REPORTOFTHB -- DIRKTOR OF THE HOSPITAL June 1919 , REPORT OF THE DIRECTOR OF THE HOSPITAL OF THE ROCKEFELLER INSTITUTE FOR mDIC!AL RESEARCH June 1919 To the Board of 6cisntlflc Dlrsctorr of The Rockefeller Institute for Yedical Rerearch Gent lemsn : The effect of war on the Hospital was to gradually change Its chief actlrltl8r from investigation to teaching and the care of patients. With ths coming of peace it har been porelble fortunately to revsrss thlr tendency, and at the present time the Hospital ir rapidly getting back to a pro-war basis. The army of flcerr assigned to the Hospital by the Surgeon Gsneral'r off ice to act (u Internor have been ordered elsewhere, and the members of our rtaf f on duty here have all been dlrcharged from the rervice. Some of . . the members of the rtaff who left the Hospital to enter the army have returned, others will not return. _ ,j... The resident staff ha8 been most o erlously p-2 !, 1 ` . r i i , . ,. .: _ . ,.. af fscted by the war and the problems conn8cted with the reorganlsation *,. . . . _. .*:. A, , ):' ..-. . . .I . . of this part of the o srvlcs are smong the moot important that we ha08 to jll . _.J.`. ^ .I _. ~`Z.' " . : . consider. In the past, as far as possible, only those men were appolnt- `_ ./ `i . . 1...Lj.l. . I .., < .` i.. i ._ : , y :.:.. .I ed assistant resident physicians who had completed their hospital service ; > : ! : -: .: I : .., I` 2 _ . . ,2$ . ._ * I 5, " and who had had special training ln the methods of 'one of the rciences j: `L` :, , ; ( I ., ,, . 1 . . . % `. ..,c `A,' I ,: contrlbut lng to medical progress. During the past two years very few ,,_ i _I. I ,I . : young men have been receiving this kind of training. Even the hospital *. ._ training received by the younger men In the army camps is not that which be8t f it8 them for the work to be undertaken here. For these reasona it is not likely that the rork done by the junior members of ths staff during the next few years will be so eatiafactory as in the past. Moreover, the older members of the staff must devote more attention thbn formerly to the question of training the young men. T6e have been fortunate in obtaining aa Resident Physician, Dr. Robert Levy, who hae had an excellent training in hoepltal work in the Johns Hopkins Hospital and who has already carried on a number of scientific inve8tlgatlon8, so that he Is fitted to direct the work of the asei8tantm in the care of patient6 and to take a aymkathetic Interest in their o clentific work am well as to carry on him own lnveetigatlon. Three aaeimtant resident phySiCiAn have been appointed to serve for the remainder of the current year. Dr. William Sadie, who ham acted am resident physician during the part year, contln- ues ae amsisttmt resident phyeician. Dr. Arthur Lyon, who was sent here by the army and ha8 been diecharged from the service, remain8 to act as aesiatant raeident physician, and Dr. James Track hae been appointed to act in a elmllar capacity. It is probable that son3 of these men will remain during the coming year. ,., ,. _. I . . . . L 4 \ . . ,Durlng &the p@riOd of reorganiratlon it haa been. , . ~ ,_ 1 (.. ,,,r thought advisable, to cu$,dqwn {am ;f,ar ,a@ -poesible, the nWnb@r. ,Of, patients I ,.: .,:: "L admitted for,itreatment T:,A B,Th$> enables ,reorganlrat ion of the research. ! t !,:. ;..,: B I activities ,$o ,.be ,undsrtakpn.!S.., The `study &of LI' ;. \.,r, `I; i ; _ I i? _ , r` i . ,' -1 &' exact knorlrdg8 of Condition8 before tho epidemic a8 rogardr the occurrence .I. .;, sl* I :.. , : `. : :.,. I(. .' ;.;. I (, ` `G . ..: . . . . 83 of B. `& luen8ae In Casbe of pneumonia or their occurrence in normal throatr. `_ : . . ;,a, _ ; ., .); , .: _ . , . - ? : ! Yt . . Obeervatione made during the paet winter 8how that the cultivation of these . . _' .I ..1 , ,.!L,; : _~ +. . L, .I 1 _I. , :' - organirrms ir mad8 with conridsrable dif flculty and that no comperl8on can be i,..:.' *.. :. ._ :; ;I i 1 made between etatirtical 8tUdi68 made by dlf ferent obrrervers or even between .,._' `. .., 7, .I. `:.. : studies made by the 888~ Ob8ertWr8 at diffsrent time8 and placea. The sod- (, ,. ium oleate medium described in my laet report ha8 proved of very great value in ieolatlng thee8 organlome, but It ha8 been found that even of this medium different lots may 8hon marked differencea in suitability for growth. Inveetigations are now under way to detera.lne, if p8sSibh, upon what the80 dif ferencee depend. By making repeated cultures and culture6 In varloua Way8, it ha8 boon possible to ieolate Influenza bacilli from the pharynx or eputum or reepiratory tract in over 85 per cent of the ca8ea of acute r-esplr- atory diaeaee, not only during the sever8 epidsmic, but in the after period a8 well. In the Ca8e8 with pnsuonia in which culture8 were made by lung puncture or at autopey, lnfluenta bacilli were cultivated f ram all, never alone hOWOVer, but always in aesoclation with pneumococcue, etreptococcue, or 8taphyIOCOCCU8. It will be remembered that during the September - Deceuber 1918 quarter, auring the height of the epidemic of influenza, a study was made by Dr. Stilluan and Miss Pritchett of the presence of B. influ- enzae in the uogths of the nomal persons aesoclated with the Inetltute, laboratory WOrk8r8, nureee, doctors etc. At that time 42 per cent of the pereonnel were found to be harboring influenza bacilli In the throats. This fact eeomed of auf flclent importance to justify repeated examinations of the I throat8 `of tdoee 8am8 persona to find whethsr this percentage' of person8 j r'emaine-d carrier;,' wheth'o'i' pbrsiatent `c'arriere occurred etc.' .,:: Consequently j ;- :I i .1 .9 .,i;s ;.,q ,: `,**`" j ' . ..y* < ., . .1~, , of the lndivfduale w8re found to be carrying influenza bacilli, In February, `i :;' : : : in March, 50 `per cent, `In April,"`36 per cent. ' ' 36 Fer