TE EFFECT OF PURE SOAPS ON THE BACTERICIDALPROPERTIES OF PHENOLIC GERMICIDES

BETTYLEE HAMPILlFrom the Department of Bacteriology, School of Hygiene and Public Health, Johns

Hopkins University

Received for publication, May 17, 1928

INTRODUCTION

It has been abundantly shown that soaps in general have alimited germicidal value, (for complete bibliography see Walker,1924, 1925, 1926, and Eggerth, 1927), and various workers haveexpressed the idea that this is due for the most part to their de-tergent properties. The literature indicates that a large varietyof organisms have been tested, at time intervals of exposure fromtwo and one-half to sixty minutes. Soaps prepared from varioustypes of oils have shown more or less selective action. Thetyphoid, diphtheria and colon bacilli, the spirochetes, the gono-coccus, the meningococcus and different types of the streptococciare easily destroyed. On the other hand, it is significant thatStaphylococcus aureus is particularly resistant. It is no doubtbecause of this selective action that efforts have been made fromtime to time to incorporate certain germicidal substances intosoaps, and thereby manufacture a product which will not onlycleanse but at the same time destroy organisms found on the skin,regardless of the type present. There are numerous commercialsoaps on the market at the present time, to many of which havebeen added phenolic derivatives. The soaps are labelled asantiseptic, and a phenol coefficient is often determined. As yetthere are no scientific data to show that the incorporation of thephenolic substances has added to the intrinsic germicidal activitywhich the soap itself may possess. The determination of the

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phenol coefficient for such a so-called "antiseptic soap" is inpractice the measure of its activity against Bacillus typhosus,which organism has been shown by several workers to be verysusceptible to the disinfectant action of soaps.Hamilton (1917) mixed cresylic acid and soap in varying pro-

portions, and reported that when the ratio between the two sub-stances was varied to obtain in the one case a complete solutionand in the other a hazy emulsion, the two preparations failedto show any material difference in germiicidal effectiveness.When one examines the table given, however, it is seen that theefficiency was actually diminished by one-half when the amountof disinfectant was reduced from 80 to 50 per cent and the soapincreased from 20 to 50 per cent. Tilley and Schaeffer (1925)found that the bactericidal efficiency of cresol was diminished inlinseed oil soap-cresol solutions if more than half the mixture wassoap. The present author, at the suggestion of Frobisher, hasshown the futility of ming large quantities of soap with sopowerful a germicide as hexyl resorcinol (see Frobisher, 1927).Rettger, Valley and Plastridge (1928) found that butyl resorcinolloses a part of its germicidal properties in the presence of puresoaps.

This investigation was undertaken to deternine the effect ofpure individual soaps on the germicidal activity of certain repre-sentative phenolic substances. Since the exact germicidal titre ofpure soaps is easily obtained, it was possible to use the soapsin such low concentrations that the bactericidal effectiveness ofthe germicides was not masked by the disinfectant action of thesoaps themselves.

METHOD

The soaps2 used were sodium oleate, sodium myristate, sodiumpalmitate, sodium stearate, potassium palmitate and potassiumstearate. Stock solutions contained 10 per cent pure saponified

2 The soaps which were obtained from Sharpe and Dohme of Baltimore wereprepared by saponifying 10 per cent of the pure fatty acids. These were ad-justed to a pH of 9.0 Throughout this paper the terms "per cent sodium oleate,""per cent sodium myristate," etc., refer to the percentage of fatty acid presentwhich has been saponified.

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EFFECT OF SOAPS ON PHENOLIC GERMICIDES 289

fatty acid. Dilutions were made from these stock solutions withsterile distilled water. The disinfectants used were phenol,meta-cresol, secondary butyl phenol, n-butyl resorcinol and

TABLE 1

Effece ofNa oleate on bactericidal activity of phenol, meta-cresol and butyl phenol at37'C. againet B. typhosus

PHEENOL YETA,-CRZOL SECONDARY BUTYLPHENOL META-CRESOL ~PHENOLPERCENTINa OLEATE Subcultures Subcultures Subcultures

Dilution miue Dilution DilutionDilution1 2 min- mnu1 2 mi- u1 2 min-minute utes minute utes minute utes

