungsart bactercider sera. Munch, med. woch., jahrg. 48, no. 
 13, p. 697-700. Munch., Apr. 30. 1901. 
 
 61
 
 62 
 
 (16) I*rm:i;~<>v A i i 
 
 I'I-IHT <Iii> natiirlirlir !iiil/.t)r.iii<liiii!iiiiiiilat .lex hnndcfl and d hnhiu>. Gent. 
 f. bakt., al>t. 1, Orijf.. IN!. :, 11... - 8M, -l.-im. Apr. 4, 1903. 
 
 (16) IVRIFKKR, K[n'ii.\iti>], ami KuiKDHEtuiBK, K[I*EN]. 
 
 l'clH>raiitil>akt*Ti(>Iy tint-he (antagoniti8che)8ultanzen nunnuli-rHera. Dent. 
 Hied. wn-h., jahty. :U, n>. 1, p. JK-S. It4-rl , .Ian. 5, 1906. 
 
 (17) PntirrKR, R[i< HAKI>], and WAMHKKMANN, A[r(;fKr]. 
 
 ('ntcr><iictiunp>n U!HT -hw weeen der choleraiinrnnniUit. /.twlir. f. hyg. n. 
 inf.-kt.. 1,1. 14, hft. 1, p. 46-63. Leipz., 1893. 
 
 (18) SACHS, HANS. 
 
 TI-UT .la.- RamnOMmHrinmt nonualer nnd iniiiiiini-atnrisrli eraenjrter amho- 
 /fpt.-rtMi IKM dt-r hatnnlym-. Deut. tned. woch., jahi^g. 31, no. 18, p. 706-707. 
 llt-rl.. May 4, 1905. 
 
 (19) Tl(iIM>lK)KK. KU IIAKD. 
 
 I'cU r di-n ulfxiii^chalt nonnaler und patholo^flcher menschlicher hlatsera. 
 Cent. f. bakt., abt 1, Orig., bd. 32, no. 6, p. 439-449. Jena, Sept. 25, 1902. 
 
 (20) Woi.KK, Al.KKKl). 
 
 Ueber grund)?etetze d-r immunitat. Cent. f. bakt., abt. 1, Orig., Ixl. :;7. n-. 
 4, j). 566-676. Jena, Dec. 12, 1904.