l S?UD!ES ON THE METHOD OF EVALUATING DISINFECTANTS AS DBWSED BY WELCH AND HUNTER Thai: for the Degree of M. S. MECHIGAN STATE COLLEGE Alma MzIdI‘ed Sic“ I943 'JHESIS Acknowledgment I wish to express my appreciation to Doctor W. L. Iv‘allmann for his helpful suggestions and guidance in this study. STUDIES N THE EETHOD CF 3 I" ATIIIu DISIIJFECT. ‘T‘l‘S AS 133V I 3233 BY K'CLCH AID LIFTER by Alma Mildred §3911 A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SC 131103 Department 0 f Bacteriology 191+} Eh £5 a 5 ‘ ‘ OUTLINE 03" 001723323 I. Historical review of methods used in evaluating germicides. A. __I_I_1_ zi__t_r_o_ methods. 3. in 11.32. methods. II. Object of study. III. Experimental. A. Constituents of the test. B. Procedure. ~ 1. Method for determination of germicidal action. a. Results of the germicidal action on human blood. ‘3. Results of the germicidal action on guinea pig blood. 2 Method for determination of toxic action. a. Experiments on standardization. ‘ (1) Preparation of antigen. (2) Length of incubation period. as (3) Concentration of red blood cells. b. Results of the toxic action on human blood. 3. Determination of toxicity indices. )4 Experiments with guinea pig blood. I V. Summary. V. References. I. Historical Review of Methods Used in Evaluating Germicides Very soon after the germ theory of disease had been definitely sub- stantiated, some efforts were made toward evaluating the action of germ- icides on bacteria. These early methods were crude. and they made no attempt at determinations on a quantitative basis. As the science of bacteriology developed, a variety of methods for evaluating germicides were introduced, all of which endeavored to evaluate the results on a definite mathematical basis. These methods may be classified into two groups, the i3 vitro and $2.1133 methods. A. 'lg Vitro Kethods The first i; vitro method introduced was the phenol coefficient method of Rideal-?alher in 1903. (l) Lbdifications of this method were develOped y different workers, of which the one by Shippen, (2) was the most worthy of note. He combined.the advantageous features of the Rideal—Walker and the Hygienic aboratory methods. Later this method became known as the Food and Drug Administration method. (3) which is now official in the United States. The phenol coefficient compares the killing power of a germicide with that of phenol against a specific organise. many workers have used manometric methods for evaluating germicides, which measures the inhibition of the metabolic activity of a bacterial suspension by a germicide. Branham (h) modified some of these earlier methods, and placed them on a quantitative basis, using the inhibition of carbon dioxide production by yeasts as the criterion for germicidal action. 231;; (5) studied the inhibition of the oxygen consumption by o -I I f \ various drugs up n Tscbor*c.1e c011. Bronfenbrenner et a1 (0/ not only applied the inhieition of oxygen consumption to bacteria, but also to V‘ a '0 M _~_ 0 W. o ‘H “ o 1 , sumption of actively gzoming organisms is urodcrtional to tne bacterial [.40 content end_thet germici es in becterioetatic concentrations inhibit cell nmltinlication, but not rate of metabolism. 1 As modifications of in vitro methole were gradually intro uc ed, it to: soon realized that the methois did not duplicate the actual con— - . . '3‘ o s ‘ T r 1_ ‘n r . A ‘.- I Q ions under thich tee test was used. it Has ielt that grrmiciiss wz1cn a r‘ ‘ . A “ '. fi '1 .5 6 n - ‘— Vere 1~e1 internn‘l; or on acous ..... nbraie 1d ne tests Lore rcc1rztr as on their ability to kill bacteria. audeft German, Letbert and “ever fin. ‘ s o _: 4 _~ ‘1 I“O_‘O I :uchsbaud end 131001, 8‘ all used met-ols itilizing the 1nL1L1t1n, ec- junction with the killing action on bacteria. Salle (;} continued the work alcnj this line, deveIOping it into a procedure for letermir ir ; the 1est d1 lution of a germiciie required to prev at the growth of embryonic chicken heart tissue durinn MS in ours to the hi hest dilution required to kill the test organism after an expo— sure of 10 minutes. Ticoret ti Hall the smaller the toxicitv index, the more nearly perfect the germicide. Later, Selle standardized this pro— ceiure, (10) perf rmin; it in the presence of organic matter. ‘ In e.further attempt to simulate practical conditions, tne ability ocytic activity by germicimi c, was introduced b;r Ive, (11) who correlated this with the phenol coefficient performs d in the T858339 0f OTEPniC matter,ani with the intraiermal injection nca“"“l h i t. —r ‘~4 f \U of p oiucing necrosis site of inocdrtion. Yelch and Vunter, (13) and ifelch and Brexe (13) used the ratio of the highest dilution of germicide inhibiting pliagoc"tosis, to the highest dilution germicidal to the test organism, for determining the toxicity index. B. .gn'zigg Kethods Although the 22,2132 methods for testing germicides more readily simulate the actual conditions of use, not as much work has been done along this line as vith the in vi tro methods. This is probably due to the fact that a large number of animals are required for the work, thus entailing expense, and also to the fact that the conditions of an exper- iment are more difficult to control. The first method deve10ped for the determination of toxicit;r 01 germicioes bye mimal inoculation was intro- duced.by Halo (1“) in 1913, in an attempt to require manufacturers to use suitable poison labels on their products, and to create some stanng_ the toxicity of their products. The to xicity coeffic ient *4) or designatin is einressed es the ratio of the least fetal dose of the germicide, to J , , ,. .I the least fatal dose of 5p phenol, consiuer1ng the latter as lCCp effec- [lo Realizing that ;n vitro tests with Her cid.es give no iniice tion what they will accomplish in vivo, Birkhaug (15 ) studied the to vicity p... 0n]. histologic changes produced by netnphen, following intravenous in- jection in rabbits. Combining both the in vitro and in 1:13 methods, Simmons (16) conta:nineted the skin of mice with anthrax spores, applied the germicidal solution, er cised the dzin, and then inverted and inserted it in the wound. '4 ilture s were made from the local lesions and be eart blood of the animals that lied. Iun ;ester andI Cent f(17) utilized the criterion of the preventio of infection by s? in disinfectants, rather than the treatment after in— fection, as a basis for evaluating skin disinfectants. The tails of .' mice artificially infected and subsequently tzeated with variou dilu— tions of dis i111fectants were inserted in the peritoneal cavity. Lbrtal- 3..) f? '1" ; J 0) Ha nterpreted as prevention of infection, and protection as evi- dence of disinfection. - ffith the orig inal purpose of ieterminin; whether the fertile e3; could be used as a means of testing the inactive tin; eerct of germi— cides on viruses, Dunham (18) injected 7 day old chick embryos with se in 111 o germicides, and noted the day of death. 