- Email: [email protected]
The effect of eugenol and oil of cloves on the growth of microorganisms
THE
EFFECT
HENRY
OF EUGENOL AND OIL OF CLOVES GROWTH OF MICROORGANISMS A. BARTELS,
B.S., D.D.S.,”
NEW YORK,
ON THE
N. Y.
PICES and their essential oils have been used since antiquity. S in foodstuffs, they were the means of inhibiting bacterial and thus preventing food spoilage.
Incorporated fermentations McCullochl records that the ancient Egyptians employed spices or their oils in embalming. Molnar2 claims cloves was first used about the middle of the sixteenth century to alleviate toothache. Prior to this time, it had been employed to (‘sweeten the breath.” It also was given as a remedy for gastric disorders and even placed in wounds to aid healing. Such usage, however, was strictly empirical. Scientific investigations of these substances could not be made until sufficient knowledge of bacteria and the methods of experimentation with them had accumulated. Thus, it was not until 188’7 that Chamberland evaluated the antibacterial properties of a number of essential oils. This author, according to Topley and Wilson,3 exposed anthrax bacteria and their spores to these oils in both gaseous and liquid states. He found that cinnamon, majorurn, sandalwood, clove, and juniper were the most effective of the essential oils. Topley and Wilson further report that Cadeac and Meunier, in 1889, determined the antiseptic potency of various essential oils against E. typhi and Pf. mall& It was found that whereas the control mercuric chloride, 1 :l,OOO, destroyed these microorganisms in ten minutes, oil of cinnamon required twelve minutes and oil of cloves twenty-five minutes. Other essential oils destroyed these microorganisms only after exposure for several days, and in some instances were even ineffective after contact for ten days. The relative antibacterial potency of various essential oils was estimated by Peck,4 1898, as follows: cassia, 1:2,100, Ceylon cinnamon, 1:2,133, cloves, 1:1,150, bay, 1:1,028, peppermint, 1:875, eucalyptol, 1:120, and eugenol, no effect. This latter finding, as will be shown later, is erroneous. MaWhinney,s in 1900, obtained inconsistent results in his examination of the inhibitive qualities of the essential oils and concluded that as germicides these oils were greatly overrated. Whether contamination played a role in his conflicting results should be given consideration. The preservation of foodstuffs by means of spices or their oils was studied by Hoffmann and Evans,6 1911, who observed that cinnamon, cloves, and mustard were all effective as inhibitors of bacterial growth. Eugenol, 1 part per 1,000 applesauce, prevented spoilage during a fourteen-month period of storage. Bachmann 1916, also studied the preservative qualities of spices and found cloves, allspice, and cinnamon effective in preventing growth of molds and bac*Department of Pathology, School of Dental and Oral SurgerY of the Faculty of Medicine, Columbia University.
458
E#ect
of Eugenol
and Oil of Cloves
459
teria. This investigator futher noted that bacteria in general were less sensitive than molds to these spices and that individual species of both groups of microorganisms varied in t,heir sensitivity. Thus, he found that B. subtilis did not grow upon an agar medium which contained oil of cloves in a dilution of 1300, while S. macrescens was only slightly sensitive to its presence. Fabian, Kehl, and Little,* 1939, investigated the role of spices in pickled food spoilage and observed that ground cinnamon and cloves were the only spices that exhibited antibacterial action in low dilution. Oil of cloves was found to restrain the growth of B. subtilis in a dilution of 1:lOO and the Staphylococcus aureus in 1:800. Castell,g 1944, also noted that in low dilutions some of the spices, especially cloves and cassia, had inhibitory qualities. He doubted, however, if such dilutions could be used in practical application. Bullock and Lightbrown,lO 1942, advised the addition of clove oil or cinnamon oil to infusions of quassia to prevent spoilage due to bacterial cont.amination. The effect of essential oils upon pathogenic yeasts and molds has also been investigated. Myers and Thienes, I*, I2 1925, isolated from