5.0 100 + _ 100 + - 100 + -5.0 150 + + 150 + - 150 + -5.0 200 + + 200 + + 200 + +4.0 100 - - 100 - - 100 + -4.0 200 + + 200 + + 200 + +3.0 100 - - 100 - - 100 + _3.0 200 + + 200 + - 200 + +2.0 100 - - 100 - - 100 + -2.0 150 + - 150 - -

2.0 200 + + 200 + - 200 + +1.0 100 - - 100 - - 100 - _1.0 150 + - 150 - - 150 + -1.0 200 + + 200 + - 200 + +0.5 100 - - 100 - - 100 - _0.5 150 + - 150 - - 200 + -0.5 200 + + 200 - - 400 + +0.2 100 - -0.2 200 - -0.2 250 - -0.2 400 - -

Controls5.0 0 + + 0 + + 0 + +0 100 - - 200 - - 2,000 - _

NaCl control 0 + + 0 + + 0 + +

+ growth on plates. - no growth on plates.

n-hexyl resorcinol. The organisms employed were Bacillustyphosus (Hopkins strain) and Staphylococcus aureus (from uri-nary infection), grown in plain broth (1.0 per cent Armour's pep-tone, 0.3 per cent Liebig beef extract and 0.5 per cent NaCl with a

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290 BETrIYLEE HAMPIL

pH of 6.8). Mixtures of soap and germicide in 5 cc. portionswere placed in test tubes in a water bath at 37°C., to which wasadded 0.1 cc. of a twenty-four-hour broth culture of the test

TABLE 2

Effect of Na oleate on bactericidal activity of butyl resorcinol and hexyl resorcinol at37°C. against B. typhosus and Staph. aureus

SUBCULTURES 8UBCuLTURESDILU- DLU-

NP OLEATZ BUT'rYL B. typhosue Staph. aureuw Bzxm B. typho8us Staph. aureuNSLAE RESOR- - . ESOIR--CINOL 1 min- 2 min- 1 min- 2 min- CINOL 1 min- 2 min- 1 min- 2 min-

ute utes ute utes ute utes ute utee

5.05.05.05.05.04.04.03.03.03.02,.02.02.01.01.01.00.50.50.50.20.20.2

Controls5.00

100200250500

1,000100200100200

1,000100200

1,000200400500200500700500

1,0001,500

+

+

+

+

0 +2,000 _

++++

+

++

+

+

+

+

+

+

+

+

100200250500

1,000100200100200

1,000100200

1,000200400

1,000200500

1,000200

1,0001,500

5,000

+++

++

+

+

+

+

++

+

+

+

++++

+

+

+

++++

+

+

+ growth on plates. - no growth on plates.

organism. Subcultures to infusion agar pour plates were madeafter one and two minute intervals of exposure, incubated at 37°C.for forty-eight hours, and examined for growth.

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THE EFFECT OF SODIUM OLEATE AND SODIUM MYRISTATE ON THE

GERMICIDAL ACTIVITY OF PHENOL, META-CRESOL, SECONDARYBUTYL PHENOL, BUTYL RESORCINOL AND HEXYL RESORCINOL

Sodium oleate has a marked inhibitory effect on the germicidalactivity of phenolic compounds (tables 1 and 2). A 5 per centsolution of this soap inhibits the action of 1:100 dilutions ofphenol, meta-cresol, butyl phenol and butyl resorcinol against

TABLE 3Effect of I per cent Na myristate on the bactericidal activity of meta-cresol, butyl

phenol and butyl resorcinol at 37TC. against B. typhosusMUTA-CRESOL SECONDARY BUTYL PHENOL BUTYL RUSORCINOL

Subcultures Subcultures SubculturesDilution Dilution Dilution

1 minute 2mmun- 1 minute 2 min- 1 minute 2u ts-

100 _ - 100 - - 100 - -

150 - - 150 - - 150 - -

200 + - 200 - - 200 - -

250 + + 300 - - 300 - -

400 - - 400 - -

500 - - 500 + -

600 + - 600 + +700 + - 700 + +800 + + 800 + +900 + + 900 + +

1,000 + + 1,000 + +Controls with nosoap 200 _ - 2,000 - - 2,000 -

1 per cent Namyristate + +

+ growth on plates. -no growth on plates.

