WW I W — ,- _’ r:- — ,1— 1 l W W A COMPARATIVE STUDY OF THE GERMICIDAL PROPERTIES OF A SELECTED GROUP OF SKIN DISINFECTANTS Thesis for the Degree of M. S. MECHICAN STATE COLLEGE Edward Howard Armbruster I942 .12’,“ “null-.114... - I r... .. .1 , I . uphf‘wwffir n‘m'rmyrf‘w r1”: ‘ Elv'bmflcofl. 4. . 1 I n 0 . . . u \ U . ‘ . ‘ VI ... ‘x I .',..0. I . . . , I . .. . V l I )3 . ‘ . . . . . . ‘ ,. .... .a. |.: ”V0.-.“ .u..af i..pr Inn-.J.n1i . .. ~ . ~. ‘ . . .,. , . . . .... . . . . , v5 rpmdc‘v‘..fl . . Ppi.. .qu". ,2! wlo ‘11.". v 1! . A. v . J , . . Vr .. .. a. . . ..u . .. r.. . A ,.. .}.l.\Jv L. t V... ea. I . . .. . . . . o I1 ‘ .‘ ,_. - ... . t: .. . . . . IA . . , .7 . ~ C u l ,. “tr- . , . ‘ K . .. l u . . v . . f. < a . .x J. - . . A . o . . . . 4- .n . .1 . . .A . . ‘ g . D . c . . . h I. V I c b ‘ . . . v u. ‘a . u. . \ . . . . . a ACKNOWLEDGEMENT I wish to express my sincere appreciation to Dr. Mallmann for his many helpful suggestions and also, to Dr. L. T. Clark of Parke, Davis and Co. for his co-operation in making this thesis possible. A COEPARATIVE STUDY OF THE GERMICIDAL PROPERTIES OF A SELECTED GROUP OF SKIN DISINFECTANTS by EDWARD HOWARD ARMBRUSTER 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 SCIENCE Department of Bacteriology 1942 Ru wsw A COMPARATIVE STUDY OF THE GERMICIDAL PROPERTIES OF ‘ A SELECTED GROUP OF SKIN DISINFECTANTS INTRODUCTION Man had practiced disinfection long before he under- stood that such things as bacteria existed. Antony Van Leewenhoek was the man to discover the "wee small animal- cules" in 1683, but it took nearly 200 years before the in- vestigators realized the significance of this discovery. It remained for Koch and his method of pure culturing to bring the study out of its chaotic state into that of a science. Koch's experiments with silk threads impregnated with anthrax spores are well known to every student in the field of disinfection. In 1897 Kronig and Paul(1) published a new method by which it was possible to make a quantitative study of disinfection. They demonstrated that disinfection by a germicidal agent was not an instantaneous act, but rather a gradual process that followed an orderly sequence. The influence of temperature on the rate of disinfection was reported by these two men at this time. Rideal and Walker(2) introduced the use of phenol as a basis of comparison for a "carbolic acid coeflicient." This was probably the first attempt to devise a practical method of testing disinfectants because the conditions under which the tests were made were standardized and in doing so, many of the sources of error that had confused earlier workers were avoided. The present F. D. A. method of determining a phenol coefficient is an outgrowth of the Rideal-Walker method. \ 146112 The phenol coefficient has been used for many years as a measurement of the efficiency of most types of disinfectants without regard to their chemical composition or the appli- cation of the compound. Inasmuch as chemical composition varies widely and application may be of totally different natures, it is apparent that no one method of testing could be applicable to all compounds. In these studies, we were interested primarily in compounds used for skin and wound disinfection. The phenol coefficient seemed a poor method of evaluation, first, because the action must take place rapidly on the skin or wound so the time factor of 10 minute test period as used in the phenol coefficient is too long. Second, it has been customary to rate the phenol coefficient on the original concentration of the product as sold and since the strength of the various disinfectants vary one from the other, the phenol coefficient frequently has been high for compounds actually of poor quality as measured by killing preperties. Therefore, the problem resolved itself into one of seek- ing a new and more practical method of evaluating disinfec- tants which are used on skin and wounds in order to fairly compare all compounds. The method had to take into considera- tion the difference in the type of action of the various com- pounds, the difference in their reaction rates or ability to kill in short intervals, and the ability to penetrate into the bacterial cell to cause death of the organism as well as pene- tration into the wound to reach the organism. The compounds selected for this study are listed below -2- with their original concentrations as well as their source. ~ All compounds, with the exception of the Phemerols were purchased on the market and all compounds are listed in the dilution as sold. The source of each compound is also listed. *Phemerol 44 (tinct.) 