1ne mir inum lethal grams per ki103ram of boiy 1He 3ht which killed more tlan half of the embryos, compared very favorably with the minimum lethal dose for man. To test the actual theragweut1c value of germicides in 1113, wh1Mul measure the toxicity for both the tissre and for the test or30.nism si- multaneously, Green and.3irkeland (19) infected the chorio-allantoic membrane of cnich e:noryos , which were later subjected to various dilu- tions of germicides. The extent of bacterial growth in the surviving embryos determined by the agar plate method, was considered as indicative f infection, and the lack of bacterial growth in the sur— 0 of the degree vivin3 etmorros as indicative of the effectiveness of the germicide in treatin3 infected tissue. II. Object of Study Althoufih the procedure for evaluating 3ermi ciies by means of the .dienol co efficient (3) is still st dard, the var'ety of results obtained on the same compound by different woncers, the inability of this method to test ger.'1iciies chemically unrelated to t11e phenolicUompounds, and to test the 3ermicides under actual conditions of use, sho ow the inadejuacy of this method. Dissatisfaction is evidenced b;r the msny attemnts to introduce otha~procedures for the testing of germicidal compounds. As most of the £2.21X2 methods require a large number of animals and often special equipment, a study of one of these methods was not undertaken. The method.presented by Welch and Hunter (12} is definitely an in vitro method, yet at the same time it involves the use of a liv- ing tissue, blood. which plays a vital part in in 1113 experiments with wound disinfection. With the use of blood, not only is organic matter furnished, in the presence of which all germicides should be tested, but also leucocytes, which are one of the important defense mechanisms of the body against infection. Because this method.could be reproduced in the average laboratory with regard to techniques and equipment, it was studied in an attemnt to determine its feasibility as a routine procedure. It was also studied to ascertain the difficulties that would be encountered in its perform— ance, the modifications, if any, that could be suggested, and also to determine if results comparable to that of other workers could be obtained. III. Eqperimental A. Constituents of the Test giggd. Five ml. of blood was withdrawn aseptically from the vein of apparently healthy individuals into a test tube containing .8 ml. of sterile 20% sodium citrate in .85fi salt solution. After inverting the tube several times to insure proper mixing, each 5 ml. of blood was di- luted to 20 ml. with sterile .85fi salt solution. Guinea pig blood was withdrawn aseptically from.the heart without anaesthesia, and collected in the same manner as the human blood. All blood was used within 3~5 hours after collection. Ormanism. F. D. A. strain to. 209 of Staphylococcus anreus (a strain .“m J' _‘ of standard resistance) was used.throu nout the experiment. Before use, '- . \——' it was transferred daily for at least 3 days, at 2H hour intervals. .45- Eylture medium. Due to the present unavailability of some of the con- stituents of the standard broth (3), the dehydrated Bacto Idsinfectant Test Hedium of the Difco Laboratories was used. This medium consists of the following ingredients: Proteose peptone, Difco 10 grrrs Bacto—beef extract 3 " Bacto—lactose 5 " Sodium chloride 2 " Ascorbic acid .25 " Distilled water 1,CCO ml. Final pH is 7, after autoclaving from 15-90 minutes at 15 pounds pressure. The medium was tubed in 10 ml. amounts. Antigen Staphylococcus anreus, (F. D. A. strain 30. 209) was grown in Kolle flasks on standard nutrient gar of pH 7.2 for MS hours at 37°C. ter smears were made from the growth in each flask to insure the purity of the growth, about 30 ml. of sterile .85fl salt solution was added to each flask, and the growth washed off by a gentle rotary motion. The washings were pooled, measured, and treated with an equal volume of a fresh sterile sensitizing chemical compound, which had been dissolved in .855 salt solution. This treated.suspension was incubated in an Er- lenmeyer flan: at 37°C. for from 2-3 hours. The flask was sh.ken at 15 minute intervals to insure thorough contact of the chemical with the organisms. At the end of that time, the mixture was centrifuged.under aseptic conditions, the supernatant fluid decanted, and the bacterial sediment was then suspended in sterile .85} salt solution, filtered through sterile cotton to remove any clumps, and diluted to give a final concentration of l by the Gates nephelometer. éntiseptics: Four germicides were used in the experiment. Ddlution of Stock pH Kama ___32£2210n . Lanufacturer Sodium orthophenylphenate 1:100 aqueous 501’ 11.15 Dow Chemical Co. ution Phemerol 1:1'000 aqueous 7.5 Parke, Davis Co. solution Tincture of mercresin 131'000 acetone- 6.25 Upjohn Co. alcohol solution 1319000 acetone— 9.6 Eli Lillv & 30. alcohol solution “ __. -- Tincture of merthiolete Sodium orthophenylphenate and.phemerol wenemade up in distilled water from the dry powder. The tinctures of mercresin and of merthio- late were used in the concentrations in which they are sold in the open market. The pH of all the disinfectants was determined by the Beckman pH meter. The chemical composition of sodium orthophenylphenate is self- evident. Phemerol is one of the quarternary ammonium compounds. tincture of mercresin is composed of a mixture of .1; orthohydroxyl phenyl mer- curic chloride and..lfi secondary amyl tricresols in an acetone-alcohol vehicle of 10% acetone and 50% alcohol, and tincture of merthiolate is a 1:1,000 dilution of sodium ethyl mercuri-thiosalicylate in an acetone- alcohol vehicle of 10% acetone and 50¢ alcohol. All of the dilutions of the germicides were made in sterile .85fi salt solution. Staining Solution. A,l£ solution of methylene blue in absolute alcohol was used for staining the blood smears. A.buffer solution with a pH of 7.2 was made by mixing 50 ml. of m/5 KE2P04 and 35 ml. of m/ XaCH, and diluting to 200 ml. with distilled water. B. Procedure 1. Method for Determination of Germicidal Action After preliminary dilutions of the germicides had been made in sterile -g- .85fi salt solution to determine their germicidal range, dilutions were made up in sterile .85w salt solution in suohvariables as to insure an accurate end.point. Two-tenths of a ml. of each dilution was pipetted into sterile Kahn test tubes, and brought up to a temperature of 37°C in a water bath. After the sterile blood had been collected and.diluted4 each ml. was mixed with .5 ml. of a 22-26 hour broth culture of Staphv- lococcus aureus (strain No. 209) after it had been filtered through a sterile cotton filter to remove any clumps. The culture-blood mixture was brought up to 37°C. in the water bath, and .3 ml. added aseptically to each tube of diluted germicide. This made the final concentration of 10%:for the whole blood. The diluted germicide and the infected blood were pipetted directly to the bottom of the tubes to avoid contamination of the walls, and to avoid loss by drying. Failure to observe these pre- cautions would introduce an error in the results. At the same time, positive and negative controls were set up, using infected and non-infected blood with sterile .85fl salt solution instead of the diluted germicide. After a 30 minute incubation period at this temgerature, with shaking at 10 minute intervals to insure uniform