.‘ & 1 , ‘ n4.- ‘ I . ‘ ' r K \ v . A L h f L ;‘ V o "‘ will ‘ ‘1‘} ‘ 1“ V l I I x y‘ ‘ ‘ I «1‘ "M1” H J 'r *‘l. .1‘ :‘l : y ‘1 w \ | 1 l l‘ .. ¥ _|U1_.x IOO (.D\IN A STUDY OF THE ACTION OF SYNTHE'HC DETERGENTS ON BACTERIA Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE Mary Georgia Bianchard 194? A STUDY OF THE ACTION OF SYNTHETIC DETERGEKTS OH BACTERIA Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the reouirements for the degree of MASTER OF SCIENCE Department of Facteriology 19142 THESIS ACKNOWLE GMEN The writer wishes to thank Dr. W. L. Mallmann, under whose direction this work was carried out, for his guidance and advice. 14611-3 TABLE OF CONTENT Page ACKNOWLEDGMENT ................................... ii INTRODUCTION ..................................... l HISTORICAL SURVEY ................................ 2 EXPER MENTAL WORK ................................ 7 Problem .......................... . ....... 7 Technique ................................ 9 Results .................................. 10 DISCUSSION ....................................... 15 COHCLUSIONS ...................................... 22 LITERATURE CITED ................................. 23 INTRODUCTI N The bacteriostatic and bactericidal activity of soaps have been known and studied for many years. This activity varies with the type of soap, with the pH of the solution in which it is used, and with the type of microbrganism. The effect of these natural deter- gents has been thought to involve two factors—-the toxic effect of the soap molecule and its surface tension reducing preperties. In recent years there has been deveIOped another group of wetting agents, the synthetic detergents. Certain of these compounds have exhibited a startling bactericidal efficiency—~far sunerior to that shown by the soaps. The literature of the last few years con— tains many references to the various tynes of activity displayed by the synthetic detergents of different types, under varying nH con- ditions, and in various concentrations. In view of the systematic variations in properties reported for these compounds, their bacteri— cidal action seemed worthy of further investigation. HISTORICAL SURVEY The study of the activity of the soaps against bacteria was . , 1 + begun in 1911, wnen Lamar noted that pneumococci treated with sooium oleate became more subject to autolysis and to complete serum—lysis O O T 2 Q w1th anti—pneumococcus serum. Lichols in 1919, as a result of his work in the army during the World War, presented bacteriologic data on the epidemiology of respiratory diseases. He observed that sodium oleate had a selective action—-killing streptococci readily in two minutes, but having no effect on the typhoid bacillus in ten minutes. Further, the antiseptic action of the soaps was lost if the reaction was changed from a pH of about 8.5 to 7 by the addition of acid. '7 At about this time Avery) noted that soaps of the unsaturated fatty acids were bactericidal for certain bacteria. He reported the the addition of sodium oleate to mediums prevented the growth of cer- tain Gram-positive organisms, principally pneumococcus and strepto- coccus, while the growth of Bacillus influenzae was enhanced by the presence of this substance. L; Walker, in l92h, found that scans prepared from the pure fatty acids differed markedly in their germicidal pronerties, with the lower members of the series possessing no (or limited) germicidal preperties against the organisms tested. He also observed their selec— tive action against different organisms. Staphylococcus aureus, for example, was not killed by any of the soaps; pneumococci and strepto- cocci were killed by the laurates, oleates, linoleates, and linolen— ates; while the typhoid bacillus was unaffected by these soaps. Eggerth conducted a series of experiments which illustrated different phases of the germicidal activity of soaps. He reported in ,R 1920’ that soap, in a concentration that was not in itself germicidal, could considerably increase the titre of acriflavine. With increasing molecular weight of the soap, the germicidal titre increased to a maximum, and then diminished. This was also true of the fatty acids, which were often more germicidal than the