cent, it' $6 pO88lble .,: ::.. i ., that the low incidence in january and the high'incldence in March:`may be In part explained by variations In the medium employed. The reeulte ehow, nevorthe- less, that a lcrrge number of the inilviduals in the Instltuto during the proeent winter have bean chronic carriers of this organism. From some individual8 all of the culturse made have been positive; from othera the ,t* culture8 have been pereietent ly negative; whllo still othar individuals, who at firet w8r8 negative, later became carriers, and vice versa. Invest iga- tiona were made to chow whether the cultivation of the organlem from the throat might depend upon some Incidental factor; for instance, whether cul- turea might be poritive and culturea mad8 a few hours later might be negative. The 8vidence 80 far obtained indicates that thie la not the caee, but lndi- cates that the technique smployed may be relied upon to 8hOW whether or not a perron la a carrier of influonra bacilli. Inveetigationr were also made to determine from what part of the naso-pharynx the org8niems were moat roadlly Isolated, or most frequently encountersi. In a setlee of about 9C individual8 three culturea were made from the throat - on8 culture from each toneil and one from the posterior wall of the pharynx. In five Individuals poritive cul- turer w8r8 obtain8d on plater from all three boUrCe8. In five individuals the culture8 from the pharynx and from one or the other tonsil wera positive. In 13 Individual8 only'the culture from the pharynx showed a growth of lnflu- . enra bacilli. In no ca8o wer8 the organism8 ieolatod f rom'the toneil and `no;" ' from `the phar&. -' . I \ _ i :' In the'e'e 90~lndividu;le,`~~~srofore, poeitivo culture8 wet8 c : :; A-4 oIkin;d ' f;om 23 or & per*csnt , % 5': 3 * 2` .a .wnaga ,*,.:*, \. ;;' __I_. * , ,i:,* L. s .a&.. and the result8 show that the organism8 were 1. * - ? ; .., ,. %, , b n&i `likely'.t6 b$ `8nc&uatered ; `i &?the * z` y' : .,.y ,I :. " ( / . 1. 1 postarlir wall of the pharynx. Cones- que*tli"~~i~.&;~, o&.~r"~~~t;;r';~ `:a;;; y,; ;b;e.g;;ions wer8 mad8, and in r .: . . ..f . I .: : ,::fi:`. `;`A. ;, ., . i' < .I :". The i : 1 ., :...;: 8tUdi88 concerning the relation;of the inhalation ,:. ' . j. of duet to the development-of.pneumonia in guinea pigs are being continu8d. A rearrangement and collect& of all th'e pathological material accumul&ed eince the opening of the hospital is baing'made. Card cataloguee are being prepared which will facilitate the use of this material. , , -84 Dr. A, R. Dochet and Dr. 0. T. Avery. During the past three monthr, in collaboration rith Dr. Cullen, a study has been made of the final hydrogm ion concentra- tion of pneumococcus cultured. Brevioua work by Dr. Avery on the fermsn- tativs activities of pneumococcl of the different types In media containing variour carbohydratea, iadioated that nd correlation oxlstod between the biochemical activities and antigsnic propertier of pneumococcus,- it being found that no biologic t7pe of this otganirm rpeclficall~ fermented any particular oarbohydrat e. Sinoo the final and optlmuk hydrogen ion concen- trationr for growth of bacterial race8 bra apparently an definite as thoso for omyme action, it resmed derirablo to fix these valuer for the diff8rsnt typos of pnrunlococcu8. In o preriour rtudf by D8mby and Atory it wa8 found that the gDt!.mm hydrogen ion concentration for growth of pnoumococcur ir a pH raluo of 7.8, and the application of this faot in the adjustment of tioatsd on artificial media for yeara, and other8 of which were removed but one or two tranafarr from active dieease pFOCO8808, all reached the 8aam final hy- . dragon ion ooncontration in 1 per cent doxtroeo broth, i.o. pH 1 5. Thi8 i8 precieely the point which limits the growth of pathogenic hsmolytic etreptocooci of human origin, when grown under eimilar conditionta. It has bettn found further that when pneumococci of different types wore grown in media containing other fermentable carbohp - drstee such aa lactoeo, eaocharoro, maltoeo, galactoee, rvffinoae and inulin, that the same final hydrogen ion concentration wae reached by all typoe. As far aa teated no type of pneumoco ccue hae been found to split mannit, arabinoas, or rhamnole. Uork ir being continued on the use In culture media of soap8 of the uluaturated fatty acfde, such a8 sodium oleate. It scmned deeirablo to dotsrmino the relation of the degree of unsaturotion to the growth aoo818rating propertie of roape for certain Gram negativs organ- isme, sepoci~lly B. influensao, ao well a8 the relation of unraturation to the bactericidal acrtion of 8Oap8 for pqoumoooaai and stroptooocci. To thie end Dr. Cullen ha8 undertaken the chemical analyrie of the various sampler of sodium oleate which are being wed in thor8 bactoriologioal oxporimonto. It hae beon found that puro oloic acid in concentration of 1:lOO In culture medium, the reaction of which hao been roadjurtod, permit8 the influenza baoillua to grow luxuriourly, while growth of pn8umococcur and etroptococcue 18 restrained at .a dilution of 1:SOOO in. solid medium and in fluid medium. .' _ s _. . ,. The initial growth-i8 $qhibited in, dilutions of oloato.as high a8 l:lOO,OOO. : It is of intrr@rt,thrt a ,cultur8 of 8,' influearae:grown .r, ip,e~eate brythiL, i, * :r 1:500 was-; found vi&lo, &terv,$hroer month8 @j.$ncubation at,.37'.. . I1.j ., , .I .i d J, 1 !3 4. i! ;5 t j: :: ::j : 3 '$ Q !j " ,( . :. ~ .