con— tact of the germicide with the blood, the tubes were removed from the bath. The tubes were again shaken to insure a uniform suspension and a H mm. loopful was removed from each tube, and transferred to tubes contain- ing 10 ml. of the Difco antiseptic broth. As phemerol and sodium ortho- phenylphenate proved to be non-bacteriostatic in action, only this ini- tial transfer was made. Hewever, with mercresin and merthiolate, a sec- ond broth tube was inoculated with four H mm. 100pfuls from the first broth tube (after mixing 3 times with a pipette) to counteract bacterio- static effects. growth made after 2% and MS hours' incubation. micidal action using human blood are presented in tables 1, 2, 3 and M, \‘D The tubes were incubated at 37°C, and observations of The results of the ger- while that with guinea.pig blood is presented in tables 5, 5, 7 and 8. A study of the results in table 9 indicates that the germicidal values for human and guinea pig blood are entirely comparable. Tdflel Germicidal Action of Sodium Crthophenylphenate for Staphylococcg§_Aureus Using Human Blood Growth After MS Hours' Incubation Series No. Final Dilutions of Antiseptic 250 375 500 625 750 875 1000 1125 1250 1375 1500 1 - - - - T T T T T T T 2 - - - - T T T T T T + 3 a - - T T T T T T T T h — — - - - T T T T T + 5 - - - - - T T T T T T 6 - - T T T T T T T T + 7 ~ - - - - T T T T T + 8 - - - u - - T T T T T 9 _ - - _ - T T T T T T 10 - - - - T T T T T T T Average final germicidal dilution is 1:662. -10... .mmmm“H mu soflpdafie ”IMHOfl.tmm Hague mmmcmsq T T T T T T T T T I I I I I I I I I OH T T T T T T T T T I T T T T I I I I m T T T T T T T T I I I I I I I I I I w T T T T T T T T T T I I + I I I I I N T T T T T T T T I I T I I I I I I I m T T T T T + T T T T T T T T I I I I m T T T T + I + T T T I I T I I I I I z T T T T + T T T T T T I I T I I I I m T T T T T T T T T T T I I I T I I I m T T T T T T T T T T T T I I I I I I H ooom ones ooma omma oooa gnaw comm ommm ooom ompm comm ommm ooom owe: come 0mm: coo: omen oflpmmwfiqu co mcoapsfien Henna . : newpmpfiocH .mhfiom m: nwpwd spyon¢ or cooam nmadm means. mfimnz4 monOOOthmcpm now HoswEmam mo coapod Hedwow1nmw r N mame .ommm “a ma moapSHfid an In stam muwpmhq 11 or I C) 'd L 0 I‘ -15- I + + + -r + + + .r + I I I I I I I I I»... hdjnoomm + II I. II I. II I It I.- Iv In I. - I. II I. II II kronvfh.ron OH + + + L. I. I + + + I I I. + I I I I I has noomm m If ' u‘ I | - II II II ' ' II II ' ' - ' I I \ooHaluahHfi-‘H m LI I. ' II II II I. 0. II I II I- I. In I. II II II txvaIFqui Hng . .r + L. .r r .r .r _. .r r .r .r I I I I I I I thdfloomm m If Ir ' ' II I f - A - II II l II II II .Iu ' I '0 knoHIMWUIHoer + + + I + + + + + I I I I I I I I I xnz;soomm + + + f + f f + k + I I I I I I I I hhfidfioomm M L. + .r .r + .r + .r + .r I I I I .I I I I WIJGWHJONJW N + L. .r f f .r .r _ + .r L. .r + + I I I I I hHfiflGoomm H \I < \I \I I \ \ IIIHII . . _\I \ \I. II \ ooow omNN oour can» ooow OUNm comm cum, uoov .INI oauu ummu goon om»: den; omozbooo: QDNN III. .III I o 0.1.1 oapPom a»-.d wO «soap dado HmdfiI mpHaHraampm mmHMnm soapupfiocH .mI5bm w: nmpmd.dpaoho dooam mam mmqfisa wcfiwb msmpnq mfioooooahxmunm pom afimmhonflg mo mpzpoqfla mo noapod Hafifiofiauww defia -12- .OOmm ”H m“ QOfipdflfid Hmvfiofiapmm Hwafim onshohq . _ . I I I _ I I I + I I I I I I + + I F + + I + I I I I I I I + I + I I I I I I I I I + I I I + I I + I r I I + I I I I I I I I + I I I I I + \ ,_ .. III nah ooow QmNm_oomw + + + F f + F + F F F f I A F a + hhfiflqoomm OH I r I I I I I + + I I + I I _ I hhfiflwhm + I I I _ I I + I I + + f + + I hhmfifloomm m I I I I I + + I + + I I I I I hhmfiwmm _ a I + + + + I _ I + I I + + I I I _ + bhdmfioomm I I + I I H + I + + r I I r I _ I hhdflflhm w _ _ a .3 I I I I I a + I + + + I I I I I m IHCdQOomm I I + I I _ I I + + I + I I I I H hhduwhm N I + _ I + I I I I + I I I + + I anchomm I I + I I m I I I I + + I I I I mumdaooom m + I + + + u I + I + + b I I I I flfimfifihm . + I + + + w I + + + + I I f + I ‘ hhflflfloomm I I I + I ‘ I I I I + + I + + I . hhcdcoomm w _ . . + + + I I b I I I + + I I + I I _ Ihdwnoomm I + I + I I ~ I I I I I I I I I I m hpdhfipm o w I I I I + I I I + + + I + I I W hhmwnoomm _ H IIII%II9I H _ x. . -. W \ .. A. . J1 .. I L. . .\. \ \ gmwm‘ouom omnm oouu Lama—Doom.om~n com: me4 coo: o-~y oomMAOmmm OOOM omfim.x I - a .01 ofiwpmwflpQ4 mo mmofipfiflwn Hmnfih mpqwarqupa nmadmm c or :oflprSUHH .mhdom m: ampwd guzoyo dooam cmadm magma wdmhnfl M&UQOQOHmnmJum now mpmaowapgmn %o mumpoqwm mo nofipod addfiofifihoo +~ magma TdfleS Germicidal Action of Soiium OrthoPhenylphenel for StFDhif lococcus Aureus USiLg Guinea Pig Blood Growth After MS Houre' Incubation Series 30. Final Dilutions of Antiseptic 250 375 500 625 750 875 1000 1125 1250 1375 1500 1 - - - - - - - I I I + 2 - - ~ — .. .. I I + I + 3 - _ _ 1 I + 1 I I I I h — — _ - - - I I I I I 5 - - - - - I i I I + I 6 - - - - - - + + I I + 7 - - - _ I I I I I I I 8 - — - - I - I I I + + 9 .. .. .. - .. - I I + I I 10 - - - - — .. .I. + I ‘1' I Average final germicidal dilution is l: 800. -16- .003 "H v 3034,),wa Hfimfiowspmu H94.“ mmagmfia II III.“ ‘1 II. ljfi'0.|lj I I I + + I I + I I + + I I hhmddoomm ) 4 + 1.! IT I. ' I. J. 1 ‘ i ' II OI II L.” 'HImwM:J-..rH. C r + I I + I I I I I + I I I I hhdfifloomm I I + + I I I I I I I I I I mndmuooom + I I I I I I I + I + I I I hhadfioomm N .r I... I I .I I. I. I I I I. I .I I \..H Hmwfidhm I I I I I I I I I I I I I I mhww: omm o + I I .. + .. .. .. .. - - .. .. I .131 a, m I I I I I I I I I I I + I I I mhmcnoomm \ If If ' + + '0 0. ll II I II II ' II L‘H )QllwuanJ-r IL I I I I I I I I + I I I I I .hhdagHooom + I I + 4. r 1 .1 + + I I + I \thdfioomm \. I I I I I I I I I I + I I I hhd H 0mm 4 I I I I I I I I I I I I I I :Hmw Qooom H I I I I I I I I I I I I I I m wawhm \\ _\.-- \ . _\I _\ J \\. )J\ \. \I J \I. ouwm uouu QIIL ooou ouI: eon: OIIJ coo: omR 001 _c Sm m ooow Ouwo oouc p O 0-- I) . .. I. II I I 03.1.1. 344:.- ...I141.VUII. Fl UWprQUfipz4 arcap:HrH rcrvm mIIcr ongc mm.mpmm soapapfiumH .uHSou m4 pmpm4 £p3ohw dooam mam mmaado Hdamp mpg n4 4 I .I AIN CHHUUOLUPhrrprrUNm MOM muwHOHApmmm «o manpodfl E mo n0flpo4 H¢6fl0fi6u¢m -17- Table 9 Comparison of Germicidal Values with Human and Guinea Pig Blood Germicide Human BloodTauinea Pig Blood Sodium orthOpherwlphenofi, 1:652 1:3CO . Phemerol 1:5 25 1:5675 Tincture of merthiolate 1:2600 < 1:2500 Tincture of mercresin 1:M550 1:J300 Examination of the tables on germicidal action discloses that neither growth nor killing action follows in consecutive order according to the dilutions, but is int,-spersed occasionally by irregularities. This may be explained in the following manner (10). It is known that the volume of a u mm. loop is .005 ml. 