corresponding soap. The lower members of the saturated series of soaps were most germicidal in an acid reaction, whereas the higher members showed greater germi— cidal action when the pH was alkaline. Lipoids were found to be active— ly inhibitory to the action of soaps.7 So far as the substituted soap compounds are concerned, he found that the toxicity of the alpha-brom soaps for all species of bacteria increased rapidly with the length of the fatty acid chain. Bayliss and Halvorson9 confirmed the work of Nichols, Avery "f and walker regarding the selective bactericidal action of the scans. They reported that the pneumococcus was especially susceptible to the action of certain unsaturated soaps, such as sodium oleate, linoleate, and linolenate. Streptococcus lactis was considerably more resistant to the action of soaps than the pneumococcus, while Escherichia coli and Staphylococcus aureus were even more resistant. These worxers 1 also noted that the ability to neutralize diphtheria toxin is a pro— perty common to all soaps. The study of the synthetic wetting agents was initiated with- 10 . _ in the last ten years. In 1935 Domagk p01nted out the bacteric1dal action of these detergents with his demonstration that the quaternary ammonium compound, long-chain alkyl dimethyl benzyl ammonium chloride 11 possessed excellent germicidal preperties. Dunn confirmed tnese observations the following year, reporting that both Gram—positive and Gram—negative organisms were readily destroyed by this compound in high dilution, which was most effective in alkaline solution. I O 12 I ‘ V O I Katz and Lipsitz, in 1935, wrote that the highly resistant Eycobacterium smegmatis was inhibited in its growth in dilutions of the sodium salt of the di-secondary butyl naphthalene sulfonic acid, 12 O J ‘ I" O and in 1937 they reported that cyclic compounds were more efzective H. n such inhibition of growth than the aliphatic compounds. The visible action of a detergent, sodium lauryl sulfate, 1n on microbrganisms was described by anliss in 1937. Cultures of Gram-negative organisms grown in lieuid media were cleared by a pro- priate concentrations of this compound, and there was at the same time a marked increase in the viscosity of the medium. The majority of Gram-positive bacteria, with the exception of the pneumococci, remained unchanged. There was no correlation between clearing action and the lethal action of the sodium lauryl sulfate, for some of the organisms which were cleared were killed while others were not. The same was true of those which were not visibly affected. 1 In 1939 Maier 5 found that the new detergent, long-chain alkyl dimethyl benzyl ammonium chloride could be used very practicably in the oreservation of vaccines and venom solutions, and that it was remarkably free from harmful effects when applied to the cornea of rabbits. Another compound, cetyl pyridinium chloride, was studied 16 by Blubaugh, Botts, and Gerwe. They resorted that both tincture and aqueous solutions of this detergent, in the absence of oresence of organic matter, were highly bactericidal for virulent organisms. 17 Freedlander observed that while certain commercial wetting agents, namely Zephiran (long—chain alkyl dimethyl beanl ammonium chloride), Nacconol NR, and Aerosol OT lOO (mono-sodium sulfonate of dioctyl suc- cinate), exhibited marked bacteriostatic effects on the growth of Eycobacterium tuberculosis, they were completely ineffective in kill— ing the organisms. Wetting agents may also be used effectively as aerosols, according to Robertson, Bigg, Miller, and Baker. | Using certain glycols, they could sterilize the air of a small chamber containing a 19 suspension of Staph.albus. Ordal, Wilson,and Berg found that the addition of wetting agents to buffered solutions of phenolic compounds did, in general, increase the germicidal activity of such solutions. The selective action of different types of detergents was , d A 20 r , observed in 1930 by cowles. fie found that, by and large, the Gram- negative organisms are not prevented from growing by the alkyl sulfates (anionic detergents), whereas the Gram-positives, for the most part, were inhibited. After studying the action of synthetic detergents on the me- 