1-1000 Parke, Davis & Co. *Phemerol 41 (tinct.) 1-500 Parke, Davis & Co. *Phemerol 45 (tinct.) 1-500 Parke, Davis & Co. *Phemerol (aqueous) 1-1000 Parke, Davis & Co. Hexylresorcinol S. T. 37 1-1000 Sharpe & Dohme Co. Iodine (tinct.) 1-40 Frank W. Kerr Co. Zepharin (tinct.) 1-1000 Alba Pharmaceutical Co.' Zepharin (aqueous) 1-1000 Alba Pharmaceutical Co. Nerourochrome 1-20 Frank W. Kerr Co. Mercresin (tinct.) 1-1000 Upjohn Co. Merthiolate (tinct.) 1-1000 Eli Lilly & Co. Merthiolate (aqueous) 1-1000 Eli Lilly & Co. Metaphen (tinct.) 1-200 Abbot Laboratories Metaphen (aqueous) 1-500 Abbot Laboratories Merphenyl Nitrate (aqueous) 1-1500 Hamilton Laboratories Merphenyl Borate (tinct.) 1-500 Hamilton Laboratories Phemerol and Zepharin were included as they are two rela- tively new compounds. Phemerol is designated chemically as a para-tertiary-octyl-phenyl-diethoxy-dimethyl-benzy1 ammonium chloride and Zepharin as alkyl-dimethyl-benzyl ammonium chlorides. *The Phemerols were supplied through the courtesy of Parke Davis & Co. -3- Since disinfectants must be active against a varied flora as is found on the skin, several types of organisms were used in this study. These organisms were: Eberthella typhosa Eberthella typhosa Parke, Davis Staphylococcus aureus Staphylococcus aureus Parke, Davis Diplococcus pneumoniae Parke, Davis Streptococcus hemolyticus Parke, Davis Pseudomonas aeruginosa Parke, Davis Michigan State & Michigan State & & & & College Co. College 00. Co. Co. 00. F. F. F. D. A.a D. A., D. A., D. A., Eberth. typhosa was chosen as it is the standard Gram negative test organism and Staph. aureus was chosen as it is the standard Gram positive test organism in the F. D. A. phenol coefficient.Dip._pneumoniae and Strep. hemolyticus were chosen as they represent two true pathogenic organisms. Pseud. aeruginosa was selected as the organism to represent the group of bacteria which are intermediates between Gram positives and Gram negative organisms. Both Parke, Davis & Co. and Michigan State College obtain- ed their original cultures of Staph. aureus and Eberth. typhosa from the Food and Drug Administration. I. KILLINC DILDTIONS The killing dilution of each disinfectant was obtained by using the F. D. A. Phenol Coeflicient Method as presented in the U. s. D. A. circular 198.”) With Strept. hemolth veal glucose infusion broth was used in place of the F. D. A. nutrient broth. This, however, was the only deviation from the standard procedure. Medication temperatures of 200 C and 37° C were used on all organisms to obtain the effect of temper- ature in disinfection by the various compounds. The Shippen modification(4) was used on the mercurials to eliminate the bacteriostatic action of the disinfectant. This consisted of making a second subculture in broth. To assure sufficient seeding, four loopfuls of broth from the primary subculture were transplanted to the secondary subculture. The results obtained are presented in Tables I-VI. The killing dilutions expressed are based on the compound as sold and not on the original dilution of the chemical agent in the preparation. The Phemerol group was the most germicidal of the select- ed group of compounds. At 20° C Phemerol tincture 44 (1-1000) was able to stand a dilution of l-5O times against §2§2§° aureus, 1-40 times against Eberth. typhosa, 1-2 times against Pseud. aeruginosa, 1-10 times against Strept. hemolyticus, and 1-60 times against Qip.!pneumoniae and still sterilize in less than 10 minutes. Zepharin which is closely related was slight- ly less active than Phemerol, but still it was superior in action to the remainder of the group of compounds tested. then Hexylresorcinolls. T, 37 (1-1000) was diluted more than 1-5, no evidence of killing action could be shown. Against Pseud. aeruginosa the compound could not stand any dilution and still sterilize in 10 minutes at 200 C. Tincture of Iodine (1-40) could stand a dilution of from 1-150 against Pseud. aeruginosa to 1-350 against Strept. hemolyticus. ‘ Mercresin was the most consistant germicidal agent among the mercurials exhibiting germicidal properties against all organisms in 10 minutes at 200 C in a diluted form. This was probably due to the addition of cresols to the compound to take care of the Gram positive organisms. Mercresin tincture (1-1000) could stand a dilution of l-lO