`> ;;.In working with medium to which hi&or conoentra- ., :. h `2. i tions of oleatr are added .it ha8 been found errrential to d8fsrmino the rrac- tion of the medium, rince ionitation of the roap tend8 to unduly incroa88 alkalinity and inhibit growth. ,I Study of Hsmol~ ic Streptococci. $inCO our b8t r8pOrt, rtudy Of the antigenie relationship of etraine of homolytic streptococcus hae been continued. The result of thir work hae been to confirm and extend the information already acquired, 80 that now it appear6 that certain dif feront typo8 of thi8 organism o xi8t and that these type8 can bo 8harply differentiated one fromthe other by moanne of agglutination and protection reactions. In all 141 rtrainr have boon rtudied and of there, 96 or approximately 70 per cant, fall into definite typo8 and 45 are a8 yet &uXI&8t#ified. ThO 96 olurifiable 8troin8 COmpri80 811 typO8; tyP0 "8 3' COmi8ting Of 28 8traine, type "S 23" of a0 rtrainm, type "8 60" of 28 rtraine, type *S 84" of 9 8train8, type o s 32" of 5 o troinr, and typo "S 273a of 6 8train8. Study of the oroo8 immune reaction6 of the firrt four typos hae boon oompl8t8d and that of the la8t two typss ita in proceee of invsetigation. . -* 1. The immune 8era ured in the &gglutination and pro- tection te8tl), were obtained by the immunisatlon of rabbit8, 8he8p and dog8. The animal8 were inoculated intravenourly with repeat8d do808 of heat-killed Organi8m8, and in mart inrtances a COrkkin number of do808 of living organ- illpI.were givixw:: It is puettiOnkbla whsther'the tire of iiving &ganirm8 ' 18 088Ontial to the prodbbtibn,Ot. 1L hot&t--#Oi&.~~ ThO"108; Of `&rimal8.With ru#h..~mothod'eithe~fram ~ener~~Fn'ibaBion~o~~loc~~int~~ti~n ot"j6i~t'r'i8'" oonriderablei L ~~o:o~pSo~nt~~fo~~~~il~atio~`, of `Lfr66hly' irol&ed~~'$n~a68rd iluman rtrainr,`~~r~tho~~ee~of the 8~li~i~fainr~att~i.`~~rr~l'di ,`of ax&al'"'&& age8, do88 not .alter in~any~~bognira$l~ way'thw ipeoitia 'qaitiw8 of 'khbi'? 8WWn. ` _ i _ . . I 's. * ' . The agglutinia and proteotive3itre of the rera ha8 remained undiminirhed for maq~.mOnthr aftrr the time of blooding. Groat : . care 18 taken in the.preparation of the organirmr to bo used in the aggluti- nation reaction. The broth 18 made from carefully 8018cted meat, and instead of the usual sodium chloride a sufficient quantity of a balanced phoephate mixture is added to give the desired oalt concentration and to ad juet the hydrogen Ion concantrat ion to a pH 7.4. Experience hae taught ue that when no sugar ir added to the medlum,S. hemolyticus grown for 24 hours in ouch a culture fluid doer not develop an acidity greater than pH 7.2, which ie just above the point at which granulat Ion appeara. Tim organirma are removed from the culture medium by centrifugaliratlon and are washed once or twice In a broth of pH 7.4 prepared in the manner dercribed above. They are then resuspended in the mame medium in a concentrtrtion approximately that attained after 24 houra growth, and are ready for uao in the reaction. The aera to be employed in the teat are mcrde up to the varioue coaceatrationr by dilution with the same broth used for euepending the bacteria. The reaction of each specific eemn le controlled by a com- plete series of like dlultionr of normal mrum of the eamo animal. Equal quantit ler of mru.m dilution and bacterial ruspenrlon are added to each tube and the reactfonr are then placed at a temperature of 55' C. for one hour. A temperature of 55' met be accurately maintained during the period of incubation and the teat met be read after the lapee of one hour, eince ,- , if they are allowed to continue longer, non-rpecific granulation oacure. :, > . 1. fr- ,If,- alumping develops in the broth oontrk, o... or in , .,$l : ._ :: ,`. `3, ' ., `. :. / ,. . `; : I :..,. L,! ,.._ ; .,.,_,_j,, more than the first two or:hres dilutions of normal serum, the reaation is to - ~*.,(L i- I c .`T >. ._ ,_<". *.I ,: *:..a , ,.,S& be regarded as unsatisfactory and discarded. By the use of this technique -L ' ,. ,, .-, / !_i . Y ` 1.: ,I . - ,.' ., I : :. . _, I ?. p it haa -been poeaible to carrTJiout reliable agglutination ferrtr of 5. hemolyt- ., .&A. .L. b ..Q,.!.. ., +*. ..-3 )< ., .>, ,_ `,: &ii, : ..ir 1, .rl' t.iiJ.. . ..A ..C A ,., /L . L icur and :to show that oonrtant type relatlonshlpr exirt *&d that the various 1. .? * 4 . 1 \ I. z. ,, ,. ;: ., .i' ., I j.: .,., i ;: L ,- *,$ typer are sharply differentiated from one another. There is little or no `. L crow-agglutination of the varioue type8 in hsterologour aera, rveq in a con- centration of 1:20, Homologour sera agglutinate organisme of the @ame type . in dilution0 ranging from 1:1200 to 1:5000, and practically all strains of the mum type react to the same point. 88 Protect ion. Although the claeaification of S. hemolyticus by means of the agglutination react Ion has been presented f lret, we have actually obtained our primary Indications of antigenlc differences between strains by means of the reaction of protection. hater each `reaction has been used to confirm the information obtained by meana of the other. In the succeesful carrying out of protection experiments, two thinga are of erpec ial import ante; first, the production of a serum of high potency, and second, the possibility of raising the virulence of the teat strains of rtrep- toaoccue to such a point that very minute doses of culture are o ufficisnt to kill white mice In a limited period of time. The virulence of hemolytio. streptoaoccl for the ordinary laboratory animalr is low when compared with an organism like pneumococcur. Doses of one cubic oentimeter or more of a twenty-four hour broth culture admlulstered intraprritoneally are required to kill guinea pigs and rabbits. Furthermore, repeated passages through these animals fail to bring about a conalderable accession of virulence. The fatal does for white rats and mice is smaller, usually in the neighborhood of 0.1 cc. of a broth culture. It has been possible to raise the virulence of many rtrainr by continuous $aseage through white* mice and rats to' such a point ' a that dome-of- iro~:O:000001.i:c.;`to.