21 tabolism of bacteria, Baker, Harrison, and Miller concluded that all cationic detergents were very effective inhibitors of bacterial metabo— lism, and that few anionic detergents inhibit as effectively. Both Gram—positive and Gram—negative organisms were sensitive to the action of cationic detergents, which exhibited their maximum activity in the alkaline nH range, whereas the anionic detergents selectively inhibited the metabolism of the Gram—positive microbrganisms, especially in the 22 acid range, and had little or no effect on Gram—negatives. They then checked the bactericidal action of these detergents and renorted that the cationic detergents, as a group, exhibited meryed bactericidal action on Gram—positive microbrgenisms and somewhat less pronounced action on Gram—negative organisms. The anionic detergents were germi— cidal only against Gram—positive organisms and were consideraol effective than the cationic compoun s. d 2? . Gershenfeld and Perlstein ’ emphas1zed the immortance of the hydrogen ion concentration in its effect on the action of detergents, showing that the anionic detergents were much more effective in acid PA. than n neutral solution. EXPERIHEHTAB WORK Problem *3 > .4 r—’ (0 m ‘J ynthetic detergents may be roughly eivi A. {N (‘D g . H 2r 0 d :1“ *3’ 2b 5') groups: (a) anionic compounds, e.g. sod'nm lauryl sulfate, which ionizes with the hydronhobic group in the anion as follows: (Ha ) + (012H25OSOI"); (b) cationic ccmnounds, e.g. laurvl pyridinium iodide, \nich contains the nyaronnobic grove 1n the cation: 7 v + - I ‘ 1 ‘ h-Clgh r + (I ); (c) non-ionizer comnounds, sucn as tne \ polyglycerol esters and other non-electrolyte ‘21 connounds. U It ha~ been observed that these comoounds differ from one grouo to another in their killing action toward bacteria. Thus, w’ile the cationic detergents have been found to be effective against almost all types of bacteria, particularly in alkaline solution, the anionic comnoands were far more ef ective crainst Gram—positives, esnecially in acid solution. Further study was indicated for a more comblete demonstration of the bactericidal action, and of the effects of chaég— ing pH. For this problem, four Gram-positive and five Gram—nebrtive organisms were selected. Grem-positive Gram-negative Bacillus subtilis Aerohacter eerogenes Alpha—hemolytic streftococcus Escherichia coli Beta—hemolytic strthococcus Eherthella typhosa Staphglococcus aureus Shigella dysenteriae Pseudomonas aeruginosa ih. netting agents were selected according to their electro— lytic structure--an anionic comsound, a cationic Ccmv and, a non»elec- U1 trolytic phospholipid, and a synthetic non-electrolyte. The anitnic detergent was Aerosol OT 10?} a chemically pure mono-sodium sulfonnte of dioctyl succinate: Ene cationic comround, Zeohiran,** is a mixture of long—chain QIEVl or L. ‘ . . . ‘ J a o I nimetqvl benzyl ammonium CKlOTlQoS. Toe cnemical structure 1s: CHO——N-—R in which 3 represents alkyl radicals rang- L'— 0 n f‘ T f‘ V 1J1? IFOL o,}1 to o it- n 5 17 13 37° ‘-yolk lecithin, with a general leci- _iosnholinid was a crude ego hin formula: 0 ‘ I O I O I rd :1 I '3” m—C)-————— O-—--- 0-{11 I O I O I I O I “U I varying sizes. CH ) p. - N \\\\OH I ‘ ‘ o . 7 **‘ The syntnetlc non-electrolytic detergent was Triton TE.' It is an octyl methyl nhenoxy polyethoxy ethanol: * American Cyanamid and Chemical Cornoration. ** Alba Pharmaceutical Company. *** Rflhm and Haas. 8 17 . ,4. , \\\ 1n MJICH n renresents -,—C - vv- . C HM O ( EH40», Can“ OH a fairly large number. These comoounds were tested for their relative bactericidal efficiencies in neutral solution, and at a n2 5 and pH 9. The nossiile inhibiting action of the two-non—electrolytes on the two electrolytic compounds we also determined. Technique The comeound to be tested was prenered in the desired con— centration in a liter of sterile distilled water. 