patients suffering with a dermatitis, a yeastlike microorganism. In comparison to 1 per cent phenol, which required sixty minutes to destroy this microorganism, they found that thymol killed in one minute, cinnamon oil in thirty minutes, and clove oil in ninety minutes. Kingery and Adkisson,13 1928, tested a variety of pathogenic fungi against various essential oils and found the most effective in fungi&da1 activity were thymol, oil of cinnamon, and oil of cloves. Oil of cloves was found to possess a phenol coefficient of 8.0 by Rideal, Rideal and Sciver,14 1928. Cinnamon, cassia, senfol, lemon grass, and cloves were found by Collier and Nitta,ls 1930, to be the most effective of 106 essential oils tested. Wide variation in the dilution of the oil which inhibited growth was noted with different bacterial species. Clove oil restrained the growth of a hemolytic streptococcus in a dilution of 1:1,600, a hemolytic staphylococcus in 1:800, a mixture of five gonococcal strains in 1:1,200, B. coli in 1:800, and the Vibrio Nasik in 1:600. More recently, Hettche and Rosenthal,‘6 1936, tested twenty-four essent,ial oils and observed that the oils of sandalwood, hop, cloves, and cassia possessed the most potent antibacterial properties. A review of the literature pertaining to the antiseptic, anesthetic, and therapeutic properties of some of the essential oils was made by Petrie,17 1938. This author tried various combinations of essential oils as sterilizing and palliative agents in tooth cavities. For shallow cavities he advocated mixtures of oils which vaporize rapidly, such as peppermint, lemon, eucalyptus, wintergreen, and aniseed in 90 per cent alcohol. Deep cavities, he claimed, should first be painted with oil of citronella and then a pledget of cotton saturated with an alcoholic solution of oils of lemon, thymol, cloves, and cassia should be sealed in the cavity. Recently, Shaw, Sprawson, and May, Is 1945, stated that zinc oxide with Bartels,lg 1946, found that low-unit eugenol destroyed penicillin completely. dilutions lost most or all of their inhibitory activity toward Staphylococus aureus when mixed with eugenol zinc oxide cement, but that high-unit concentrations, though affected, still possessed definit,e growth-restraining action.
460
Henry
A. Bade&s
This review of the literature indicates bacteriology, mycology, medicine, dentistry, certain essential oils to possess antibacterial component, eugenol, is one of the more active
that investigators in the fields of and food preservation have found properties. Oil of cloves and its of these oils.
EXPERIMENTAL
The following methods were employed in testing the antibacterial qualities of oil of cloves and eugenol: 1. Agar cup plate. Melted infusion agar was cooled to 45’ C. and seeded with Staphylococcus aureus or B. coli. Plates were poured and when the agar was solid, small holes were bored in the center of each plate with either a lipless test tube or a cork borer. Oil of cloves or eugenol was placed in these agar depressions. A clear zone surrounding the test substance was indicative of restraint of microbic growth. 2. Infusion agar, 100 ml. amounts, was adjusted to pH 6.0, pH 7.0, and pH 8.0, respectively. Oil of cloves or eugenol was added to each lot to give a concentration of 0.5 per cent. Plates were poured and after solidification, onehalf of the medium was removed and replaced with plain infusion agar of the same pH reaction. The surface of the medium was inoculated in parallel streaks with B. subtilis, Staphylococcus aureus, B. coli and B. pyocyaneus. Each streak of inoculum was begun on the plain infusion agar portion and carried over onto the agar containing the clove oil or eugenol. Antibacterial properties were indicated by the restraint of growth on the clove agar and the plain infusion agar neighboring it. (See Table I and Figs. 1 and 2.) Tasrx
I.
Tm
EFFWC
OF Ckovs
OIL
AND EUGFNK
IN
AGAR
MEDIUMS
OF DIFFERENT
PH
REACTIONS GROWTH MICROORGANISM
REACTlON
FXTGENOL
subtilis subtilis
pH pH pH
6.0 7.0 8.0
B. B. B.
pH pH pH
6.0 7.0 8.0
Staphylococcus Staphylococcus Staphylococcus
pH pH pH
6.0 7.0 8.0
B.
coli
B. B.
coli coli
pH pH pH
6.0 7.0 8.0
tttt tttt tttt
s‘ubtilis aweus aweus aurew
ttt ttt ++-It++ tt tt
INHIBITION AGAR
IN
MM.