against Staph. aureus, 1-70 against Eberth. typhosa, 1-30 against Pseud. aergginosa, 1-15 against Strept. hemolyticus, and 1-40 against Dip. ppeumoniafi. Merphenyl Borate tincture (1-500) showed no killing action against Strept. hemolyticus at 20° c and could stand only a dilution of 1-3 against Staph. aureus before its action was destroyed by dilution. When tested against the Gram negative Eberth. typhosa, this compound could be diluted 1-225 and still kill in 10 minutes. Mercurochrome (1-20) showed no evidence of kill against Strept. hemolyticus and it had to be used full strength against Staph. aureus. Against the Gram negative Eberth typhosa, this germicide exhibited most action, killing in 10 minutes at 20° 0 at a dilution of 1-50. - 5 - - agent hnosa in high '3’ §EEQE° Metaphen organisms in :st effective com- ~any of the three te period. Against d kill while against 11 if the solution showing fair activity 9E8, was ineffective «als, as a group, exert a Gram negative organisms y weakly effective against -erthiolates showed their :cteriostat than that of a :n subcultures show growth not when a Gram negative test the case when Gram positive would seem to indicate that be applied to the testing of est organisms and the high 7 - Aqueous Metaphen (1-500) was another selective agent which sterilized Eberth. typhosa and Pseud. aerpggnosa in high dilutions while being unable to kill Staph. aureus, Stggp. hemolyticus, and pip. pneumonigg'when diluted. Metaphen tincture (1-200) killed all Gram positive test organisms in a dilution of 1-2 in 10 minutes at 20° C. Aqueous Merthiolate (l-lOOO) was the least effective com- pound tested, showing absolutely no kill on any of the three Gram positive test organisms in the 10 minute period. Against Eberth. typhosa a dilution of 1-2 effected kill while against Pseud. aeruginosa it was impossible to kill if the solution were not used full strength. Merphenyl Nitrate (1-1500), while showing fair activity against the Gram negative test organisms, was ineffective against the Gram positives. These data show that the mercurials, as a group, exert a strong selective action against the Gram negative organisms while they remain ineffective or only weakly effective against the Gram positive organisms. The Merthiolates showed their action to be more like that of a bacteriostat than that of a germicide. In no instance did the Shippen subcultures show growth where the primary subculture did not when a Gram negative test organism was used. This was not the case when Gram positive test organisms were used. This would seem to indicate that the Shippen modification cannot be applied to the testing of mercurials with Gram negative test organisms and the high l-7- _values obtained by the method are due in part to bacteriostasis. It may be that the disinfectant is adsorbed on the organisms and held there very tightly. In this way the organisms would be prevented from multiplying but at the same time not be killed. If this hypothesis is true, the F. D. A. method can- not be used to compare the killing dilutions of mercurials and non-mercurials against Gram negative organisms. A second explanation for this failure of the Shippen modi- fication would be the possibility that the germicide kills the majority of the organisms and only a very few viable organisms are carried into the primary subculture. This would make it extremely improbable for the second subculture to receive any organisms from the primary, even by using four loopfuls as in transfering by the Shippen technique. This possibility was shown to be false in the rate reaction tests which follow. If these compounds were to be compared on the basis of the F. D. A. phenol coefficient, Iodine, instead of Phemerol would be considered the most desirable compound. The phenol coefficient of Iodine against Staph. aureus at 20° 0 is 2.9 and the phenol coefficient of Phemerol 44 under comparable conditions is 0.7. This would indicate falsely that Iodine is 4 times more active than Phemerol 44, while on the basis of effective dilution it can be seen that the Phemerol 44 is diluted 50,000 times and Iodine only 8000 times which makes Phemerol over 8 times more active as a germicidal agent. The mere fact that one manufacturer puts his compound up in a l-lOOO dilution while his competitor retails his at a - g - l-500_dilution is no criterion of the germicidal effective- ness or reserve strength of the compound. This is proven by the results against Strept. hemolyticus