190 m1. of the solu- tion was then transferred to a sterile 230 m1. Erlenmeyer flask. The 3 NaOH or 331 immedi— c<. r,-, p: 5.3: H. 5 solution was adjusted to the desired pH b‘ ately prior to use, as determined by indie tors on a snot nlote. Tne pH was checked again immediatelv after the aidition of the nrotn cul— ture to be sure that the value had not changed. -1 ,o from 23—20 C. To 1’3 m1. of detergent solution was adied 1 m1. of a filtered BH—hour broth culture. The broth used wes a Tryptose medium containing 20 rm. Trvptose, 5 gm. FnCl, and 1030 ml. water. The mixture was shaken thoroughly, the pH checked, and after 1, 5, and 13 minute inter- vals a 1 ml. sample was transferred to a 99 ml. seline blank, so as to obtain a 1—100 dilution. Further dilutions of l-lQCC, 1—10,?“0, and 1-10 ,900 were then made. 1 m1. of each of these dilutions was trans- ferred to a sterile Petri dish, and M50 C. ago was noured into the filate. The agar used was a Tryptose egor, containing 2% 3m. Tryntose, 2 rI‘m. dextrose, 5 cm. XaCl, and 15 em. agar to 1000 ml. distilled water. L) *3 J FJ t) The plates were rotated, allowed to solidify, and incubated for ”3 O I O Q ~ 0 O O 0 hours at 37 C. At the end of this time tne dilution nlate containin from 30 to 330 colonies was counted in its entirety. At the time of each test a control count was made by adding 1 ml. of the broth culture to 99 ml. of sterile water, diluting in blanks, and platiné out. Results Several counts of surviving cells were made for each organ- ism tested, and for each solution of varying concentration and pH value. These counts were averared, and the result divided by the con- trol count to obtain the percentage of survivers. This figure was then subtracted from 100 per cent for the percentage of organisms “illed. It was felt that these percentage figures showed relative values better than the numerical counts themselves. Obviously these values cannot be considered except as showing the trends of the action taking place when there is not comnlete killing. The cationic detergent, Zephiran, was the most effective bactericidal agent of the comncunds tested. At a concentration of 1-10.030 it killed both Gram-negative and Gram-positive organisms at pH values of 5, 7, and 9. By using a higher dilution, (Table I) how- ever, (1—ioo,ooo) it was found that Zenhiran failed to kill more than 80 per cent of the taphraureus present at a pH value of 5 in 10 min- utes, though it was more effective against Pseud.aeru;inosa and Esch. ‘ggli: At a pH of 9 all these organisms were killed. Aerosol OT (Table II), the anionic detergent, was not effec- tive at such high dilutions as Zeshiran, so that it was necessary to use a concentration of l-lOOO to demonstrate its selective action. At a pH of 7 this comnound was comnletely effective against the strongly 11 OOH OOH OOH OOH OOH OOH OOH OOH OOH ..amoaHmsgmm .Osmmm OOH OOH OOH .....mmsmWopma .opmd OOH OOH OOH OOH OOH :.OO ..........HHoo .somm OOH OOH OOH O.mm H.OO m.Om O.mH O.HO O.mm .......msmpsm .gampm ammo ammo ammo ammo ammo .psmo ammo ammo ammo pmm Hmm pmm pom Hum pom mom pmm Ham OH O H OH O H OOH m H, smHmmmOO wmusOHz mmpsaHz mapsaHz mmg ng mmO mg O24 mmOmomxm HO HOHH HO szHOHOzO Oszmd> mmmzm ZoHebqom Z¢mHmmmN BZMQ mmm HO0.0 wm QMHHHM mEmHz<¢mo ho madeszmmm H MHm mmnz: zOHOOHOm OO Homommd HOMO mam H.O Hm OOHHHH mzmHOHOmO HO mmHeszmmm HH mHm¢H 13 Gram-positive E: subtilis, partially effective against Staph.aureus, and hardly active at all against the streptococci. Aerosol's comnlete ineffectiveness against gram-negative organisms, with the exception of Esch.coli, was apparent at a pH of 7. At a pH of 5, however, a 1-1000 dilution of this compound was relatively effective against all organ- isms tested, though complete killing action in 1 minute was demonstrated only with Staph.aureus and the beta~hemolytic streptococcus. At a pH of 9 Aerosol was less effective than at a DH of 7. 26 It was shown by Baker, Miller, and Harrison that phospho- lipids such as lecithin, cephalin, and sphingomyelin prevented the inhibition of bacterial metabolism which was caused by synthetic deter- gents. They also found that lecithin made germicidal concentrations of the detergents ineffective. In checking this inhibitory action, a 1.0 per cent crude egg yolk lecithin was used with a 0.01 per cent Zephiran solution. No bactericidal action was apparent against Stanh. aureus or Aero.aerogenes. The lecithin was also ineffective as a kill- ing agent by itself against these organisms at all pH values. This inhibitory action is not characteristic of all