'Let us assume that the particular germicide we are investigating is that of phemerol in a l/2,200 dilution. In the germicidal test as actually performed. this gives a final dilution of 1/5,SCO.' If one leapful or .005 m1. of this l/5,500 dilution of phemerol were inoculated into 10 ml. of broth. the final dilution of phemerol in the broth would be 5%9 x 5,500 or a dilu- tion of l/ll,OC0,000. If a minority of organisms registed the 1/5,EOC dilution of phemerol, their likelihood of survival in the broth would be very great, as the final concentration of phemerol wouli be so dilute as to be non-germicidal. Let us consiier the fate of the bacteria in this particular dilution of phemerol. Suppose the number of bacteria present in 1/10 ml. of broth 5 culture is 5 x 10 . This diluted in the germicidal test would give a final dilution of l x 105 organi ems. If 99;? of these organisms were killed by 'qu, -15- etvw3cik. l a resistant minority of 103 organisms would still be left. One loopfu would contain .005 x 1C3 or 5 bacteria.per ml. As this number would be added to 10 ml. of broth, the final dilution of org iisms in the broth would be .5 bacteria per ml. According to the laws of chance, the possibility of transferring this number of organisms each time a locpful was transferred, as well as the possibility of such a reduced number growing in the broth, is quite remote. Due to this factor in the critical killing dilutions of a germ- icide, the appearance of irregularities in the growth of consecutive di— lutions can be reaiily explained. In those cases where a secondary trans- fer is made to overcome bacteriostatic action, the likelihood of trans— ferring a few viable org_nisms is still further reduced. To insure the constancy of action of the staphylococcus strain that was used, its resistance to phenol was determined according to the stand~ ard procedure (3} each time the test was performed. The results of these tests are shown in Table 10. Even though this strain was supposed to show constant resistance, nevertheless there was considerable variation. At times the gmwth in the broth was quite floccular, showing a reversion to the rough form. This deviation in resistance would naturally influence the germicidal end.point, which is one of the drawbacks to any method employing the :henol coefficient or a modification of it. waever. when the results were computed, this was taken into consideration, as those that deviated too markedly from the normal were eliminated. n . ,.. - \ .‘ , D. A. Dtrain n0. ng/ to 5H Phenol v 1" _. ‘ "' ... 0% Time ijccure to 9p Phenol “....w 5 mins. 13 wins. 15 wins. o y ... — a. 10;, + .. ... 110 + r + 110 + 1 + 1C0 — - - 1‘0 + - - 90 - - - 100 - - _ ll '3' c- .— or :v - - " 100 + - - GO _. .5 - 110 + + + 90 - - - lCC + - - 113 + - - go .. .. .. 1C3 + _ - .1 r4 0 + I I 90 + - - 100 + - _ 110 + _ _ lCC + + + 110 + T + 90 + - - lCO + - _ 110 + - ,. ——-— ...... — ALO-—--———.———-i 2. method for Determination of Toxic Action After preliminary dilutions of the germicides had been made in sterile physiological salt solution to determine their toxic range, dilutions were made up in sterile physiological salt solution in such variables as to insure an accurate enl point. One-tenth ml. of each dilution was pipettei directly into the bottom of a sterile Kahn test tube, followed by the addition of .2 ml. of standardized antigen. This mixture was brought up to 37°C in a water bath, after wnich .2 ml. of diluted blood (previously warmed to 37°C) was added. This made the final concentration of whole blood at 10%. At the same time, a control tube was set up, using sterile .SEfi salt solution instead of the dilution of germicide. After an incubation period of 30 minutes at this temperature, with shaking at 5 minute intervals to insure uniform contact of the germicide and antigen 'ith the blood, the tubes were renovet.from the water bath. By means of a Wright capillary pipette, a 1r0p of the suspension was removed from the bottom of each test tube, and smeared on clean grease- free glass slides, according to the usual technique for making blood smears. Drying of the slides was facilitated bv means of an electric fan. The smears were stained for 1 minute with 15 drOps of the methylene blue so- lution, followed by the addition of twice the amount of buffer solution in mixing the solution on the slides, air was blown :25 for M minutes T) ai on them several tines. The slides were then washed in tap water, anl allowed to dry by draining. In each of the critical dilutions, the number of staphylococci phago- cytized by the polymorphonuclear leucocytes were counted in each of 25 cells. Only those cells were counted in which it could be definitely determined that the organisms were phagocytized and not just lying on ration were f-ZJ ...-lo (3" (1.) top of th cells. Those cells showing evidence of elininated from .he count, unless all of the cells in filet part We H1 r dilution showed evid e'1-ce of disinteoration due to the toxicity of the germicide. The degree of phagocytosis is interpreted as follows: 30 organisms engulfed Abser.ce of ph eocytosis 0—20 " “ Sli :ht phab ocytosis 20—hC " " Hoderate " 20-MO " " barked " Bel ore adOpt the above technique, some experiments were set up in an attempt to obta n optimum and clear cut phagocytic action. This consisted in deterriinim ng the chemical mos t suitable for the prepare ation of the antigen, the length of the incu ation period. and the concentre tion of red blood cells. In these comparative tests, human blood was used errcl dsively throu ‘ o -.t a. Enperiments on Standardization \ o l) PreLaration of Antigen As St afhvloc cue agreus is one of the most resistant vegetative organisms, it is necessary to artificially opsonize it in order to induce phaéocytosis. One percent solutions of chromium potassium sulphate, ferric ammonium sulphate, and tannic acid were made up in .SSp salt so- lution, and sterilized by oas si1= thro ,3h 3 fritted Ml ass filter. Three 0 q -\ c". ll‘E’wS different lots of antigen were male, in which the Stash 'loc_cm“ suspension was treated with each of the above compounds. The tests were run according to the proceétre for determining toxic action, but with the use of . $5 salt Mlu ion instead of the dilutions of the germiciles. These results are shown in Table 11. Not only did the leucocytes phagocytize a greater numoer of stavly- lococci with the use of the chromium potassium sulphate tr ed antigen, but the organisms remained s>jarate and distinct, ani did not form in -21.. clumps, as with the ferrlc amnonium sulphate the fact that iuring the 2—3 hour paration of the antigen, sone oxilation took ferric i Effect on Cpsonization of (final concentration) by Three ncii greatel rntiQ' treated enti place, wi th This formed sucu a gelatinous conting around the 10; Human Blood Different Compounds incubation period reqnirei for the pre- the formation of or- frec trem of this yellow coating by ‘J r.’ (3&3 454 - 000 I _,'_ 6'5" ‘40 3o 7 C he 30 M0 3 no I ~ ~1~4~4 NNN-q NN-J-J-xl ‘oaiiEj ~q—q ~4—4 car” ?)() finmber of Organisms Phagocvtizei.per Cell Chromium Po- Tennic Ferric Ammo— tpssium Sulpiate Acii nium Sulphate 7M0 O O YEC O O i. x 7,0 7%0 2 730 C C 7E0 «O 12 w, ‘- "?'C ‘}c 6 ! 