`O.;00OOdd01'dc; oi `broth'cultuk at=6 auf-' ' " ' -. I : ., _ ( ' : .1 .a:, f'.. ,__ n& `hav;ybben;hbl;:to bducr';;&. `id'ih* ~-*g" h:; ' alluded to above of ,iuch',potency that'6.5 cc. administered intraperitoneally is auf f icient to protect a"white' mouse aiainat `one hundred `thousand. lethal ' doses of a highly virulent streptococcus. In order to produce such a serum many animals muet be wed; only a few of which may give the desired result. Theee seemingly difficult conditions must be attained in order that suffic- isntly `long-range protection tltere may be carried out to insure the relia- bility of the information obtained. Protection against one or two lethal doeee of a series of &rains of streptococcus by a monovalent merum ie aub- ject to such a variety of interpretations that the evidence so gained cannot be considsred of much value in judging accur;ltely of antigenic ralationehips of tha different attains. Tha technique obearvad in the protocols hae been as followa: Ths potency of all sera haa baen tsstad for the hcmologoue strain of organism and only euch sera utilized as gave a sufficiently wide rmge of protect ion to insure rispmdable results. For Infect ion virulent streptococci hava been used which have been grown for approximately eighteen hour8 in either plain broth or ascites broth. In the inoculation of animals, the tech- nique advised by Neufsld has bean followad with only a minor variation. The test animal8 have bean injected lntraparitoneally with 0.5 cc. of sar~m twenty- four hour8 before the conduction of the experiment. Tentative trials have shown that if ths merum Is given eimultaneously with the infecting dome, no protect ion results, and that to enaura 8uccesa the merum must be given at least eight hourr before infection. On the following day a series of viru- lence controls are Inoculated intraperitoneally and the serum animals injected in the same manner with doaea of culture ranging from 0.001 cc. to 0.00000001 cc. of broth culture. Animals surviving for a period of five days are considered to be adequately protected. By the use of thia method it has baen poreible to test the antigenic relationship of a considerable number of virulent straina :. of S. hemolytlcur. In every instance in whfch it has been poseible , to, carry s, I. ri. * out protection tests the aame type specificity has been manifest that waat;Tvi- .> , . . ,. -I... .' : ,.. _.. `_ 1 .I.( Thie work has cleared up a number of polntr about !. . .,, . . .i ; . ..3 s .: Str~ptocQCCue hemolyticue that' have been in dispute for many years. In the, . first place, S. hemolyticus is not a unit type as was previously SUppOSed, but probably coneiete of a number of types, at least six of which have been Identified. It has been pQ8alble to produce sera of considerably hig,her potency than any hitherto reported. Prevloua investigators have stated that freehly isolated human strains change their antigenic properties on animal paneagsn, and that the latter proce'dure developee what hi*% been called "animal virulence" and gives a common antigenic character to all strains. KS have fou&d no evidence to support this contenticn, in fact immune eera produced with human attains that kave never been passed through animals afford a high degree of protection against strains that have received many animal paeshges. In addition, the antigenic difference between strains of F. hemolyticus which have been passed through animals Is quite as distinct as that between strains which have not been so passed. It appears from this work that there is some rslation- ship between fermentat ive and ant igenic characters. All the members of type "S 60" ferment mannit, and' there are no mennit fermenters in any of the other groups. There are, howevar, two strains which ferment mannit which are un- classif led. By this work a method has been developed for the identification and classification of S. hemolyticus, and a satisfactory method has been found for the titration of antistreptococcus serum. Thus the way has been cleared for epidemiological investigation and for study of specific therapy and prophylaxis. B It,, wt.11 :bs& recrllgd that in, a previoue report attention was called to,work by Dr., Dochet iadicating.that antipneumococcus Immune serum i. ' caused some inhibition, of growth of the homologous ,organisms,; This sffect:was ' : correlated with, the so-called sntiblartic immunity. Later observation. by Dr. Blake throw eome.doubt on these conclusions and the matter has remained unset- tled. It seemed possible that the question might be definitely answered by using the technique developed by Dr. Barber for the isolation and growth of single cells. Y Dr. Barber has made an extended series of observations obrerving th8 division time and rate of growth of single pairs of pneumococci when placed in immune serum and under diverse other conditions. The conclurions from these observationa are: 1. In homologour oetum pneumococcus becomes invested with a thick capsule and tends to grow in chains or roogloea-like masees. 2. In hsterologous serum the growth is similar to that in broth. 3. Growth rate in homologous 88rum during earlier generations is apparently exactly the same a8 that in the heterologoue serum or normal horre serum. In connection with the experiments on pneumococcus the growth rate of thir organiam ram invertigated. In hanging drops when conditions are most favorable, the generation time ir approxFmately 30 minutes. #`here one pair or similar small sowings ars made into test tubes containing 5 to 10 cc. of medium, the rate is practiaally the oame, about 30 generations in 15 hours. From the time when the first appreciable cloudiness appears In a test tube until the tube ie distinctly cloudy, the rate lo alro 30 minutes. During the first half hour after isolation in a hanging drop one generation is formed and two during the first hour. Some experiments indicated that a somewhat more rapid growth (generation time 26 to 28 minutes) takes place in capillary tubes. This' observation