non-elec— m trolytic detergents, however. While Triton kn appears to prevent Aerosol from acting against Pseud. aeruginosa, Stagh. aureus, and Aero. aerogenes even at an acid pH, it has no effect on the killing action of Zephiran with these organisms. Triton XE (Table III) also annears to have some effect as a bactericidal agent itself, but the concentra- tion used was extremely high. 1h H.Om H.Om O.Om O.Om m.Om m.mm O.HH m.Hm O.Hm ...Omoch5tOm .Osmmm m.HO «.am m.zh O.O~ O.H~ H.Hm m.mm m.mH :.Om .....mmamm0hmm .opma O.Hm 0.0 O.MH O.Hm O.mm O m.MH O.H m.mH .......mama:m .Opmpm a. sh O...” atom. an a...” g a... OS OH O H OH O H OH O H Eqummao mmpScHE mmusmHE wouquz Own Ham mum mm 924 mmbmomxm %o QQHB mo szHBHQzOo OZHMMdfi mmmmb ZOHBDHOm HZ EOEHMB HERO mmm O.H Wm QMHHHM mzmthmmO Mo mmamm ommm HHH mgmdb DISCUSSION A number of factors should be included in a consideration of the data presented. These'factors include: (1) the surface-tension reducing properties of the detergents; (2) the cha ge on the ion con- ’ taining the hydrophobic group; (3) the p. i .1 of the solution; and (h) C? n) the general and srecific char c eristics of the microorganisms. The hydrophilic-hydrOphobic balance of the molecule and the specific chem- ical structure of the molecule were not considered in this study. FIT Surface tension. .ne effect of surface tension as a factor in the growth of bacteria has been studied by several workers. Larson, 7 27 n Q 1 A Q 0 Q Cantwell, and fiartzell Iound that most OI tne common bacteria snowed some Trowt‘ ' d' "‘ e ‘ f t ' W - l s 2 " c h g \bfl in a me lum Jnos- sur ace en51on "as as -ow as 3 dynes. It was noted by Ayers, Rune, and Johnson2 taat £223,921} and Aegg aerogenes grew fairly well in a medium with a surface tension of 35 vnes. They observed that a particular surface tension could not be considered a critical tension whicn exhibited its effect solely through its influence on the permeability of the cell. If such were t on value should exhibit the *J. all dearessants at a certain surface tens same effect. They concluded that the nature of the surface tension depressant as well as the actual surface tension value in dynes must 2? Gibbs, “atonelor, and Sickels ' concurred with this 30 view. Frobisher be considered. also concluded that there seemed to be little rela- tion between the ability of reducents, such as sodium oleate and sodium ricinoleate, to kill or inhibit the growth of Eberth. tgohosa, and their ability to reduce surface tension. It was found in this exteri- n y.” ment that lecithin and Triton NE, which lower the surface tension aqueous solution considerably have little or no effect on the organ- isms tested. The difficulty with all such studies lies in the fact that it has been possible to measure only the air-solution interfacial tension, where in the case of a lowered surface tension there is an increased concentration of the substance in solution, 1.e. nositive adsorption occurs. In reneral, if there is positive adsorption at the O" I on interface, there will be at the solution—container inter— Ho ir-‘olut 0) U face. But it remains an open question whether there is a correlated lotering of the surface tension at the bacterial cell-solution inter- face, and thus a higher concentration of the solution on the bacterial surface. It has, however, been necessary to assume that a detergent which lowers surface tension at the solution-air interface will do th same for the solution~bacterial cell interface in order to exnlain, e.a. why the bactericidal action of a non-toxic solution such as hexylresorcinol is enhanced by small quantities of sodium oleate.31 Fluids of very low surface tension may, under some circumstances, permeate or penetrate into minute crevices or interstices filled with air more readily than fluids of high surface tension. Assuming that there is a lowered surface tenSion at the solution-cell interface, the solution is probably adsorbed on the surface of the organisms, so that the solution exists in higher concentration at the most effective point. There would then be reason to believe that the surface tension reducent could induce changes in the permeabilitf of the membrane which would facilitate the entrance of toxic molecules