7&0 to 7ND \ 1C MO 740 ‘to 1 O \. OMWOOWOOOOO-{Z’OONKH -22... (2) Length of Incubation Period As all reactions necessitate a certain time period for the various constituents to be in contact with each other in order to bring the re— actions to completion, it \Jas thought advisable to perform the test for tox'c action'usin; incubation teriois o: 30, M5 and 60 minutes, with the The test was performed according to the previously des- L - I“ - -- three entibens. cribed technitde, with the use of nhvsiologicnl salt solution inst end of I! .) t1 e dilutions of germiciies, and with the different incubation per rio is es the only variant. These results are shown in T lee 12- Although the re was a slight increase in the number of ors mis sphar gocytized for the lon:er incuostion periods, it was felt thet the slightly increased numbers did not sufficiently effect the results to werr.nt the additional time involved in perfor. uin* t1 e test. Action of the three comp pounds in effecting phagocytic action, remained relatively the sexe with regard to each other, regardless of the time period. (3) Concentration of Red.Blood Gen As it wes found ov Velch and Brewer (13) that a final concentration of MOS red blood cells proved to be too much organic matter to test the ger i cidal activity of most germicides , they employed a final concentration of 105 red blood cells for the germicidal tests, and a final concentration ‘of hofi red blood cells for the toxicity tests (12). They stated that it was necessary to use different concentrations of red.blood cells in the two phases of the test, in ortier to obtein definite end.points for a greater number of germi1cides used. since toxicity indices are based on the ratio of toxic action to germicidal action, it seemed logical that the same final concentration of red blood cells should be used for both tests. In view of this fact, toxicity tests were set up in duplicate with various dilutions of the four germicides, using human rel.blood cells in n o o ’ ’ - \ '3: feet on Opsonizntion of 1C7; Pittman Blood (final Concentration; by Tires Different Cozqnounds at Three Different Incubation Periods Chromium J * ..-- Perri—E— “HT—”h“ tassiun_31lnhate- Taneic A311 , -Ammnn11 Sulnhn‘ - 3 mins. HE, rSins. 6’3 mine. EC mirs. 145 mins. ‘20 mins. 30 nine. 145 ruins. 8U nine. 7&0 7h0 7M0 0 0 720 0 1 0 7pc 15 a o 7ho 7h0 0 0 2 7ho 7hc 7&0 7M0 7&0 7h0 0 o 2 7nc 7N0 7&0 o 7M0 7:0 0 7M0 0 7M0 5 7&0 7h0 7&0 2 12 o 1 7&0 26 740 7&0 o 7ho 6 2 0 7h 7ND 7M0 7&0 7h0 7&0 7&0 C 0 10 7&0 7h0 7M0 7h0 <0 7h0 7:0 0 7kg 7to 7:0 7h0 10 19 o o 7M0 7M0 7M0 7M0 7h0 C 7&0 5 7M0 C 7ho 7h0 730 0 7H0 7ho 7 0 2 7M0 7h0 7W0 7M0 7H0 0 O 0 7L0 7L0 7&0 7¥0 0 0 0 18 0 0 7hc 7M0 7h0 7H0 20 u 0 o 7143 714:. 71:0 7150 7‘40 7’10 0 3 O fie we we fin 7M: 30 o 0 fin 7uo 7h0 7u0 10 7&0 o o 0 5 710 7to 7&0 30 35 7 0 0 O 7 7&0 13 0 30 7&0 o 1 0 7D0 7h0 ,MO 30 7h0 7h0 8 0 8 7h0 7h0 7h0 7h0 7h0 '7h0 0 0 7%0 7ho 7bc 7&0 7&0 7M0 0 c 0 0 730 YHO 7”0 8 7¥0 7h0 3 o 0 7M0 7t0 7N0 7ho 7M0 7M0 3 o 20 7&0 7&0 720 7h0 7&0 "*0 O 3 C 0 I! ‘ 4 I ’1 1 0 ‘ fl fir al concentretions 01 lbw and toe. Otherwise, the tests were per:ormed according to the 3reviousl;-.cr lesc Wii ed method for determining toxic action. These results ere shown in tables 13, 1h, lb and lb. Exenination of these tables shows that tde toxic end point as well as the point at which the germicide is no longer toxic, is much sh erper l with the use of the final concentration of lCo g numen red blood cells, than with the MC} red blood cells. This any oe explto inei by the fact that the quantitJ of germicide and the n mber of organisms is constant, but with a greater number of polynorphonucleer leucccyees in the h0£ blood cell suspension, here will Be a fewer number of oncteria phegm - CJtized per cell. Wi h the 40$ red.blood cell suspension, more organic netter is nresent, and iue to the e ester viscosity, the germicide is not brought into such direct contact with the cells, either due to the mechon ~ff FJ. O I.) H ( ct of inr3 roper mining, or due to the decreased amount of electro- (I) lyte in the suspension. In the more concentrated dilutions of the germi- n v es, the polyuiorph ionuclenr leucocytes are affected by the toxic action *5. {JJ of the germicide due to its extreme concentration, but in the more critical dilutions, the other effect s are more obvious. Tebl e 13 Effect of Final Concentrations of 10% and of M03 Human Blood Cells on Toxic Action of Sodium OrthOphenylphenate 103 Red Blood Cells [ [nos Red Blood Cells Final Dilution of Germiciie Con— Con- 500 1000 1500 2000 2500 3000 3500 n000 r01 500 1000 1500 2000 trol 0 20 0 7h0 710 710 710 35 0 7h0 7&0 0 38 7&0 30 700 700 700 0 7h0 7h0 7h0 0 0 30 23 7&0 710 710 5 710 730 ‘8 ‘8 0 7&0 32 30 710 720 7&0 %; 20 38 22 7u0 a q C 730 20 7&0 7&0 7h0 710 g: 25 28 7h0 to .9. .2 0 0 0 7110 7320 711.0 7140 .53 23 7110 7110 711.0 :5 1.3 1 10 71:4: 7110 7‘40 71:0 71:0 1;} no 0 71.0 7‘40 £3 t5 0 30 0 7&0 7M0 7H0 7h0 i5 no 7M0 7&0 700 3 i5 0 0 u s fin we 7M23 25 we fix flw .5 .53 28 15 O 71: 7110 7‘40 7110 fi 7% 35 7110 7’10 .3339 0 3 1m #0 flw W0 flm.§$ 0 E0 7“) 2 “§"%; 0 5 6 'wc an fin 7M>mfi 0 rm flm rm 39 .93 17 0 0 7110 7110 7‘40 730 88 38 12 16 7110 (1.53, 0,53 0 7110 35 7110 7310 7110 7?.20 of? 0 71:0 7110 7110 .53: £3,523 0 20 0 71.10 30 7110 7&0 3,11; 1 0 7110 7110 u ,, 0 7&0 1 7t 7M0 7b 710 .3 0 7h0 710 7h0 g 5 15 7‘10 0 7110 711.0 7310 7110 g; 22 71:0 71:0 7‘40 8 8 25 0 1:0 7140 71.10 7110 7110 8 8 0 7110 35 o O 0 0 0 35 7h0 7M0 7h0 (3 5 ho 7u0 7h0 +’ +’ 0 0 7b0 5 23 710 700 +’ 0 2 7h0 7h0 :3 :3 0 Lt 1 30 7110 7’40 71:0 ,9: 7140 1: 71m 7110 :3 :3 0 0 0 71.10 713.0 7110 7340 :2 7310 711C 71:0 7110 2; 3 26 0 35 71:0 35 7‘40 7110 :3 0 110 7110 711.0 0* 9 0 7’10 13 0 7110 7310 71:0 9 0 7110 71.10 710 £3 .3 0 32 38 7&0 71" 710 7‘40 .3 o 7’10 mo 7110 Table 1& Effect of Final Concentrations of 10% and of hOfi Human Blood Cells on Toxic Action of Tincture of Eercresin 10¢ Red Blood Cells MO; Red.Blooi Cells Final Efilution of Germicide 5000 10000 15000 20000 25000 5000 10000 15000 20000 25000 30000 0 0 35 7&0 7&0 1 & 0 7&0 16 7&0 0 0 0 7&0 7&0 0 3 0 20 23 7&0 0 0 0 7&0 7&0 0 0 L0 0 6 7&0 c 0 0 3 7&0 0 0 13 0 37 7&0 0 0 0 7& 7 20 5 3 7&0 2 7&0 0 0 c 7& 7&0 8 g 1 30 22 7&0 0 0 0 7&0 7&0 0 6 0 7&0 7&0 0 0 ,1 7&0 32 3 0 3 0 0 5 0 o 20 0 7&0 8 0 3 17 6 7&0 0 0 1 28 7&0 15 0 & 1& 36 7&0 0 0 & 7&0 7&0 10 3 7&0 0 7&0 7 0 0 2” 7&0 7&0 13 0 3 5 c 7&0 0 0 7 7&0 7&0 0 0 7&0 ' 12 &0 c 0 22 7&0 7&0 11 0 0 16 7&0 7&0 C 0 3 30 7&0 0 6 1 11 0 0 0 0 0 7&0 7&0 7 0 0 26 1 7&0 o 0 0 17 7&0 3 0 0 0 35 7&0 0 0 0 7&0 7&0 0 8 & 7&0 &0 6 0 0 7&0 7&0 0 1 0 1 9 ho 7&0 0 0 0 0 7&0 10 0 1 7&0 7&0 7&0 0 0 7&0 0 7:0 0 0 5 1 90 0 c 0 0 0 7&0 1 2 5 1 7&0 7&0 0 0 0 7&0 7“0 6 1 0 1 18 7&0 0 0 0 7&0 710 9 I: 0 7&0 71.0 7&0 0 0 25 7&0 7&0 2 1 0 9 35 7&0 -37- r.) 1.— A5 C... m. {1.8.11 ? ‘ Jerthiolate } l incture of MC .: m .1 E1001 Cells 9 3 CCALLPvccrPCP—CnCnLCCCPVCCCflLCfiCrs ‘1 ‘I. l. ‘ nix/L ‘1‘1 .‘-‘H._ Ah‘g‘“ \1 71717171171 71 7171.117. 7.117.17.1171717 717. .7.1....-. 1..ro3:..01C07-..-/03JL.CCmL.1..0 ”rt/.10 ...-07.0.0 1A.... A.L. 5L A.\uu.\11.u ILA-1.1.6. \113.-1..-.. r.._A L. A.....\- 7171 71 71L-.7 -71..1 #1 71 C 7oayn~ Avw ~371Aw Ad «linunUC OLA.~OCAUCC\H #1. Age Ml...- q. .J‘ ..k\.. \L \.L 1‘ILL. ‘1 1+ L141 ‘. \IL \11.\11u ‘1 1.— \1..~. 71 71117171 7171 71 717171 71 7- 7; EJOOCCAVO A....VCCCFVOYCOYPLJqu/I—n/AVOO 71/ \1». \11.T\H4.\11..\..1LT fifiJA/b 71. ‘111 A..... 7.J\.».\IL1 TCOCO TJKJO LQLOFvaoOA AUO 300m. 0.. a; 1.1... Maw 91 214111.19 14.14 MWMW 1.-.»- 111. flv AW 0 1 90v PU 7J1 7../C 716 2/0 C C rh/Oh O C 30 A; 1.1» 12 .2 2 3 .L..) 14111 1412 7 7 7 O «:7... 971C 030 10 O 2 30C 2 2 9300 114 2 P. 11 11 111. 7.1.. 