/ CLOVES1
tttt ttt ttt tt ttt tt tt tt tt Unaffected Unaffected Unaffected
H. pyocyaneus
Unaffected
Unaffected
Unaffected Unaffected
TJnaffected
*Inhibition of 16 to ++. B. pyoc?lalteus grew or oil of cloves. tTwo manufacturers’
-
ADJACENT
Unaffected
20 mm. designated by + + t +; 11 to 1.7 mm. by +++; 6 to 10 well even upon the portion of medium containing either the brands
of
TO”
1 CLOVESzAGARt
tttt ttt ttt tt tt tt tt tt tt
R. H.
pyocyaneus pyocyaneus
ZONE
AGARt
mm. by eugenol
cloves.
3. Agar in test tubes, cooled to 45O C., was seeded with various microorganisms and poured in plates. Small amounts of eugenol alone or mixed with zinc oxide or zinc oxide alone were placed on the surface of the medium. Zones of growth inhibition about the test materials indicated sensitivity of the microorganisms. (See Table 11,)
Fig. l.-Forty-eight er half plate (control Note, B. pyocyamus other microorganisms
Upper half plate (test portion). infusion agar plus 0.5 per cent eugenol. Lmvhours’ incubation. A, B, wbtilis; B, Staphylococcus aweus; C, B. coli; D, B. pyocyaWUS. portion), plain infusion agar. grows on the test portion. Note also the absence of growth along heavy inoculated streak 1lneS of on test portion and adjacent control portion. (See results, Table I.)
462
Henry
A. Bartels
Eflect TABLE
II.
EFFECT
ON GROWTH
of Eugenol OF
NIOROORGANISMS ZINC OXIDE
OF EUGENOL, GROWTH EUGENOL
MICROORGANISM
Staphylococms aweus Staphylococcus albzls Staphylococcus citreus Serratia mmcesoens (Col. strain) (Feb. strain) Monilia albicnn.~ B. subtilis IC’. typhi E. coli B. Friedliinder B. protezls B. pyocyanms (Col. strain) (New York University strain) (New York State Laboratorv strain) ‘Area measured was from the periphery
463
rind Oil of Cloves EUGENOL-ZINC
INHIBITION
AND
IN MILLIMETERS*
EUGENOLZINC OXIDE
ZINC
OXIDE
65 5
: 5
i 0
(i
5 5 7 5 7 5 3 4
i 0 0 0 0 0 0
: 0
0 0 0 development.
6 9 5 9 : 5
of test
OXIDE,
: 0 material
to region
of colony
4. Infusion agar plates. were inoculated in parallel streaks with four test microorganisms mentioned previously. In the center of each streak was placed either zinc oxide or zinc oxide with eugenol. Interruption of growth along the streak lines in the vicinit,y of the test material indicated inhibitory activity. (See Fig. 3.) 5. Eugenol, 10 per cent, in aqueous suspension, alcoholic solution, or detergent (oxydon) suspension was added in variable amounts to 10 ml. volumes of infusion broth, which previously was inoculated either with Staphylococcus aureus or B. pyocyaneus. RESULTS
The following information was obtained from the preceding experiments : 1. Microorganisms vary in their sensitivity toward eugenol or oil of cloves. E. typhi and Monil& al&cans (Candida albicans) were affected to a greater extent than the other microorganisms tested. B. pyocyaneus (Ps. aewginosa) was the most resistant. Three strains of this bacterium obtained from different sources (Columbia University, New York University, and New York State Laboratory) were tested and all showed similar resistance toward oil of cloves or eugenol. Other studies are being made on this subject. 2. Variation in hydrogen-ion reaction between pH 6.0, pH 7.0, and pH 8.0 had little effect on t,he inhibitory qualities of either oil of cloves or eugenol. 3. Zinc oxide has no apparent antibacterial effect in the dried state on Even when added to infusion broth in 1 per cent amounts, microorganisms. it did not cause destruction of either Staphylococcus aureus or B. pyocyaneus during a three-day period of exposure. 4. Oil of cloves or eugenol when incorporated into a paste with zinc oxide still possesses definite inhibitive properties. 5. Eugenol in alcoholic solution was more effective than when suspended in water or detergent. This no doubt is due to t,he fact that the aqueous and de-
Fig. 3.-Forty-eight hours’ incubation infusion agar. A, coli. Note that zinc oxide does not restrain growth, whereas growth of microorganisms with exception of B. pyocyaneus.