at 20° C where Merphenyl Borate (1-500) was unable to kill in 10 minutes while Phemerol 44 (1-EOQ could stand a dilution of 1-10 and still kill. A bactericide which is to be used in skin and tissue disinfection must be effective against all types of organisms in a diluted form if it is to be considered a good germicide. Mercurochrome, Metaphen, Merphenyl Nitrate, Merphenyl Borate, and Merthiolate do not possess this property as they are unable to kill Strept. hemolyticus in a diluted form. The only instance where a selective compound could be safely used is in the case of a specific instance where the type of organism causing the infection is known and a selective acting compound can then be used if it is effective. This is also the reason why a series of different types of organisms must be used for determining the germicidal properties of a compound. Killing in a diluted form is another vital factor in tissue disinfection. In a wound the compound may be diluted several times by the serum present. A compound like Merthiolate, while a good bacteriostat, may prevent further multiplication of the organism but it would have insufficient killing action when diluted with the serum and unless constantly applied, the infection could become active once more. When the temperature under which the killing dilution tests were made was raised to 37° 0, all compounds increased -9- in their activity with the exception of Iodine, which remain- ed at the same level as in the 20° C test. - lo - TABLE I THE KILLIKG DILUTIOHS OF VARIOUS DISIKFECTAWTS IN 10 MINUTES ON STAEHILCCOCCUS AUREUS (P. D. 02432 Disinfectant Orig. Conc. Killing Dilution 2ooc 31°C Phenol 1-70 1-80 Phemerol 44 (tinct.) 1-1000 1-50 1-130 Phemerol 41 (tinct.) 1'500 1'50 1'225 Phemerol 45 (tinct.) l-SOO 1-80 1-300 Phemerol (aqueous) 1'1000 1'50 1-150 Hexylresorcinol S. T. 37 1'1000 1’4 1'5 Iodine (tinct.) 1-40 1-200 1-200 Zepharin (tinct.) 1-1000 1-20 1-90 Zepharin (aqueous) 1-1000 l-20 l-90 iercurochrome l-2O l-l l-2 Mercresin (tinct.) 1-1000 1-10 1-10 Merthiolate (tinct.) 1-1000 l-l l-2 Merthiolate (aqueous) l-lOOO no kill“ l-l Metaphen (tinct.) 1-200 1-2 1-5 Metaphen (aqueous) 1-500 l-1 1-3 Merphenyl Nitrate (aqueous) 1-1500 no kill* l-l Merphenyl Borate (tinct.) 1-500 1-3 1-5 Shippen modification used on all compounds showing bacteriostatic properties. *No kill indicates original concentration failed to sterilize. - 11 - The literature reveals that discrepancies in the F. D.A. Phenol coefficient method have been observed in numerous instances. Among the authors reporting are Meyer and Gather- coal(5), and Vicher, Meyer, and Gathercoa1(6). They report- ed that they were unable to secure uniform results in the F. D. A. Phenol coefficient using Staph. aureus as the test organism. The latter authors report a study in which 19 strains of Staph. aureus exhibited a day to day variation in resistance. Table VI shows the difference in resistance of the F. D. A. test culture of Staph. aureus at 200 0. Both Michigan State College and Parke, Davis and 00. obtained their culture of the standard test organism from the Food and Drug Administra- tion. It can readily be seen that the college strain is much less resistant to all compounds tested. The Parke, Davis strain of Staph. aureus shows the Phemerol to be only 2/3 as effective as shown by the college strain, and the Zepharins are shown to be only 1/2 as effective whereas, on Phenol the resistance was approximately the same. Reddish(7) stated that weak cultures will give results different from those obtained with cultures of normal resistance as based on phenol. The question now arises as to which culture is to be considered as normal. Phenol cannot be relied on to determine normalcy of an organism in relation to its germi- cidal resistance unless the compounds are closely allied structurally to phenol. It was observed on Eberth. typhosa .L P. D. A. test culture obtained from the college laboratory, -17.. that the 10 minute killing dilution on phenol was 1-100 and the 10 minute killing dilution on aqueous Phemerol (1-1000) was 1-20. Several days later it was noticed that the same organism had become more resistant to phenol, requiring a 1-90 dilution to kill in 10 minutes. A check was made at this time on the aqueous Phemerol (l-lOOO) which should kill at 1-50. As the resistance of the organism increased toward phenol, it decreased in relation to Phemerol. This action was noted primarily on the Phemerols and the Zepharins. This