will be conf inaed. Another phare in the growth of paeumococcus is being investigated, the lag, or in lone cas8s failure to grow, of sing18 pair8, or eiuilar small Iowings, mad8 into test tubes containing relatively large quanti- ties of broth, when controls' in hanging drops or on agar show growth. Twenty- eight to thirty series, including some 325 test tube cultures have been done. General summary of results: Of 8ome 325 transfers spproximately 50 per cent have grown. In some later 8xp8riments with plain broth nearly 90 per cent havs grown. A pair, or even a half-pair, transferred when actively growing, may continue growth in the test tube at an undiminiehed rate, at all events, may accomplish about 33 generations in 15 hours. Presence of serum increases probability of growth. .rently does not. Some series indicate that contaot with the sides or bottom of the test tube or with some solid substance promotes growth. Thir has not been definitely proved. The period of growth at which oellr for sowing are taken is undoubtedly an important factor. Washing in broth of cello f rou an old culture apparently promoter their chances of growth in test tube or hangin& drop. It is doubtful whether the quantity of fluid to which cells are transferred is in itself an important faotor. Growth has rucceeded further in 125 cc., where one pair was #own. This matter will be tested A with large quantities of medium. Dr. D. D. Van Slvke Improvazent in ths McLean-Van Slrks Blood Chloride Method. The micro-method of McLean and Van Slyke for the ;a tzzication of blood chloridee required thrse flltratione - one after coag- ulatior, of the proteins with heat and magnesium aulfate, a eecond after treatmact of the filtrate with charcoal, and a third after precipitation of the zhlorids rith standard rllver nitrate. In the f lnal filtrate the excess :Jilvcr was determined by our iodometric titration. A modification of the method war made neceeeary by ;..a lack of :hlorlde-free magnesium rulfate and charcoal during the war. With the assletance of Lt. Jamer Dor.leavy, one of :l G ir.ot ruztorr in the U. $3. A. clam in clinloal chemistry, the necessity : 5r tksee reagents was obviated, and at the admb time the three filtration8 i5er8 condone&d into one. By adding picrlo acid to the standard ellver nitrate a eolutiot nae obtained which precipitator both the protein8 and the chlorides of the plaema`simultansourl~. The detrxmlnatlon ir therefore eiaplified to a einglr filtration, yd tit'mtlon of the filtrate. Method for Tit@loo- of Osanic Acids In UrlaQ. ' ' While Dr. Pa'lmsr raa on the etaf f a method for a burette until a pH of 2.7 ii reached. At pH - 8 all organic acid. occurring ir. urine are in the form of ralte, at pH 'I 2.7 they are iron, 93 to 100 per cent free. The amount of HCl added to chingo the reaction' from pH I 8 to pH s 2.7 therefore Indicates approximately 96 per cent of the organic acids present. If ueak nitrogenous bases were present tky xould also be estimated in the titration. The only such base occurring in urine in signif icant amounts ie treat inine, ahich is included quantitatively in the titration. Consequently the organic acid titration is, for exact results, to be corrected by subtracting the amount of creatin preeent in tene of MflC solution. The total output of organic acids in a normal adult apraars to be 500 to 800 cc. of Y/l3 acid. In diabetic ketcnuria the output rises parallel with the acetone bodies, and may reach several t ties the nor::.al value. The titration is extremely simple and may be used as an approximate measure of the ketone excretion in diabetics when facilities are ,not present for the exact determination of the ketone substances. It anrears possible that information on several other problems may be gained with the met. cd. Titration Method for &asma Bicarbonatp. With Dr. Stlllman a& Dr. Culleq a method has beer. devised for determining ths plasma bicarbonate by simple titration. The results are quantitatively identical with and equal in accuracy to those ' obtained by determining'ths CO2 combining power of the plasma. The new method, . i' * / .* .- , 3 : ,, i j _ . 1 . <. ' -i although' `rends red &siblr only by the p#ysic+chemical studies on blood re&t ion i .i I_ -1 J>, , ; `: pub;ished'in the `iast few years, -`.*' . ,,.. A!..' `_`. I,,.. J' is ext rsmely rimpi' and requires `no * B"pei ial " ' , . >`d apparatw . The blood plasma (2 cc. ) is mixed in a relatively large flask with ._ hydroxide lie run in f&n a burette'until the"color equals that of a standard phosphate solution of pH 7.4, the reaction of the blood in viva. The use of this endpoint assures that the proteins bind only the ssme amounts of acid and alkali as in viva, and do not affect the accuracy of the titration. The difference bstreen the 5 cc. of B/59 HC1 added tc, the plaernrc and the 1/50 I'raOH required to titrate back to normal blood reaction repressnts the am0ur.t of acid nautralizad by the plasma bicarbonate. Tha titrat ioc *has an advan- tage over the gasometric CO2 mathod in that the titration requires no epecial apparatus, `although it la no' quicker, and does require accurate standard solutions of acid, alkali and phosphate. Study of the HemozloSin Challgpe in Pnerunonl~. Dr. Stadie ha3 added further obaarvatlons to his previous results, all confinring the latter in showing that s'ranosis in .Jn_e.u.