B.
pyocyaneus; B, zinc oxide-eugenol
B.
~02~: paste,
C, Staphylococcus large or small
aureus~ amount.
D,, B. mhlbits
E@ect of Eugenol and Oil of CEov.es tergent suspensions either separate out in the form of globules or sediment at the bottom of the tubes of broth, and so act only in a restricted area of the broth, allowing growth to occur in the upper part of the tube. 6. Oil of cloves and eugenol were comparable in their antibacterial properties. Peck’s observation that eugenol was without effect was due possibly to use of an inferior brand of eugenol or to the presence of a resistant microorganism in specimens of saliva which he employed as inoculum. Our results with B. pyocyaneus indicate this latter possibility. SUMMARY
Bacterial species vary in their individual sensitivity to either oil of cloves or eugenol. B. pyocyaneus (Ps. aeruginosa) was markedly resistant. Variation in hydrogen-ion concentration had no appreciable effect on the activity of clove oil or eugenol. Incorporated in a paste with zinc oxide, eugenol, or oil of cloves still retains definite antibacterial properties. REFERENCES 1. McCulloch. E. C.: Disinfection and Sterilization. Philadeluhia. 1936. Lea & Febiger,v pp. 16, 482, and 510 Cloves-Oil of Cloves-Eugenol; Their Medico-Dental History, Dental 2. Molnar, E. J.: Items Interest 64: 521, 662, 745, 876, 971, 1942. 3. Topley, W. W, and Wilson, G. S.: The Principles of Bacteriology and Immunity, ed. 2, Baltimore, 1936, William Wood & Company, pp. 117. 4. Peck, A. H.: The Essential Oils and Some Other Agents; Their Antiseptic Value, Also Their Irritating and Nonirritating Properties, Dent. Rev. 12: 593, 1898. 5. MaWhinney, E. : Antiseptics and Disinfectants, Trans. Illinois State Dent. Sot., pp. 125, 1900. 6. Hoffmann, C., and Evans, A. C.: The Use of Spices as Preservatives, J. Ind. & Eng. Chem. 3: 835, 1911. F. M.: The Inhibiting Action of Certain Suices on the Growth of Micro7. Bachmann. organisms, J. Ind. & Eng. Chem. 8: 620, 1916. I F. W., Kehl, C. F., and Little, N. W.: 8. Fabian, The Role of Spices in Pickled Food Spoilage, Food Research 4: 269, 1939. Thermophilic Bacteria in Foods and in Various Ingredients Entering 9. Castell, C. H.: Into the Manufacture of Food, Food Research 9: 410, 1944. K., and Lightbrown, J. W.: 10. Bullock, The Preservation of Concentrated Infusions, Quart. J. Pharm. & Pharmacol. 15: 51, 1942. H. B., and Thienes, C. H.: The Fungicidal Activity of Certain Volatile Oils, 11. Myers, J. A. M. A. 84: 1985, 1925. An Unappreciated Fungicidal Activity of Certain Volatile Oils, J. A. 12. Myers, H. B.: M. A. 89: 1834. 1927. A.: Certain Volatile Oils and Stearoptens as Fungi13. Kingery, L. B., and Adkisson, cides, Arch. Dermat. & Syph. 17: 499, 1928. 14. Rideal, S., Rideal, E. K., and Sciver, A.: Investigations Into the Germicidal Powers and Capillary Activities of Certain Essential Oils, Perfum. Ess. Oil Rec. 19: 285, 1928. 15. Collier, W. A., and Nitta, Y.: The Effect of Ethereal Oils Upon Different Species of Bacteria, Ztschr. f. Hyg. u. Infektionskr. 111: 301, 1930. H. O., and Rosenthal, P.: Investigation of the Bactericidal and Bacterio16. Hettche, static Activity of Volatile Oils, Arch. f. Hyg. 115: 302, 1936. 17. Petrie, J. M.: The Essential Oils in the Treatment and Sterilization of Live Dentine, Dent. Record 58: 250, 1938. 18. Shaw, F. E., Sprawson, E., and May, H. B.: Use of Penicillin in Dental Pulp Canal Therapy, Brit. M. J. 1: 551, 1945. The Effect of Zinc Oxide Eugenol Cement on Penicillin, Ann. J. 19. Bartels, H. A.: ORTHODONTICS AND ORAL SURG. 32: 344,1946. 20. Ruehle, G. L., and Brewer, C. M.: United States Food and Drug Administration Methods of Testing Antiseptics and Disinfectants, Circular 198, 1931, U. S. Department of Agriculture, Washington, D. C. L