present in the solution. This possibility is borne out by the fact that when an excess of sodium oleate is added to hexylresorcinol, it inhibits the bactericidal action of the latter substance. This is believed to be the result of the formation \J ('0 adsorption of the soap on the surfaces of the cells, with t; of a protective film or coating. These results may be comnared with ‘ those in this experiment in which an excess of lecithin prevented a germicidal concentration of Zephiran from actin, against th bacteria. ~ Aside from its possible conjunctive action with toxic molecules, the *4 0 VO" H. d (D (D H H) rface tension va S C: ue seems to be of little importance in determining the germicidal action of the detergent. Aerosol CT solu- tions, for example, have a considerably lOWer surface tension value than Zepniran solutions. It is interestin? to note, however, th"t in an acid solution, in which Zephiran is relatively inei face tension value was noticeably higher than those obtained with neu- tral and alkaline solutions. 0 L) H. O The chnrje on the ion containing the hydrOjE group, The factor of charge on the hydrophobic group of the deter ent appears to be of considerable importance, as has been shown by Baker, miller, and 21 Harrison, and by the results of this study. Zerhiran ionizes with the pOsitive charge on the ion which contains the long-chain hydro- iobic group. Aerosol OT ionizes with the negetive charge on the long— chain hydronhobic group. Lecithin does not ionize, except as a zwitter- ion, while Triton NE is a non-electrolyte. The simplest explanation for the bactericidal efficiency of Zephiran might be considered in terms of the mutual attraction between the negatively—charged bacterial cells and the positively charged hydrophobic groun. For a clearer concept of this factor, however, it is necessary to consider first the pH of the solutions. The pH of the solution. The effects of the :3 on the action 13 of various compounds have been noted previously in connection with the '29 7. 0 7 ' ~ A J a *1 action of soap. Kligler,’ Stearn and atearn, and others have noted that increase of pH favors tae disinfecting power of basic dyes and 3 decrease of pH, that of acid dyes. Stearn and Stearn are inclined ‘JJ to believe that the hydrogen ion concentration directly affects the bacterial cell, altering the protOplasm so as to render the o'aanisms more susceptible to the action of toxic substances. 1.1+ Osterhout/ has shown, in his work with large plant cells, that electrolytes pass through the non-aqueous protoplasmic surface chiefly in the molecular form, since its low dielectric constant would not permit much dissociation. It is true, he says, that some disso- ciation takes place and that ions can enter to some exten , but the con— centration of the ions is very small. If this phenomenon can be ap- plied to the bacterial cell, it is seen that the undissociated electro- lyte should be a more effective bactericidal agent. Zephiran's great- est effectiveness is in an alkaline solution, which would favor the formation of the undissociated molecule, whereas Aerosol's greatest effectiveness would lie in the acid range, wherein its ionization would tend to be reversed to the molecular form. Aerosol OT and Zephiran are both strong electrolytes, however, and it is hard to imagine that their ionization could be prevented to any extent in the relatively weak acid and base solutions in which they were used. Furthermore, if an unionized molecule is more effective in penetrating the bacterial cell, why do not the non-electrolytes lecithin and Triton NE exhibit more action? There may also be some question as to whether Osterhout's observations on plant cells are directly applicable to all living cells. r ) Stearn and Stearn report that the bacterial cell behaves 19 rsten, and that, while all bacteria are negat1velv Charged, each organise has 1ts o n particular isoslectric roint. They point cit the t Ior ar -y or CJanism, rega*dless of the value of its iso- electric point, cations should be retained to a greater extent at high JH values than at low ores, and the reverse should be true for anions. It has been s}1ovn that the anion of Aerosol is more efiective as a bacte *‘5 O .4. .4 