- scnl~ is due to incouulata oxvgmtian o! thd arterial blood, and that a lon oxygen saturation of the arterial blood ia of prognostic Importance. tile Dr. Stsdie, for the study ofApleth3moglohiq question in pneumonia, hae also devised a method for determining methemoglobin, which, whlla not pet adequately tested, app'esre promieing. Tie unchanged hemoglobin is eetimated from the combining power of ths blood for oxygen, dotermined gaso- metrically. The unchanged hemoglobin plue me&hemoglobin ie estimated by trsat ing the blood first sith potaseium ferricyanide, which turns all the hemo- glob in t o methemoglob in, then with potassium cyanide, which tune all into cyan- hemoglobin. The latter is detsrained calorimetrically. The differenos botresn this value and the hemoglobin .eetimated. from the oxygen capacity represents tie methemoglobin. .it. _' : .. `A: I , ,.., . The Dietr:butlon of Arsenic in the Body after Intravenous'In~t'ctidn ' " t -.pf A 189. and the Yodo !and Rate,`of Excretioq. " .' This work, which was in pf-ogrees"at the time of the -.`,i "`- last resort, has been completed with Dr. Stadia. Tha following eummarizee tha rssults: 1. The ercrstion of A 189 in the urine of man, ae measured by the total areenic , is practically complete at the end of twenty to thirty days. 2. The total srcretion of arsenic in the fecee is equal to or greater t&n the excretion in the urine. 3. Arsenic ie excretsd in coneidernble quantitiee in the stomach. 4. In the excretion of arsenic after intravenous injection of A 109, rabtite reesmble man in tl:at they eliminate about equally through the kidneys and lntertinee and require about three reeks to free their bodies of the injected armonic, when the latter har been given in large amounts. When the araor.ic excretion ie complete, the arrenlc content of the organs of the rabbit ia no hlghor than In controls which have recieved no A 189. There Is LL~ JO- depoeition of arsenic in the tirruee. 5. In the blood, A 189 Is abort snt irely In the plasma and serum. Concentration In the blood falls off very rcr?idly after intravenoue inJection. At the and of an hour, the average concentrutlon ir only 2 to 10 mgme. of arsenic per liter. 6. No arsenic was found in the rplnal fluid of five treated came. 7. In the tisauas of rabbit8 injected with non-fatal doees of A 199, the highest concentration of arssnic is In the liver, the kidneys and mueclee coming next. The brain contained very little. ".. ; !* ,,> 8. t After dome of 50 mg. per kilo (sub-lethal) no difference was s i, ._ L .- ,. observed in the distribution of arsenic in the tissues, whether the A 189 was ,,( ' _ ._ -. a_ , . c i *, I . 'L Y . i 8. , s? iA . (r i _ : ,` X. : `+ 4. * : injected with d`ne or three moleculee of dodium hydroxide. 1 . .:* ! ..,, . : i ,. 4k.. .I &!&, .e 1 L i ..`,, .i :c .I i C' ,d 1 LA... ,:. , :. .i &.. " f f! 1 i 3w ,Q ; ,,,,,-9. Injection into a rabbit during two to five minute6 of 150 mgme. of .,a &.a .l.r w..-&9;it,,d+J$:, .1:.t,&?~.&T:* Ah&: ; +;.:s!, + ,! ,., ^ .;I 4 * b " ,I.. ..i.: "s31 A 189 `der kilo with one mol4cul4 of sodium hydroxide kille the rabbi;`;;& an . 2`. 6 `!$ L ` i, .., 9.k.c `a 3 I-t " .,. :A+! ., : . ..-15 h a i , .^ : *_ . ,, -. 8 1 ,. / `A;:; `, I : ". , ?' ,. : `..,<.t .`)" extresely high concentration of arrenic in ths lunge, apparently due to floc- `. il ) II , ,.. ` +i .=;' sir -.:i !.SC. !.b.k,.:l' ..i, .) * culation of A X9 in the pulmonary capillaries. In two experiments ths injec- :_ ._: . `. tion period was prolongad to 30 minutss, and the rabbita were uninjured. This i I. t - suggest6 that reduction of the alkali from 3 moloculee to 1 molecule does not _. . .-. `. .> increase the toxicity of the drug, excspt insofar ae it favors inmediate floc- culat ion in the capillaries. / : 10. Solutions of A 189, when mixed with" blood eerum or plasma, give rise to a precipitate over a wide range of dilutions. The eolubility of A 189 in serum ir such that a liter of raturated serum contains 0.35 mgm. of arsenic. The eolubilitp io much lore than that of salvaroan. When the drug ie mixed with a large volume of rerum the eolubillty is not affeoted by the amount of alkali (1 or 3 molsculer) with which it fe dissolved before mixing with the 11. Sinoe, after injection of A 189 in therapeutic doeeta, from 6 to 15 mg. ?? o mrnic per liter are found in the blood, the serum of which can dissolve only 0.35 mg. per liter, it seeme that the injected drug, unlers lmmediatrly altered to a more soluble product, must qirculats In the form of a suspension, a rslativ~ly small part being in true rolution. That the arsenical fraction at least ultimately doer alter into a more soluble product . lr indicated by the fact that the arsenic concentration in the urine may reach 10 mg. per liter. study of the Th-otic Effect of A 189 on Human SrDhLliq. Thir work was begun in January 1918 by Dr. Stillman. Dr. Stadlo joined the prbblem about three month* later. During the paet fall Capt. Klauder &Id Lt. Bortuine of the Sanitary Co&s were detailed to _ ,: ,_ .`A a,;:; r;;.:..!;b;: ` ::c :.,7$Ly) ,' ,i;;: ,,f "T: 1. Without rrsrawy. (. `( I .ri,, _<... , , *, 8 >,- ,;:. . ,..`. r I :t ,`; * .+ :, * a. 10 mg..`of A 189 per kilogram every 5 daya. ,,. Therapeutloall~ effiknt but abandoned for iess intensive treatment because ' of abdom1oal prim; b. 7.5 mg. of A 189 per kilogram every 7 days. : Therapeutically efficient, almoet no cramps; but 3 cases of exfoliative der- mat it ia. Therefore abandoned for the following: 2. With mercury. a. 7 mg. of A 189 per kllogrwa every 14 days, with mercury either as I$$12 Intramuscularly, or a0 metallic mercury Inunctions. No rerious toxic symptoms of any kind in 11 patiente treated. Raeeemnnn reaction disappeared after the admlnlrtration of but half as much A 189 (20-40 mg. per kilo.) as was required when A 189 WV given alone. ?wo cases during treatment developed mucous patches which cleared up on continuation of the same treatment . II. -tic ru . Eighty uasea of syphilla have been traated with A 189 until they were either free of lesions and Vassarmann reaction, or showed toxic rymptoma suffioient to make cessation of treatment advisable. Of the 80 oases, 21wen primary, 37 secondary, 3 tertiary, 8 Intent, and 11 . central nervous syphilis. Time did not pennlt a serloue attempt to `cure the central nervous cases, although 