0000OCOOlCOAULLOOOOIOOCOOOAV 0000OOCOOOPVAvOflunJflvOOCOOOOOO lls on Tonic P" n A u Effect of Final Concentration of lCfi ani M09 lood m _;1 Colls 10;:Red Flood Dilution of Germicile Final 0SusieOflvCAbChLOA-LOAUOOrNC wcmwnmCmLVh I: o. L1 ... . . ‘If IJl‘H 1‘11 \H. ‘1‘. ‘1‘ ‘1 7 77417741777: 77LIT7L177$W7741 00FA....OPNUCYOlflVAVCCAVflFUOCLAUPLALCAL LU, \H.‘ \IL. Alf." ‘ILT ‘11. ‘ \1 \.-.. 7J‘1L1. ‘IL 1*‘t‘1u‘l1w1‘avf1 77$1 7;! 71 7 7177- 77717 7177;17 0021120:JrOOFCO/bnwco:mehw717COO 9.211 134 7:... 1L 71 71 71 7171 Ger-CKODYJHDAJMH l.JrUAV~vALOC71.O/OOA./1 C/PU 7. 71 71 COCOCIO2200014001LOOCCFJOO7JOO Table 16 ’1 N L V. (‘U' - . Viv ‘43) AIL‘q ‘1 of N I“‘ L . :Lemerol 32'1 O '- n1-) ‘OVJLL -= . -11 3' Blood Cells on To O C O 0 2 O COCOOCB. LlOCOOCrOan C1111..uCO 1m l C 11-11 1A1.-1... 1. 1111.1 -..L.1.....-..:1..-1-1O-.1.l.11 Law 7- 717171 17- 77171711? 71717. 71771 71 ma L C lCCCCnLSCIJZZL-MLCM.nw 811.1 n-LoLlCnLCC C 1 11 1... C 71 7 77 71 7.1.17.1 7 O ..L. M-L.Wunu200_n-LCCOOOMJCOOOOLULJOOlO“JO 1L _ C 71.... nu nw nu mu n. nu nu nu nL nw nu nu «u nw nm nu nu nu nL nu mL . nu Au 0 nu n1). ML. \|1\HL ‘1. ‘1LT \.|H ‘. \l ‘1»? 1.1 1‘11 ‘11”. ‘11”. \1LL \1-L.1\ ‘ LN..- ‘.|.1‘1H..ol 0 7.717171 7-./”17.7 711711717171 7177171717171 71 r; «I. C CCQITCCCR- OnLCCCCLDCLIOrDC1 00 AU \ILf. ‘1.1-1‘1.1. \L ‘L \1U-‘Ha1 1‘1. ..‘d ‘L ‘5 1*1‘1L2‘lL 1th”; ‘11. Mu.71 7117 7 71717171 71717- 1717.71 71 71 7171 -L a...” 0. AL “ nu nu nu nu nu nu nL nu nu nu nu nL nu nu nu nu nu nu nu nL nu nu nu nu nu L...— .-.-L-.. b. Results of the Toxic Action on Human Blood -oxicity tests were performed with the four 5ermicides, with the aloyted moiif‘1 cations. The results shown in t.hles 17 , 13, 13, 20 and 21 ere based on the overa5e of the results obtained on five series of tests with each germicide. The toxic end.point was selected as the lowest dilution of the germicide in which all pha.ocytic action w sinhibited. In those cases m} ere one or two cells showed less than five organisms a: pha5ocytized, those dilutions were also considered as toxic. as that slight amount of phagocytoy s was co W11 red ne5lifl ble. The non-toxic end point was selected as the highest dilution of the germicide in which over 70§ of the cells showed marked phagocytosis. The de5ree to which ph -agocytosis takes place may be interpreted in the following manner (11). In those dilutions in which pha5ocytosis was completely inhibited, the leucocytes vere killed immediately by the germicide due to its extreme concentration, so that the normal function of the leucocyte was not possible. In those dilutions in which phagocy- tosis \as slight , the killing action of the germicide did not take place immediately, so that pha5ocytic action we initiated. However, after a her time fine 5 ernicide be5an to ac, and st0pped further phagocytic activity by the leucocyte. In those dilutions in which phagocytosis was marked. file germicide had been diluted to the point uhere it no lon :er interfered with the nor 0.1 phagocytic activity of the cell (20). Complete inhibition of phagocytosis annot be interpreted as complete destruction of the cell, as this state can also be due merely to loss of function. From a study of table 21, it rill De note i tiat there is a HrrIel variation in ghagocytic activity of the controls, from 55-32” of the cells showin5 marted pha 50 cyto 0813. Sometimes this decreased phagocytic activity 4.... did not affect the final results materially as in Series 2, but in some cas3s it affected the results markedly. rne do“ on vnich Series 3 was performed, a d‘plicate series was run, vit11 h ood fro m two civilials as the onl" Vsm sale fact or. The control on Series U showed tint 925 of the cells lispl.*e i ri.rr_ced phagocytosis. while the control on the drplicate series only shovel 52). This iecreased phagocytic activ- ity affectei the final results to such an e::tent th t the ese results hal to be discarded. Mi ce leucocytes a.re constan ly unierroinr destruction in the blcoi unier norral coniitions, he presence of a few cells in the control test: sho~inb comgmle eleck of p1agocytic activity couli easily be a tributed to this phenomenon. AJot‘ner ooservotion mafie while performing the toxicity tests, was that in the more concentrated dilutions of the germiciies, the red blood {.0 cells were lysed, r24 tr e leucocytes shoxei eviience of lisir mt egrntion. particularly in those dilutions that proved to be toxic. However with ‘ 75 he erol, a surface tension depressant, the leucocytes ani their nhago- cyti c activity were nna Hi.cted, even thor 11 coeguletion of the plasma, snl l,sis 3f the rel blood cells too : glece. -31... Toxicity Tests With Series 1 Series 2 Series 3 1250 1500 750 3250 3500 3750 1500 1750 2000 3250 3500 3750 hooo 500 1750 3500 , 0 2 0 710 7&0 7h0 0 is 0 7h0 0 7h0 7h0 0 30 7&0 o 0 16 710 30 1 0 0 7hc 7h0 0 7u0 u o 26 7L0 0 0 0 710 71 7h0 0 0 0 0 0 7h 0 0 1 7M0 0 0 26 o 0 7h0 0 0 0 0 7h0 o 7h0 0 0 0 0 o o 710 710 710 0 0 o 7u0 7&0 7M0 0 0 7L0 30 0 o 9 no 7h0 7u0 0 27 7h0 0 7h0 7n0 7h0 0 11 7&0 0: 0 15 33 7&0 7ho 0 0 2 7u0 7h0 7ho 7h0 0 0 710 0. 0 0 7&0 7h0 0 0 0 o 7&0 7M0 7h0 7h 0 u 0 0 15 35 0 he 0 0 23 0 7h 7h0 0 7M0 0 0 0 0 o 0 0 7h0 7M0 0 0 0 0 710 7h0 0 0 0 710 o 0 7h0 7&0 7M0 h 0 0 28 0 7&0 7&0 710 0 7&0 25 0 O 0 71:0 71:0 7110 0 C 0 O 7110 71;0 71: 5 0 7‘40 0 1 .7h0 710 0 7h 0 36 0 7h0 0 7ho 7h0 0 17 7h0 0 0 0 7110 7110 711.0 0 0 0 7140 7140 0 o 0 0 710 0 0 12 7&0 0 7h0 0 38 o 7h0 0 710 7h0 0 710 0 0 1 0 0 7h0 7hc o 0 30 710 7&0 7h0 7&0 0 3 0 o 0 h 7M0 7M0 710 o 0 12 0 7M0 0 0 0 19 710 0 0 0 0 7&0 2o 0 0 0 0 0 7h0 7&0 o 17 720 0 0 0 7&0 0 7u0 0 0 7h0 0 7h0 0 7u0 0 7&0 0 0 3 o 7M0 25 7h0 0 0 15 o 0 16 o 0 0 740 o c c 7h0 1 7ho 0 12 0 7ho 0 7h0 7h0 0 23 0 0 0 0 no 2 7&0 o 0 0 0 7M0 7h0 22 0 7%0 t 0 0 7 0 7h0 7ho 0 0 7h0 7h0 7&0 0 710 0 5 0 0 1 27 25 7&0 7ho 0 18 0 7&0 7h0 7M0 7&0 0 C 3 0 0 0 12 7M0 7h0 0 0 0 7&0 7 0 7M0 7h0 0 6 0 Toxic l: 1230 moxie l: 1500 Toxic 1' lfOC Eon~tcric l: 3750 Eon-toxic l: 3750 Ton-tn ic l: K,/CljcrchC7/.Cabcccccfi.CCCC1.C7J 5'1 +1r ' -. 'v V ovi m J. C [L e C ...-.. C w... .1.— y S W CCAVCOCCthntc.UCF.CCA..CCP..CCA»CAL 3.1. >17 Pv \ A MN. n~ no r» no m0 mw n0 n0 :0 mo me no my my C m. me. no mo n1. .0 mu .01. mo m1777777 7771ww wfiflrrrrflTYW 0 MW mw my mw my n00 Ono n» nu n0 n0 .3 mo mw no nu no ms nu nu mu no as no ‘1 \IL ‘1. ‘h \1; I ‘1... \Ilu 1 1 \VIA— \I.. my 71 17-7171 7.1 7.71777 71? 77.. C . . my OCanCOCCOOOCCCOCOW.«JOOOCCC \HI. \HH ‘1. \H. \1 \I. ‘1“..1 \I ‘L ‘1. \IIH."\1P. .ol._. 7-71 7.771717. 71w! 71 771 7. 717 717171 O .— C 1.53.0. 2037.11..- 05311 .15014 .{rn nun/011.3 C 11 .1. Ru 1|. 9. D m.“ Wk lO._OAVC7../O307JOOOVCCOO._hvol\u-fiullc F .... 9,. m. ...L o. 3 0 MW CnvCCOOPvOOn;CCh.COQ.OOAvOCh.CCnu O 1 flu C COOOCOOOA.COCCCCC CCCCCOOCC FJ .1. 11. \d. 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A..-V A... 04 ....-. n : 22500 toxic l Toxic l: 7500 $ 5 won-- 1:lCOCC cxic l ; 2COCO Toxic t -0 NO? Lie 1 12500 lutOXiC 1: 30,000 Tor H01 AL C A..-A flu _.A)A A..-.. 0 co P-C l a... C .... :0. «i n.“ O C t 3... a .L n 0.. 8'7 T C. A 9A fig 2.x n-C SJ .. l K O C t ...