Bacillus typhosus in one minute. After two minutes the organ-isms are killed. The same concentration of soap prevents theactivity of 1:200 dilutions of these four compounds as well as ofhexyl resorcinol after two minutes exposure. A 4 per cent solu-tion of the soap gives the same results. As the concentrationof the soap is decreased, this inhibitory action re present,provided the amount of the disinfectant is likewise decreased.

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A 0.5 per cent solution of sodium oleate%is sufficient to destroy thebactericidal activity of so powerful a disinfectant as hexyl resor-cinol in a 1:1000 dilution. This amount of the disinfectant is atleast five times as much as is required to kill the organisms inaqueous solution in the same length of time. Frobisher (1927)

TABLE 4

Effect of 1 per cent K. palmitate on bactericidal activity of meta-cresol, butyl phenol,butyl resorcinol and hexyl resorcinol at 37'C. against B. typhosus

META-CRESOL SECONDARY BUTYL BEUTL RESORCINOL HEXYL RESORCINOLPHENOL

Subcultures Sub- Sub- Sub-cultures cultures cultures

Dilution 1 2 Dilution 2 Dilution - 2 Dilution 1 2min- min- min- min- min- min- min- min-ute utes uto utes ute utes ute utes

100 100 - - 100 - - 100 - -

150 - - 150 - - 150 - - 150 - -

200 + _ 200 - - 200 - - 200 - -

300 + + 300 - - 300 - - 300 - -

400 + - 400 - - 400 - -

500 + + 500 + - 500 + -600 + + 600 + - 600 + -

700 + + 700 + + 700 + -

800 + + 800 + + 800 + -900 + + 900 + + 900 + -

1,000 + + 1,000 + + 1,000 + -1,200 + +1,400 + +

Controls with nosoap

200 - - 2,000 - - 2,000 - 5,000 -1 per cent K. + + + +palmitatecontrol

+ growth on plates. -no growth on plates.

has reported that small amounts of sodium oleate enhance theactivity of disinfectants by virtue of the ability of the soap tolower surface tension, thereby causing a more rapid destructionof the bacteria. In repeated experiments in which sodium oleatewas used in our work, there was a suggestion that this might betrue for phenol but not for the other disinfectants. Concen-

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trations of less than.O.5 per cent sodium oleate seemed to have noeffect whatsoever on the bactericidal action of the higher phenolseven in very weak dilutions of the disinfectants.Sodium myristate is slightly germicidal for Bacillus typhosus

in a 5 per cent concentration. For that reason 1 per cent solu-tions were employed. In this concentration, its inhibitory action

TABLE 5Effect of 1 per cent K stearate on bactericidal activity of meta-cresol, butyl phenol,

butyl resorcinol and hexyl resorcinol at 87'C. against B. typhosu8

MZTA-CRESOL 5BCONDAiY BUTTL BUTTL NUSORCINOL HUXYL RUSORCINOLPHZNOL

Subcultures Sub- Sub- Sub-cultures. cultures cultursDilution Dilution - Dilution Dilution1 2 1 2 1 2 1 2

min- min- min- min- min- min- mini- min-ute utes ute utes ute utes ute utes

100 _ _ 100 100 - - 100 - -150 + - 150 150 - - 150 - -

200 + - 200 + - 200 - - 200 - -300 + + 300 + - 300 - - 300 - -

400 + + 400 + - 400 + -500 + + 500 + - 500 + -600 + + 600 + + 600 + -700 + + 700 + + 700 + +800 + + 800 + + 800 + +900 + + 900 + + 900 + +

1,000 + + 1,000 + + 1,000 + +Controls with nosoap

200 - - 2,000 - _ 2,000 - _ 5,000 - -1 per cent Kstearate control + +

+ growth on plates. - no growth on plates.

on the sterilizing power of meta-cresol, butyl phenol and butylresorcinol is very marked (table 3). Butyl phenol and butylresorcinol are reduced in activity at least 75 per cent. Meta-cresol is less affected. A comparison with 1 per cent sodium oleateindicates that the germicidal activity of the compounds is re-duced less by the addition of sodium myristate than by that ofsodium oleate.