3 of them were curqd, both in symptome and in the Wassermann reaction of the spinal fluid. Of the 69 other oases the treatment was successful in 66, both I, Wassermann react ion ind lerionr disappearing. One primary case free f ram Wasoermann reaution at the beginning remained free. 2 Thi 3 `kiiidhg` &WS' failed to't&ate suf fioient A 189;to make ,. ..' .j them Rassumanbfr~~~' " .' . ._ T& tr&&nt ;br'i&pprd in one ease becausr of abdoa ! lnal crainps and blood'y `&ols; and in another because ofexaoerbation of an already existing retrobulbkr neurit is. The third failure to uomplete the treatment was due to exfoliative dermatitis which resulted fatally. It is a point of interest that thr 3 cases of tertiary ryphilis wers cured, by A 189 without mercury, of lesions and Wassermann reaction as rapidly and with as little drug (50 mg. per kilo.) as were the primary caees. Kith so few cases one cannot, of course, say whether such result8 may be expec- ted again or are exceptional responses. The reeulte above summarized were obtained while the optimum mode of admlnistration was being worked out. Consequently most of the caees repor- ted were not treated according to what preeent experience indicatea as the plan involving minimum toxic symptoms with efficient therapy. III. -ted mode of admlnistret. The results eummarired above under "Experiment s in Yode of Administration " indicate that the optimum treatment with A 189 probably lies betnoen Plan 1 b, which appeared too lnten- rive (occasional toxic symptoms) and Plan. 2 a, which appeared not intensive enough (occasional recurrence of lesions durlng treatment). It wae, there- fore, plannod to teat the following: 7 mg. of A lf39 per kilogram body weight every 10 days, the h 189 being prrpared for Intravenous inJection by solution in 1.00 molecule of FlaOH and 300 parts of 0.9 per cent sodium chloride solution. Simultan- eous treatment with meroury. went in the Van Slrke Method for the )Ivdrolvsie of PSqtelns. Miss Hiiler. \' This method was publiehed in 1911,` and has in the' `. past few years been much used in proloin analyeee, particular& `oi protein .' ' . . `, foods. The amino acids are divided into two -groups', the hex&b bases `and .' ' ' ' . . ,,( j. the mono-amino acids, by precipitation with phospho-tungstih `acid. T;e .: :. , I: 1 :: ~ bases, arginine, hist ldine, and lysine , are determined by methods which have ;-. : 1-L .:.: . ..a--- 1, 7. :.; ?, ! + .~ I .`.: , ,, 1.: . . .I The moqo-amino fraction is div%d' ;, Ji,m) stood the test of use satirfactorily. ;. I( .: . .:. _. II .i,, Into two sub-groups by deter&in&ion of t&l nitrogen an~"fh6;!baminb' nitGo- !. , .)S 4_. gen . The sub-group containing non-a&o nitrogen consists of the threb"&nino'~~' acids, proline, oxyproline, and tryptaphane. This non-amino nitrogen has b&n" determined by difference between the total and amino nitrogen of ths mono-amino fraction, and like all determinations by difference was subject to a summation s of errors. Hire Hillor has been working out a direct method to dioplacs the determination of thir group by dIff8renc8. The amino nitrogsn is destroyed by nitrous acid And the nitrour acid is gotten rid of by reduc- ing to ammonia with a rim-copper couple. After boiling off the ammonia the only nitrogen left la that of the non-amino type, and thir Is determined by Kjeldahl. The method has been developed rufficiently to give accurate rerulto with purs Amino acids, ard ha8 been applied to the analysis of gsla- tin and cAa8Ia with good resultr. Ylrr Hiller ir now working on a new method for the s of hI8tIdlrlQ In hydrolyzed proteins. The hsxone baaee, argi- nine, lycine, And hirtidlns am repsrated from the other amino acid8 by precip- itation with phoaphotung8tIc acid, and all the nitrogen, except the two non- amino nitrogen atoar of hI8tIdIne, Ir elimiaatad by treating firet with alkali, then with nitrour Acid, the rsridual nitrogen reprorenting the hlstine. The method ir not yet complete. PreDaration And Te8tinQ of DarPx)cholIc Acid ae the hOUmOCOCCU8- Pia80lvin~ &ibrtanc8 Qf Bill. _ Ml08 Hlllqr hA8 purif Ied by ,recryrtallirAtion a con- I. : , eiderable amount of dsroxycholic acid,..whtih hae been rtsted by:IIalr ,to be ., L ,!; the 8UbrtAnC8 rs8pOnriblO for the Ability of the bile to dI88OlVe pnrumococci. With Dr. AY8rx the 8Ub8tAKXA ir AbOUt to be teeted In regard to it8 8OlVAnt power for ~~~~IOCOCC~. ' : .; ,. ,m . `.= ,, rj & 7 c. i,`. it,. `. I .A VO8On a VblUnt@i& `l&88 i8ir A& "f rti "HO,,,; %A; - 1 extended thd work on God hiL80; to &bon mdnoxidhi We' h&e .found that .ths gaeometric method for blood oxygen prsviouoly worked out In thir laboratory &An be rucceuefully Applied t'b carbon monoxide without Any change except the addi- tion of a 8hort rtep at the bnd of'the determination. Carbon mon&ids And oxygen are extrActed And meaeured together,' Thbn the oxygen ie absorbed by letting 1 to 2 cc. of pprogAllol solution flow slowly Into the apfiaratue. The residual gas, after correction for the slight amount of nitrogen gas dis- solved by blood, ie meaeured as carbon monoxide. The method has given entirely reliable results for blood with all possible proportions of carbon monoxide . . . ..d oxyheraoglobine. Guinea pigs were found to become ucconecioua when approx- lsately 3/4 of their hemoglobin was changed to carbon monoxide hemoglobin. It appears to be the firat carbon monoxide method auf ficiently aiaple and accurate to make clinical application pOe8Ibls. Dr. Salvesen is about to apply this method in an attempt to Improve Haldane's technique for &eter&pIna the blood volume In afiimale and man. The rubject I8 allowed to abrorb Into hie blood a known baount of carbon monoxide gas, and the amount present per QC. of blood 18 Jetarmined. The result indioatee the total blood volume. Dr. Salveren also hopee to check up more accurately the