is another indication that the Phenol coefficient can- not be applied to all types of disinfectants. Reddish(8) has stated that the Phenol coefficient should be confined to compounds closely allied to phenol and that unrelated com- pounds could not be compared with any degree of accuracy by this method. - lg - II. AGAR CUP PLATE METHOD Penetration must play a major factor in the destruction of organisms on the skin or in a wound. It is obvious that to kill an organism, the compound must first penetrate the cell to be an effective germicide, therefor the property of penetration must be determined for a comparative study of disinfectants. The Food and Drug Administration presents a procedure known as the Agar-cup-plate method for the measurement of penetration of germicides. In this study, he method as outlined by the U. S. D. A. circular 198 was slightly revised to obtain more accurate results. A flask containing 500 ml. of plain nutrient 1.5 per cent agar was seeded with 2.5 ml. of a 24 hour culture of the test organism and distributed aseptically in Petri dishes using approximately 40 ml. to each dish. When the agar had solidified, a layer of paraffin was poured over its surface so as to seal it from air. This was done to prevent errors resulting from the compound volatilizing into the atmosphere above the agar and later redissolving in the mois- ture on the surface of the agar. After the paraffin had solidified, a cup 1.5 cm. in diameter was cut aseptically from the center of the dish. This cup was then filled to the top of the paraffin level With SGGdéd agar in such a manner, as to seal the interface between the paraffin and the agar. In the center of this fill a cup 1 cm. in diameter was cut so as to leave a collar - 19 - of agar to prevent capillary seepage between the agar-paraffin interface. The bottom of the cup was sealed with a drop of agar to prevent capillary seepage at this point. Under these conditions, the penetration measured would be from the cup out towards the edge of the dish rather than from the surface of the agar to the bottom. Into each cup, 0.3 ml. of disin- fectant was placed and the dishes then incubated for 48 hours. At this time the paraffin was removed and the plates observed. _ 20 - TABLE VII , TESTS MADE ON VARIOUS DISINFECTANTS BE THE AGAR CUP METHOD Staphylococcus aureus (P. D. 02482) Zone produced in centimeters Disinfectant Orig. Conc. Plain Agar .Dextrose Agar Phemerol 44 (tinct.) 1-1000 1.1 0.4 Phemerol (aqueous) 1-1000 1.0 0.4 Phemerol 45 (tinct.) 1-500 1.3 0.4 Hexylresorcinol S. T. 37 1-1000 0.5 0.4 Iodine (tinct.) 1-40 1.0 1.0 Zepharin (tinct.) 1-1000 1.8 0.6 Zepharin (aqueous) 1-1000 1.8 0.6 Merthiolate (tinct.) 1-1000 complete 1.1 Merthiolate (aqueous) 1-1000 complete 1.0 Mercurochrome 1-20 3.2 0.6 Metaphen (tinct.) 1-200 3.3 1.2 Metaphen (aqueous) 1-500 3.3 1.2 Merphenyl Nitrate (aqueous) 1-1500 3.4 1.2 Merphenyl Borate (tinct.) 1-500 complete 1.2 Mercresin (tinct.) l-lOOO 3.8 1.2 Control 0 0 - 20 - TABLE VIII TESTS MADE 0N VARIOUS DISINFECTANTS BY THE AGAR CUP METHOD Eberthella typhosa ( Lab. strain ) Zone Produced in Centimeters Disinfectant Orig. Conc. Plain Agar Phemerol 44 (tinct.) 1-1000 0.7 Phemerol (aqueous) 1-1000 0.6 Phemerol 45 (tinct.) 1-500 0.8 Hexylresorcinol S. T. 37 1-1000 0.2 Iodine (tinct.) 1-40 1.0 Zepharin (tinct.) 1-1000 1.1 Zepharin (aqueous) 1-1000 1.2 Merthiolate (tinct.) 1-1000 3.2 Ierthiolate (aqueous) 1-1000 3.2 Mercurochrome 1-20 2.6 Metaphen (tinct.) 1-200 2.5 Metaphen (aqueous) 1-500 2.5 Merphenyl Nitrate (aqueous) 1-1500 2.0 Merphenyl Borate (tinct.) 1-500 3.2 Mercresin (tinct.) 1-1000 3.1 Control 0 - 21 - As can be seen from Tables VII and VIII, there is no correlation between the killing dilutions and the penetration as measured by this method. One would conclude that the Merthiolates and Merphenyl Borate are the superior compounds because they have a higher rate of penetration than any of the remainder of the disinfectants. This assumption must be false because Merthiolate was unable to kill Staph. aureus even in concentrated form in the dilution test, while here it completely prevented growth of the organism. Phemerol 44 (1-1000) produced a zone of 1.1 centimeters from the cup and Zepharin produced a zone of 1.8 centimeters against Staph. aureus. As these are related compounds of a non-mercurial nature, the element of bacteriostosis may be eliminated. The killing dilution studies would indicate that Phemerol 44 should be the more active in