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EFFECT OF POTASSIUM PALMITATE AND POTASSIUM STEARATE ON

THE GERMICIDAL ACTIVITY OF CERTAIN PHENOLS

The potassium soaps of palmitic and stearic acids were usedbecause of the high gelation properties of the two sodium soapsof the two acids. One per cent solutions were tested, and thesame general inhibitory action displayed by sodium myristateand sodium oleate was shown to be present (tables 4 and 5).Potassium palmitate decreases the action of butyl phenol, butylresorcinol and hexyl resorcinol at least 80 per cent. Potassiumstearate acts as an even more inhibitory agent. When theseresults are compared with those for sodium myristate, it is ap-parent that as the fatty acid series is ascended, the soaps of thehigher acids become progressively more inhibitory, provided theyare tested in the liquid or disperse phase. This point will be takenup later in detail. When potassium stearate is compared with thecorresponding soap of the oleic acid series, sodium oleate, there isa negligible difference in the inhibitory activity of the two soaps.This would seem to indicate that the amount of saturation of thefatty acid has no marked influence on the degree of inhibitionexerted by the soap.

INFLUENCE OF PHYSICAL CONDITION OF PURE SOAPS ON THEBACTERICIDAL ACTION OF PHENOLS

Early in the work, the sodium soaps of palmitic and stearicacids were tested in 0.5 per cent solutions with varying concen-trations of the disinfectants. The results were most irregular,but on the whole appeared to indicate that these two soaps hadless influence on the bactericidal action of the germicides thansodium myristate or sodium oleate. No reason for this was ap-parent until the question of the physical condition of the soapswas taken into consideration. Solutions of these soaps of 0.5per cent concentration form soft gels very quickly, even at 370C.In making up a series of test dilutions it was necessary to meltthe stock solutions and measure out the quantity desired. Bythe time the soap was mixed with varying concentrations of thegermicide and the test made, some of the final mixtures were

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geled. This often gave results which made it appear that the soapinterfered with large amounts of the disinfectants to a greaterextent than with smaller amounts. It occurred to us that thismight be due entirely to the physical condition of the soap, andexperiments were devised to determine this point. When thetests were run before the soap had geled there was marked inter-ference with the disinfectant. When run after a gel had formed,there was less interference (see table 6). The extreme signifi-

TABLE 6Influence of physical condition of Na palmitate on germicidal properties

of butyl resorcinol, 870C.

A* BtPUR CDNT DILUTION OF

NPALMITATU BUTYL B. typhou B. typhoe8uNa BU~~~3ZSON&CINOL _ _ _ _ _ _ _ _ _ _ _

1 minute 2 minutes 1 minute 2 minutes

0.5 700 _ - _ _0.5 800 ++++ +0.5 900 ++++ +0.5 1,000 ++++ +0.5 1,200 ++++ ++0.5 1,400 ++++ ++0.5 1,600 ++++ +++ _ _0.5 1,800 ++++ ++++ - _

+ = growth. - = no growth.* A: Butyl resorcinol kept at 370C. Na palmitate added. Tests run before gel

formed.t B: Butyl resorcinol and Na palmitate mixed. Allowed to set for two hours at

room temperature. Temperature raised to 37°C. and tests run.

cance of this is realized when one considers that the detergentproperties of soaps parallel the amount of colloidal soap present(McBain, 1920).

DISCUSSION

The data in the foregoing pages bring up for considerationpoints of both theoretical and practical importance. Althoughno satisfactory and complete explanation for the effect of soapson the germicidal properties of phenols can be offered from astudy based solely on germicidal action of the disinfectants,

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the results stimulate speculation. Four different possibilitiespresent themselves:

1. The occurrence of a true chemical reaction between the soapand the phenolic compound, resulting in a non-germicidal sub-stance.