experimente of Haldane on carbon monoxide AbeOrptIOn, which ions the eole baeie for the wxen wtloa the=, which portulatee that oxygen may be act Ively recreted by the lung8 into the blood, 80 that .the oxygen tension In the arterial blood may exceed that of the alveolar air. Krogh ha8 roverely damaged thir theory with hi8 tonometer experiments, but no one ha8 yet demon- strated any fallacy in Haldane' experimental ,relultl, or repeated them with,, , improved technique 8uch.8.8 rotid juotify either their rejection or their accep- ?? ? ? I returned to the Horpitd of The Rockefeller Inati- tuts on February 1, 1919, after an abeonce of, exactly one year. I , From.Feb- L .: ruary 1 to Marbh 12, 1918, I ~a8 OXA duty in the Office of the Surgeon Oensral. In thir post, the preliminary organitation of U. S. General Uoepital 89 engaged a large portion of my attention. On March 15 I railed to join the American Expeditionary Forcer. After conference8 in England, 1 became an assist-t of Major (later Brigadier General) Thaysr and bore the title of Senior Consultant in Cardio-Imacular Diaeaaea, A. E. F. Experience with a great many Invalided soldierr showed that the cardiac complaints, which it wae my duty to etudy and to eug- gset management for, depended not on a structural disease of the heart, but represented an etpfm8ion, the uaual expreeaion, of a peycho-neurotic or an anxiety state. To lllelt all caeea of cardiac complainta to an affection of thir nature is however incorrect. Improperly managed convalsecentr f ram acute infcctlour dlrsar@who complain in a rimllar faehlon are rufferiag frcm a different condition and require dlf ferent management. Thir lr alro trw of men who hare been obliged to undergo exertion beyond their ability to por- form work. They belong probably to the group of cama of "Wart Strain". There group8 were confused In the minds of medical officsrr and required study to reparate them rationally with a view to proper therapy. On the nature of these rariour affections, informa- tion wan gathered alro by Dr. Franclo 8. Peabody at the Lakewood Hospital. The experiencer and obrervatione of both of UB are now being collected, and ~111 be published u a book. We hope the book may ehow what advanuar have been made during the war of 1914-18 in understanding thimr rather compli- cated o ub ject, To think it rhould alro present whatever rerioue contributionr , I, _. ,,? . . ..i.* -; 1 _ 8 have been made by other obrerverr. ,.4. ;: In jeot'ing `the. D;;g A. l89'$miI &i+ In )ebt'iiiQ siilt' Solution' hib=tiii'cc&platad: Porty-orie `e~perim&itr"r~rb do&: .`( It ras'f'ijtid that 3O'mgm.'per'k~." oi' \" . rabbit'a'relijht' iste,'r'ithoui'effect on; thb heAiti'~f' four rabbiti'. ""`lhea'- 50 mgm. were injected, 9 of 14 gabbitr ahoweb'ti' altebation on'th6 mech&h~of the heart beat; one of there rabbit8 died. When 100 mgm. were injected, 9 " of 10 rabbit@ rhowed alterations; 9 of them rabbit@ died. One of there which ; died war.the one rhowing nb',effect on the heart.`. I I Injecting ralt solution (0.85 per cent) caured irrmgularitier in 2 of 7 rabbitm. There rabbit6 all recovered. When E/2 llaOH war added to the malt molution in thm mamm amount am im added when A 189 im prmmmnt, 4 of 6 rabbits showed changer In the heart. Onm of theme rabbltm died. The conclueionm drawn from theme experlmentm arm as followm: 1. A 189 is toxic for the heartm of rabbitm, when injmctmd at the rate of 50 mgm. per kilo. of rabbit'8 weight. 2. Rnbbitm arm killed when 100 mgm. per kilo. of body weight arm. injmctmd. 3. The adminimtrat ion of the drug im followed by a variety of cardiac irregularitlsm. 4. The injmotioa of malt molution ad of malt o olution to which N/2 BaOH ham been added may be followed by cardiac irregularities. II. Km have made daily eleatrocardiogramo of a nrurber of aqimalm, Into which Dr. Hoguchi ham iajectmd the mpirochaete of yellow fever. It ham been obmervsd that in patients the heart rats in thim diemame is low. Electrocardiograma have been made primarily to obtain ourvmm of the rate in the experimental animala, and to ascertain the mechanlmm of the brady- cardia, if prmrent. III. Uith the cooperation of Hajor General Shankr it ham been pommiblr to mtudy 200 o oldlerm, ohimfly of the Infantry, who have served overream. So far 143 o oldimrm have been examined. A phymical exam- ination 18 made beforo and after a standard exercire teat; an eleotrocardio- gram is taken in the rmcumbmnt and in the erect poritionm, together with the curve of rempiration; and an X-ray plate is made at two gletsrm, In inspiration, the breathing being normal. A certain number of platem will be made in expira- tion am well. It im expected that information will be obtained on the follow- ing main pointe: . _I_l.-X1- .--.. -.--".-_I -- -.. -.. ._-.I .-.-_ `--~~-.--C.I a. The mire of the heart. b. The pomltion of the heart in the chart. c. The type of elsctrocardiogrmm ammociatmd with diffmrmncsm in alma and mhaps of the heart. d. The influence of rmmpiration on the rlmmtrocardiogram, account helng taken of the porltion of thm hmart in the cheat. m. The effect on the electrocardiogram of pomition, 1.6. of lying and standing. IV. Dr. Levy hao o tudied with Dr. Cullen thm methods of Frmmsrvlng GStrophanthin in solution. It ham been shown that thim drug deterioratmm in potency if mtsrllirmd in glamr containera. yimldlng alkali. This dmtsriorstion im accompanied by change in optical rotation. It ham been fotind that thim deterioration may be prevented by dimmoloiag the drug in a buffer mixture (phoephatmm) at the neutral point (pH 7.0). Dr. Levy lm almo engaged in mtudying the GStrophan- thin prepared by Jacob8 and Hmidelbergsr. So far it ham been mhown that thm act ion of t him drug im identical with that of GStrophanthin (Horck). Thim work ham been preliminary to further o tudiem on the pharmacology of strophan- thin.