penetration than the Zepharin. Again this was not borne out. Further evidence that this test does not measure pene- tration is the fact that the mercurials show less activity against the Eberth. typhosa than Sgaph. aureus in this test, while the opposite was shown to be true in the killing dilu- tion studies. Harris and Prout<9) believe that the agar cup-plate method shows diffusion and recommend this test as a means of determining the degree of diffusion of a compound. It has been observed that the addition of dextrose to a gel will retard diffusion through the gel(1o). Table VII shows a comparison between plain nutrient agar and 0.2 per - 22 - cent dextrose agar, with Staph. aureus being used as the test organism. The zone of diffusion by Phemerol was decreased from 1.1cm. on plain agar to 0.4 cm., Zepharin from 1.8 cm. to 0.6 cm., Merthiolate from a complete zone to 1.0 cm., Metaphen from 1.3 cm.to 1.2 cm., Mercresin from 5.8 cm. to 1.2 cm., and Merphenyl Borate from a complete zone to 1.2 cm. These data substantiate the assumption that the agar cup-plate method is a measure of the diffusion and not pene- tration; that is the preperty to go into a water phase and diffuse through a colloidal medium rather than the measure of the pr0perty of a compound to penetrate organic matter. ' - 25 - III.RATE REACTIONS The problem now was to determine a procedure to supplant the agar cup-plate method for measuring penetration and this was done by the rate reaction test. Anderson(11) stated that the rate of kill could also be taken as a measure of penetration. From a study of rate reactions it is possible to compute the reaction ratejtemp- erature rate, and dilution rate of a germicide. It is possible to determine killing action in a time interval of 15 seconds, which makes this method especially applicable to the evaluation of skin disinfectants, in which a short exposure period is essential. The knowledge of the rate of reaction of a disinfectant is important as this will limit its application. A compound to be used on the skin or in a wound must have a rapid rate of reaction in order to exert killing power in the limited time it is in contact with the organisms it must destroy. The dilution rate is also essential as it must be known as to whether or not the compound will destroy organisms in a short period of time even in a diluted form such as exists when in contact with the serum in a wound. The temperature rate is a third factor of major import- ance in testing germicides. While a high temperature rate is a desirable property in a compound which is used on skin or wound , it may also be an undesirable property if the compound is to be used on a cold surface such as metal. In the latter instance the concentration of the disinfectant -é4- 7 would have to be increased in order to sterilize duerto its decrease in activity at lower temperatures, while in the former case it may be possible to dilute the compound and secure disinfection because of the greater activity at elev- ated temperatures. Lastly, the rate reaction would be a measure of bacterial penetration, as a compound which kills the offending organisms must penetrate the cell in order to destroy them. In testing by the rate reaction method, 10 m1. of the dilution of disinfectant was pipetted into a medication tube and one m1. of a 24 hour test culture was then added. Prior to the addition of the test culture, it was filtered through .a sterile cotton filter to remove clumps of organisms. At intervals of 15, 30, 45, 60, 90, 120, and 180 seconds and 4, 5, and 10 minutes from initial seeding, a 0.5 ml. sample was pipetted into a 99.5 ml. dilution blank. This blank contained 1 percent peptone and 0.85 percent sodium chloride in distilled water. The special peptone dilution blank was used to aid in overcoming the bacteriostatic action of the disinfectants. . Further dilutions were made in sterile saline and plated on 1.5 per cent tryptose agar. To determine the number of organisms present at zero time, a medication tube of 10 ml. sterile water was seeded in a similar manner as the dis- infectant tube. One sample was plated out as before and all plates were incubated at 37° C for 48 hours before counting. Staph. aureus and Eberth. typhosa obtained from Parke, - 25 - Davis and Co. were used as the test organisms. Medication temperatures of 200 C and 300 C were used to determine the effect of temperature on the speed of action. 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