-A ~ .1... .... w a ... A .C O wAmA ”'— Ta‘ol e 21 Controls of Toxicity Tests vtized per Cell ‘I 3 u. — -. ' q 1 - juaber of OTOinSmS Paagcc Series 1 Series 2 Series 3 Series M Series 5 7&0 7ho 7uo 7M0 7L5 rm rm 7% 7m 7m 7&3 20 7&0 7u0 7&0 7&0 7 o 7ho 7hc 7uc 7ho 7&0 7&0 25 7‘40 0 711.0 7&0 7‘40 7M0 7uo 7M0 7M0 7uo 7M0 o 7hc o 7no 7M0 7ho 7M0 7ho 7ho 2 7uo o 7uc 7L0 7&0 7&0 7hc 7M0 3 7M0 0 7M 7M0 7M0 MO 730 7hc 7&0 7M0 7M0 C 7M0 7M0 7&0 7&0 7&0 2 7&0 7L0 71K) 713-0 73.10 7M0 O 7&0 27 720 o 7ho 7&0 o 7M0 7uo 7M0 o 7hc 7&0 7M0 7ho 7M0 7&0 o 7ho 7&0 7‘40 28 714-0 71; 71:0 7M0 7ne 7ND 7&0 o 7uo o no 7uo 7ND 7‘40 0 7&0 7‘40 7140 7uo 7uo 7&0 7uo 7ho 3. Determination of Caxicity Indices Using the same method for calculating toxicity indices as that pro- posed.by Selle, (10) the toxicity indices were calculated on the basis of the average results obtained in the germicidal and toxicity tests. Tcse {able 2° end hose chtnined.by Welsh and Eunter (12) ...... ‘A-l 3 ‘40 .‘5 results are srou and Welch and Brewer (13) in table 23. A study of these tables shows that the results obtained were very come parable and the amount of deviation was no greeter than that which would be expected in the hands of different workers. Although they did not rake any studies on phemerol, the results which they obtained on zephiran are comparable, since both are quarternary ammonium compounds. The slight differences obtained in the results with sodium orthOphenylphenate may be due to the differences in hydrogen ion concentration of the two solu» on ..40 ticns. They do not give the hydrogen ion concentration of the solut they used, while the one I used had a pH of 11.15. Some of the higher 3 orthophenols an insoluble in water, but are soluble in an excess of alkali. *3 (Cl) waever, an excess of 318111 reduces the as micidal power. e fact the 91 ei’icient germiciie is a comp (D C '0' Rd ,4 These tables emphasiz and pouni which is non-toxic in the dilution in which it is germicidal also the import? ce of using germiciues only in the particular dilution .Jo in which the" are germ cidal. If germiciies are used in the dilution in which there is absolutely no toxicity toward tissue, they are no longer germicidal. Toxicity J's/3 Germicidc :ermiciial Hon-toxic Toxicity Zfllntion Dilution Indéx Sodium Orthophenylphenete l: 1: 652 l: 3750 1.7 Phemerol 1: 1: 5325 1: 11200 . 2:6 Tincture of merthiolete l: l: ZDCO 1: 30500 h.h2 Tincture of mercresin l: l: #550 1: 30000 2.3 Toxicity Indices of Velch, Brewer and Hunter Sodium Orthophenylphenate Phemerol Tincture of merthiolate Tincture of mercresin T') 3130 t ( (no ) 8 )CQKNO r \ U.) i O O l4|d+4t4 <3 .0 O. O. O. N l—‘KJJKO II I! II II ieterminei 1. n n “. Experiments with Guinea Pig Blood As guinea pigs are cosy to handle end readily available laborator" 0 animals, studies were attempted using their blood, with a View toward solution. The tests were performed in every way as in the procedire out— lined for human blood, but in the srears of blood samples from three diff— erent guinea pigs, only from or e to two cells showed.phegocytosis, and Itized from two to five organisms. Since these samples of guinea pig blood showed so little phegocytic activity, it mes thoufiht t’nt thes eguinea pigs might be abnormal, so irmeiiately upon withlrawal of the blood from one of the pigs, 3 blood smear are made, ani stained with “Wiqht‘s stein. On the basis of 200 leucocytos counted the following differential was obtained. Dolfrorroonucl,ars 2C1 1W .Whocvtes 750 Ensinophiles 2R Mononuclears 2; hang? of the red blood cells showed r._e.r.ted enisocytosis and ooirilo- 0mg OCCOLI'nON-J“ Ci“ :kow-Qf fishy-row.“ oval fol Chem—r. Li‘.u,am& cytooiSJ/an occasional normoblest has found. l.any of the lymphocytes also showed an abnormelity in the form of an oval inclusion cell, which was sometimes smooth in alipeerance, and so.neti:nes so disintegrated that only the cell wall remained. L'SUM.11’ 01113 one body was founi in a cell, but occasionally several small ones were found. These bodies took the acid stain. As judged by the standaris for noraA a1 values of human blooli, thi (0 smear showed evidences of a pathological condition, both with regard to the leucocytes and the erythorooytes. This belief was further substanti- ated by the fact that all three guinea pigs died about one month later the leucooytes and the erythrocytes. This belief was further substantieted by the fact that all three guinea pigs died about one month later) Following that time interval, another guinea pig was bled, and efforts were again made toward performing the toxicity test. At this time 100 leucocytes were counted, of which 13% showed phagocytosis. The action was very weak, however, es 18% showed slight phegocytosis, while only one cell phagocyti7ed 3b organism. At the same time, another control was to (D . ~ . .. f . . t up in whicn the 1ncuboti-n periou was extended to o0 minutes. This ,I' ' time only 23 l of the cells showed phagocytosis, most of which showed only slight phagocytosis, and only he showed marked phagocytosis. As the nor- mal phagocytic activity of the cells was so low, it was not deemed advis- able to continue the toxicity tests using guinea pig blood. These results are not in agreement with those of Welch (22) who states that the average of tMaoontrol tests from ten normal guinea pigs showed that 565 of the cells exhibited marked_phagocytosis (730 organisms per cell). A blood smear was also made from this guinea pig, and on the basis of 200 cells counted; the following differential was obtained: r" Polymorphenuclears h2.jo .4 Lymphocytes 55-5fl Large mononuclears l p An occasional red.blood cell showed anisocytosis, aohromia, and poly- chronatophilia, and an occasional normoblast was seen. Esny of these lymphocytes also showed the presence of the inclusion cells. Klieneberger (23) gives the following average normal values ani the range of normal values for guinea pig blood: ! Kormnl Values tense of Values ,4 Eb 79}; 3.3.0. 5,270,000; w.3.c. Hbe3—107fi; 3.3.0. u,u30,ooo_ 15,000. 0,150,000; v.3.c. b.90o-13,hco.e _ fi‘d’ :fifferential Differential —~ r. f 'I :4 rolymorptonuclears 38.9 3 Polymorpnonuclears 9-M80 1‘7:th cvt e s 1443 . 5 5'5 1'3“th cy t e 3 33" S 8 [9 j ” 4 . . ’ Boeinophiles 13 W Boeinophiles l-lC; . ,. f , ,4 Lononucleors .5 p hononuclears 0-1.5o Besophiles .SSw Basophiles O—lp Transitionnls .be Transitionals .5u3p 1 4L... The blood findings of the normal guinea pig show (23) that the red blood cells exhibit considerable anisocytosis, frequent evidences of polychromstOphilia, an occasional stippled cell, and very rarely an erythroblest. The hemorlobin and red cell values are approximately the same as in human blood, but the leucocytes are slightly higher, with the lymphocytes in the majority. In the blood smear, lymphocytic cells are quite regularly found with eccentric nuclei and oval inclusion cells. In the same animal, cells of this type are found with a small nucleus and a large inclusion body, and cells with a large nucleus and a small inclusion body. It is not certain whether these cells belong to the lynphoid or myelocyte system, .nd whether this change in the size of the inclusion cells is a cycle in the development of the cell or not. ibwney (2h) states that a peculiar body, oval in shape, and sugges- tive of a phagocytized erythrocyte is