2. A protective colloidal action on the part of the soap for thebacteria as suggested by Frobisher (1927).

3. The ability of the soap to remove the phenolic substancefrom the solution in a definite partition-coefficient ratio.

4. A combination of physico-chemical factors which may ormay not have been expressed in the first three statements.

It would be a difficult matter to present any evidence to sup-port the first statement in a paper of this type with results basedon strictly bacteriological methods. However, in a long seriesof detailed experiments, which are not tabulated in this publica-tion, an effort was made to show a definite molecular relationshipbetween the amount of soap required to inactivate the phenolicsubstance and the amount of germicide present. The destruc-tion of viability of the bacteria was used as an index of the pres-ence of free germicide. As might be expected from a technicso unsuitable for this type of determination, the results wereirregular and indefinite in their conclusions.The theory of Frobisher offers an interesting and simple ex-

planation. The protective colloidal action of soaps has beenamply demonstrated by Papaconstantinou (1925) who assigneddefinite "gold numbers" to different pure soaps. If this idea isapplied directly to the case of soaps and phenolic substances, wewould pre-suppose a suspension of soap-in-water and phenoliccompound-in-water, to which would be added the bacterial cul-ture. The bacteria would be immediately surrounded by a filmof soap and hence protected from the action of the germicide.This theory might be easily evolved from working with phenolsonly in true solutions. However, when one handles super-saturated solutions of these compounds, other facts are observedwhich render so simple an explanation untenable. For instance,1 part of butyl phenol added to 99 parts of water is not a truesolution, but may be shaken into a fine suspension of oil globules

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in water. If one drop of 10 per cent sodium oleate is added to 1cc. of this mixture, the oil globules immediately disappear, andthe solution becomes clear. The same holds true for emulsions ofbutyl resorcinol and hexyl resorcinol. This can not be due to thefree alkali present in the soap, since it requires less sodium oleateto obtain a water clear solution of the germicide than sodiumcarbonate or sodium hydroxide. Furthermore, when solutionscontaining 1 per cent butyl phenol and 5, 4, 3, 2, 1 and 0.5 percent sodium oleate, respectively, are tested for germicidal activity,the first four solutions (containing the higher concentration ofsoap) are inactive in one minute against Bacillus typhosus. Theremaining two are germicidal. In the last tube, globules of oilare plainly visible, indicating a supersaturation of butyl phenolboth for the soap and for the water. When the experiment isrepeated with 0.5 per cent butyl phenol, the general results arethe same. The germicidal action of the disinfectant is evident ina concentration of soap slightly more than is necessary to hold thebutyl phenol in solution.The condition seems analagous to that of the well known

phenol-alcohol-water combination in which Reichel (1909) hasshown that although phenol in alcohol is non-germicidal, by de-creasing the amount of alcohol or by adding NaCl the partition-coefficient ratio of the phenol between the alcohol and the wateris increased for the water, and therefore more phenol is in solutionand the bacteria are killed. Efforts to increase the water solu-bility of at least one of the compounds, hexyl resorcinol, in thepresence of soap by adding sodium carbonate, sodium hydroxideand sodium chloride were unsuccessful. The recent work ofCooper and Sanders (1927) with sodium stearate and phenollends support to this hypothesis. These workers find a definitepartition-coefficient ratio between the concentration of phenolin water and in the soap. By increasing the amount of phenol,the increased phenol uptake with sodium stearate is associatedwith passage of the soap into solution.

It seems unlikely, however, that the actual explanation of theinhibitory effect of soaps on the bactericidal activity of phenols isfully accounted for by either, or both, the second and third

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hypotheses, since there are other factors which should be takeninto consideration. The very fact that soaps are colloidal elec-trolytes must necessarily complicate any simple explanation.The problem is one of theoretical importance and interest, and ourresults suggest that its final solution lies in the hands of thephysical chemist.From a practical standpoint, this work indicates the unsuita-

bility of phenolic compounds as germicidal agents in soaps. Agermicidal soap is expected to perform a double function. Itmust cleanse the surface, whether this surface be the skin, floors,or walls, etc. In skin disinfection, it should be expected to de-stroy whatever bacteria are mechanically washed off in the lather,as well as those remainuing on the skin; at the same time it shouldbe capable of some penetration into the depths of surface irregu-larities. In this discussion no consideration will be given to thequestion of penetration.Our experimental results indicate that the only possibility of