9) e I1 ( Ho O H. 0- solution, while the cation of Zerniran is more effective in alkaline solu ions- -which nould apnea r to sear out the h;.nflo hesis of Stearn and Stearn. One possible explanation for this behavior lies in the fa t that a bacterial suspension may be considered to be a negatively-charged colloid. Cations, then, would tend to be attracted to the surface of a negatively charged particle. The addition of H+ ions prior to the addition of this detergent might act to neutralize this negative charee on the bacterial cell (or even give the cell a positive charge), however, so that there no longer existed such a strong mutual attraction between the cell and the ca- tion of the detergent. An anion, on the other hand, Should be more effic ent in an acid solution, for it should be . mch more rea adil y attre cted when the bacterial cell charges have been neutralized, or even better if the cellh as become positively charged due to the ad- sorbed layer of positive hydrogen ions. It would be more difficult Icr n anionic detergent, then, to act throu.h an electrical attraction mechanism than for a cationic deterjer t, and its only possible action would take place in an acid solution. This vould explain in p rt th relative inefficiency of Aerosol O? as compared with Zephiran, and wculd also partially explain the almost co.xslete lack of bactericidal action of lecithin and Triton JL. D.) (J The general and specific characteristics of the microbrgan— iggg. One of the most interesting results of this and of other studies on the bactericidal activity of the synthetic detergents has been the relative specificity of action of the anionic detergents for Gram—posi— tive organisms, at least in neutral solution, as contrasted with the strong killing action of the cationic compounds for all organisms. The contrast in sensitivity to toxic substances between Gram-positive and Gram-negative organisms has been noted many times. Stearn and 33 . .. Stearn wrote: "The pOpular idea that Gram—pos1t1ve organisms are more sensitive to basic dyes than are Gram-negative is borne out only in the sense that the former find themselves at one end of a gradual series showing gradations in sensitivity rather than in a very distinct group showing sensitivity of a different order of magnitude from any member of the other group." In their study they found a gradual de- crease in sensitivity to basic dyes from the strongly Gram-positive through the border-line organisms to the strongly Gram-negative, the order being: 2: subtilis, Staph. aureus, Shir. dysenteriae, Esch. coli, Eberth. typhosa, and Aero. aerogenes. 30 Mallmann, Botwright, and Churchill reported that the slow oxidizing agents, potassium dichromate, and sodium azide, exerted a bacterio’Static effect on Gram-negative bacteria. By the use of dif- A ferent dilutions of these slow oxidizing agents the gradations in U character from strongly Gram—positive to strongly Gram-negative or an- isms was again shown, the order being: Staph. aureus, b. cereus (com- parable to B. subtilis), Pseud. aeruginosa, Each. coli, Eterth. and Aero. aerogenes. In this study it is likewise apparent that there exist grada- tions in sensitivity to th .e anionic dete gent, Aerosol OT , which are to be seen in the per cent killed figures in Taole II. On the basis of these figures, the gradations from strongly Gran—nositive to strong- rj‘ til .J. (n a‘ Q I L , btnfn. lureus, alone-stren- y Gran-negative would be: B. s‘l tococous, beta-strentococcus, Shig. @Lsenteriae, Esch. coli, Eberth. tvnhosa, Aero. aero:enes s, and Pseud. eeruginosa. _l’_ - The results of these three studies cannot be di rectlgr corre- lated, but the imnortanoe of the general and sneciIic characteristics of the microbrganisms is evident. Further work along this line may throw more light on the ouestion of the Grem—stain n3 characteristics of bacteria. The synthetic detergents appear to act in several ways to kill microbr g'eni ems. Because of th ei r surfs ace—tension lo:erin; action they are probably adsorbed in a fairly high concentration on the bac- terial cell, and may permeate the cell we all more readilya is consequence. on or revul- cf Ho Either aiding or opposing this actionn may be the attrac