found in the lymphocytes and mono- cytes of guinea pig blood. Usually one is present in a cell, and some- times several may occur. He ascribes the name of Kurloff bodies to these inclusion cells, and states that they have also been described in the lymphocytes of will Cavidae. -hl- As the above references show, the normal guinea pig blood deviates considerably, particularly in the differential count, and the inclusion bodies in the lymphocytic cells, and the evidences of anemia are normally present. It cannot be assumed then, that the guinea pigs I bled showed any pathological condition. However, these findings do not help to ex- plain the weak phagocytic action of guinea pig cells. Klieneberger (23) states that a thorough anatomical study, ad a study of the diet and liv- ing conditions would have to be made, in order to explain the frequent anemia found in the guinea pig, which is otherwise difficult to explain. If these are consilered as possible contributory factors to anemia in the guinea.pig, might not they also in some way affect the phagocytic activity? ' . Although Spink (25) et al in their experiments could not show that ascorbic acid influenced antibacterial activity, yet in a review of ex- periments performed by other workers, results were shown that the activi- ty of the complement was directly related to ptimal concentrations of as- corbic acid. The experimental work of haccari (25) and Ardy (27) offers further proof that the opsonic power of guinea pigs is increased by the injection of vitamin C, and that in scorbutic animals the opsonic power is almost zero. Velch (22) showed that a thermolabile substance in the complement of the guinea pig was an essential factor in producing phago- cytosis in the method for determining the toxicity of germicides. It is generally known that guinea pigs require vitamin C in their diet, for without it, they soon show evidences of nutritional diseases. On the basis of the evidence furnished, he conjecture could be made that the .0 . ' m; AUQ 17> iailure of guinea pig cells to show normal phagocytosis ie—aeedéé—te—eub— \...:<_. ‘v‘ urvw3~o C. HOWQVLA” Tod-TL)“ fad-00f ‘5 mQQAQA 'T; 50L..- g—TZN‘T’EAQ. TLJJs $1.; T -ua- Toxicity indices could not be calculated on guinea pig blood, as I was unable to perform tests showing the toxic action of germicides on guinea pig blood. -h}- IV. Summary The method of evalun in" germicides according to their toxicity Q indices, that is, the hirhest nossi blc germicidal efficiency with the least nos ible toxicity for human cells, is a meth ed that can be repro- duced in most laboratories with fairly consistent results. This is sub- stantia ted by the si ilarity in results obtained in this study and that obtained 7y "elch, Brewer, pni Hunter on the sane compounds. It reproduces to some extent the actual conditions encountered in wounds by testing germiciies in the presence of organic matter, blood, ‘v he'body, but is also an re 0 .533 Q; P“ b C!- which not only is a tiss‘e normally important defe.sive necharism against bacterial invasion by means of the glusé ocytic activity of t e leucocym . Other actual conditions simulated are the temperature (bo‘y ternperature, at which the test is performed, H) (1’ )‘J ..l (D ani tne use of physiological salt solution as a diluent for all 0 Actual conditions as they natu:ally occur are not sin ulated, for the dermi iies are not tested accoriin; to their internal action, on mucous surfaces. or on the other tlSSlleS of the body. However, by test- ing *ermicides according to their toxic aetion on one of the body tissues they are classified on an entirely different basis than when crassified solely by their phenol coe fficie nts s. as the toricity index is entirely independent of the phenol coe1fficient, the fox nor met} d of rating; places tation the use of aermicides. b". O .‘3 a: narrower lim 3v the retention of a modifiei.phencl coefficient method for testinb the ger rzicidal action, all t‘e deficiencies s of the phenol c3efficient "ethod are encountered. Among the most important of these deficiencies may be mentioned the lini ation of testing vita only a few organisms occurring in nat‘i rel infections, variability in resistance and nuMbers -1114. q of organisms, proportion of culture to u silifectant, inaccuracy of loop, inf 1nence of coraposition of medium on growth, testing 1n the absence of -.J organic matter, anl the inapplicability of use on compounis unrelated to phenol. Yith regard to the actual performance of the test, the en nd points for leternining toxic action are very snarp, ani fairly consistent, but of maki1? thu smears, staining, and counting the ingested L: the proces U) bacteria is not only time—coz.sumi 3, but very tedious. However, the use of slides furnishes a.permanent record which can be referred to ani c eckel at any time. The necessit" of havin-; freshly drawn human blood available, t is not constant 1n its nhazo3"tie abilit , are other J. L.- 13 ,‘Z‘ {1: 4. 9’ ;) ’ b ’1) 0 1+ + 3‘ ‘3 1 f H. : 01001 is available at any 1:) obstacles in this test. Although uinea Pi time, its weak phagocytic activity) anu the ii ents as coxpared to that of human blooi, lees not make its use feasible 1 for usternining toric action, or for use in simulating the activities 0 R 'I nd reactions of human blooi. The possibility of vitam1n v r1e1ficie ncy as an s:? lanation of use; phagocytic action, offers the opportunity for further investigation along this line. In conclusion, I believe that this method serves cnlv as a tentative in vitro method for classifying germicides on the basis of their gerniciial ani toxic effects, preoarrtory to testing then'by in vivo methols in ex- perimental and naturr 11 i1 fections.. \N f" -u'h J V. References T111991, S. and 217311321313 J. S. T1e 9111.11 1:11: ‘501 11 of 31 cinfs:ctr1nt1. JOLLI‘. 2103;. 5:111. 1113:“... 3.11113e11, L. P. A Fallacy in the 3131:;11311 1 ethois of: mxining Dis- infectants. 11.1.1. Jorr. P1111. Health, 18: 231, 13213. . A ‘H . (‘8‘ I, -1 A . . ’TV‘ ' L‘ ’ “‘1‘ "Q ‘ " d? r‘ ’l ‘ Paella, G. L. .1. 121111 gravel“, v. 1.. 1.1.111e11 mates 20011 11:11 4N1: .~..1- Dept. Agra Circ., 1.70. 193. 13-171. Brelfimm, Sara 3. 11m Izzrproved Techniaue for the Comparison of Anti— szebtics by Yeast Fer111entation. Jour. Infect. Dis... ’4sz 1112, 1929, ..11y, J. C. T113 Exraluetion of aer111c111. b;t-eI-Lez1o:netr1c f-iethod. I Bronfenorenner. J., .-ershe", A.. 3., 9.1111 Doubly, J. A. Eva]. 11211131011 of Gemniciies by e. .Lpo1.1etric laethol. Proc. Soc. Exp. Biol. 11.111 lied. , 38: 210, 1938. Greig, Largaret a. an;1 fibogerheiie, J. C. Evaluation of Germiciies 1.1;; 211.21 1or.1etr1c 21et11011. Jour. Beet” 1#1: 557, 1911].. 3:13.16, :1. J., and L323.“ “.15, A. S. A. Com :11‘15011 of the Resistance of Eatery-1, 9.11.1 23111111157011 0 Ti sue to Germicidzal Substances. froc. Soc. Ho (0 Cn- ‘1 ' ’1’ .12.; o £11101. 8.1%}. Feed. 32: 005 9 H K 1) \1 J 1'} 0 \ . ‘ Selle, A. J., 11.011119, II. A.. (111:1 Schec 11neister, I. L. A ll'ew liethogl ‘9 l «1 for the gvalxut 1011 of Germicizlal 311‘.uste11ces. Jour. Bact., 331- 1937. Selle, 15.. J., 211c0211ie, W. A.. Sche 11meister, 1. I... an". Foorrl. 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