producing a germicidal soap with a phenolic compound, whichwill destroy the bacteria mechanically removed from the skin,lies in the addition of an excess of the disinfectant. The use ofthe lower or cruder phenols is ruled out because this class ofsubstances is not only poisonous but extremely iimtating to theskin in the concentrations which would be necessary to destroybacteria. Theoretically, it is possible to use a non-irritatingsubstance such as hexyl resorcinol in large enough quantities toyield a germicidal soap, but from a practical standpoint the costof such a product precludes its general use at present. Thefact that the disinfectant action of phenol is interfered with tosuch a pronounced extent by the disperse phase of the soap mustalso be taken into account. For example, a cake of soap con-taining as much as 5 per cent of a phenolic compound of highbactericidal efficiency would not be bactericidal when used forwashing the hands. McBain, cited by Fall (1927), has listedseven factors which are necessary for detergent action. Amongthese factors are the necessity of having the soap in solutionand the necessity of having the soap in colloidal form. It shouldbe emphasized that this colloidal or disperse phase has been

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shown to be more highly inhibitive to the germicidal action ofphenols than the soft gel phase. Obviously, as the soap isdiluted, the germicide is not only diluted, but at the same time itsactivity is interfered with more extensively. This fact alonealso eliminates the practicability of obtaining a liquid soap withgermicidal properties which could be attributed directly to aphenolic compound incorporated with it.

CONCLUSIONS

1. The marked inhibitory action of sodium oleate, sodiummyristate, potassium palmitate and potassium stearate on thebactericidal properties of phenol, meta-cresol, secondary butylphenol, n-butyl resorcinol and n-hexyl resorcinol has been demon-strated. The relationship appears to be more or less quantita-tive and indicates the impossibility of producing a germicidal soapby adding small quantities of a phenolic compound to soap. Theaddition of large quantities of a phenolic disinfectant is not fea-sible, since the cost of production precludes the use of any but verycrude and irritating phenols. Phenols are therefore unsuitableas disinfectant agents in the production of germicidal soaps.

2. The germicidal activity of a phenolic compound is inter-fered with more extensively by the disperse phase than by the gelphase of soaps.

3. Several theories to explain the inhibitive action of soaps onthe bactericidal activity of phenols have been discussed. Evi-dence has been introduced to show that the soap removes thephenolic substance from the solution and thereby interferes withits bactericidal activity, since this activity is dependent upon thesolution of the phenol in water.

REFERENCES

COOPER, E. A., AND SANDERS, E. 1927 Relations of phenol to proteins and othercolloids. Jour. Phys. Chem., 31, 1-22.

EGGERTH, A. H. 1927 The effect of pH on the germicidal action of soaps. Jour.Gen. Phys., 10,147-160.

FALL, P. H. 1927 Detergent action of soaps. Jour. Phys. Chem., 31, 809-849.FROBISHER, MARTIN, JR. 1927 Studies upon the relationship between surface

tension and the action of disinfectants, with special reference to hexyl-resorcinol. Jour. Bacteriol., 13, 163-182.

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HAMILTON, H. C. 1917 Facts and fallacies of disinfection. Amer. Jour. Pub.Health, 7, 282-295.

McBAIN, J. W. 1920 Third Report on Colloid Chem. Brit. Assoc. Adv. Science.PAPACONSTANTINOU, B. 1925 Protective action of soaps on Zsigmondy's gold

solutions. Jour. Phys. Chem., 29, 319-322.REICHEL, H. 1909 Zur Theorie der Desinfektion. Biochem. Ztschr., 22,

149-200.RETTGER, L. F., VALLEY, G. AND PLASTRIDGE, W. N. 1928 Studies on the dis-

infectant properties of certain alkyl phenols. Centralblatt f. Bak-teriologie, (in press).

TILLEY, F. W., AND SCHAEFFER, J. M. 1927 Germicidal efficiency of cocoanutoil and linseed oil soaps and of their mixtures with cresol. Jour. Infec.Dis., 37,358-367.

WALKER, J. E. 1924 The germicidal properties of chemically pure soaps. Jour.Infec. Dis., 35,557-566.

WALKER, J. E. 1925 The germicidal properties of soap. Jour. Infec. Dis., 37,181-192.

WALK1ER, J. E. 1926 The germicidal properties of soap. Jour. Infec. Dis., 38,127-130.

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