sion of the hydroohobic ion of the deter:ent b7 t1 e negstive charge on the becteriel cell, which is influenced by tne presence or absence of hydrogen ions. And finallr, the effect of cert in deter ents on ,_ I H. u’) 3 J 4 .J \‘_ l ‘1 (1‘ J ”3 lifferent microorgn1 denendin; on th 0 general and sneci- fic characteristics of the or anism, part 1c cul1rly u11et1er it is Gram V edditian there must be considered the '15 O U1 (1' H0 < (D O ’1 c.) E I S D '2.) C' H a: 0) H Li ‘1 nature of the toxic substance itself, Which was not a fwctor studied CONCLUSIOKS The cationic detergent, Zenhiran, in a 0.01 per cent solution kill— ooth Gram-positive and Gram-negative organisms at pH values of 5, 7, and 9. In a 0.001 per cent solution it was considerably 744 less efficient at a pH of 5 than at a p? of 9. The anionic detergent, Aerosol CT, in a 0.1 per cent solution kill- ed only strongly Gram—positive organisms at a pH of 7. At a pH of 5 it not only killed all Gram-positive orsaniems, but was also quite effective against Gram-negatives. The non-electrolytic detergent, Triton YE, showed some killin' against both Gram—positive and Gram-negative organisms 1n a 1.0 oer cent solution. A combination 1.0 per cent Triton—0.01 per cent Zephiran solution sh wed comolete killing action. A combination 1.0 per cent Triton—0.1 per cent Aerosol solution showed no killing action. The naturally occurring phospholipid, lecithin, showed no killinv ’1 1-) :5 Ho U) s U) 0 action against both Gram-positive and Gram-negative o combination 1.0 per cent lecithin-0.01 per cent Zerhiran solution showed no killing action. [0 Km! [J .— IJIM'Q \“WTY'j‘V: CITED ‘ sonsAgA. \J-k..a Lamar, R. V. Action on the pne rnococcus and its exnerim121t11 infec- tions of crn'lpen sodi um oleate a11d anti W me_1ococcus seru1. J. Exp. hed. 1911, 13:1. nichols, H. J. Bacteriolo ic data on the enidemiolojy of resniretory diseascs in the Army. J. Lab. and Clin. ted. 1913-90 5:502. elective medium for B. influenzee. J. Am. Uei. Ass'n. ,r‘ y c o n ‘V H ' A. ‘l J E. aerm1c1oal pronerties 01 soaps. J. inzect. D1s. 172~, Eggerth, A. The bactericidal action of acri‘ine dyes an‘ the ad1uv ho effect ofs serum. J. Infect. Dis. igzo, 33:3. Lrjerth, A. The effect of pH on the germicidal ac ction of soans. J. hys. 1920, 10:1h7. (D (D :3 F4 *0 fertn, A. 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Preservation of tiolOgica fluids with alkvl cinetnyl benzyl ammonium chloride. J. Eact.19?9, 33:31. Bluhaugh, L. V.. BOttS. C. W.,em1d Gerwe, E. Study of germicidal Fronerties of cetyl ny1fiii nium c‘dloride. J. Br1ct 1940, 33:R1. 17. 18. 22. 23. 2h 25. 26. F1) 4 Freedlander, B. L. Bacteriostatic action of various wetting age ents upon growth of tubercle bacilli in vitro. Proc. Soc. Em:v Biol. and Med. 19M , u1151. Robertson, 0. H., 3155, F., Miller, B. F. , and Baker. Z. Steriliza- tion of air by certain glycols emnloy ed as aerosols. Science, 1991, 93:213. Ordal, E. J., Wilson, J. L., and Borg, A. F. Action of wetting agents on microbrganisms. J. Bact. 1991, 92:119. elective bacteriostatic action. OW1es, P. B. Alkyl-sulfates: tlidr s 8,11:33. Yale J. of Biol. and Med. 19 3 Baker, Z. Harrison, B. W., and Hiller, B. F. Action of synthetic dete rgents on metabolism of bacteria. J. Exn. med. l9h1, 73: 2'9. Baker, 2., Harrison, R. W., and Miller, E. F. 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Surface tension and bacterial growth. J. Dact. 1926, 11:393. Frobisb er, M. Relations of surface tension to bacterial phenomena. J. Infect. Dis. 192 6, 33:00. Frobisher, M. Interfacial tension and bacterial growth. J. Duct. /' 1927, 3:103. 1 Kligler, I. J. ganic comnounds. Osterhout, W. J. V. The a cells. ?ot. Rev. 1‘} Stearn, E. W. and Stearn, A. 7 110. 2. Rallmann, A study of tr rn, A. E. of dyes a V5;. L. , E'Ot‘.',ri'::ht’ Inf. becteriostatic effect of 19h1, 69:215. Med. . University of iissouri Studies, I? 4.4., slow -e antisentic J. EX? 1913, It) \)I properties of certain or- 27:363. 1 ‘ of nydrogen ion concentra- ia. Am. J. Pub. Health, I \ ’37 1’3), The selective Infect. Dis. ‘(~- V3‘1‘L I ~ ‘I‘_ 6:- 1 ‘1 .0 1.... 1. .. .3111 -. In." ...} 11.1..11 V1111? . .1 o( . ‘1‘“? ‘101‘.’ - 1 ... 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