‘
EFFECT
HENRY
OF EUGENOL AND OIL OF CLOVES GROWTH OF MICROORGANISMS A. BARTELS,
B.S., D.D.S.,”
NEW YORK,
ON THE
N. Y.
PICES and their essential oils have been used since antiquity. S in foodstuffs, they were the means of inhibiting bacterial and thus preventing food spoilage.
Incorporated fermentations McCullochl records that the ancient Egyptians employed spices or their oils in embalming. Molnar2 claims cloves was first used about the middle of the sixteenth century to alleviate toothache. Prior to this time, it had been employed to (‘sweeten the breath.” It also was given as a remedy for gastric disorders and even placed in wounds to aid healing. Such usage, however, was strictly empirical. Scientific investigations of these substances could not be made until sufficient knowledge of bacteria and the methods of experimentation with them had accumulated. Thus, it was not until 188’7 that Chamberland evaluated the antibacterial properties of a number of essential oils. This author, according to Topley and Wilson,3 exposed anthrax bacteria and their spores to these oils in both gaseous and liquid states. He found that cinnamon, majorurn, sandalwood, clove, and juniper were the most effective of the essential oils. Topley and Wilson further report that Cadeac and Meunier, in 1889, determined the antiseptic potency of various essential oils against E. typhi and Pf. mall& It was found that whereas the control mercuric chloride, 1 :l,OOO, destroyed these microorganisms in ten minutes, oil of cinnamon required twelve minutes and oil of cloves twenty-five minutes. Other essential oils destroyed these microorganisms only after exposure for several days, and in some instances were even ineffective after contact for ten days. The relative antibacterial potency of various essential oils was estimated by Peck,4 1898, as follows: cassia, 1:2,100, Ceylon cinnamon, 1:2,133, cloves, 1:1,150, bay, 1:1,028, peppermint, 1:875, eucalyptol, 1:120, and eugenol, no effect. This latter finding, as will be shown later, is erroneous. MaWhinney,s in 1900, obtained inconsistent results in his examination of the inhibitive qualities of the essential oils and concluded that as germicides these oils were greatly overrated. Whether contamination played a role in his conflicting results should be given consideration. The preservation of foodstuffs by means of spices or their oils was studied by Hoffmann and Evans,6 1911, who observed that cinnamon, cloves, and mustard were all effective as inhibitors of bacterial growth. Eugenol, 1 part per 1,000 applesauce, prevented spoilage during a fourteen-month period of storage. Bachmann 1916, also studied the preservative qualities of spices and found cloves, allspice, and cinnamon effective in preventing growth of molds and bac*Department of Pathology, School of Dental and Oral SurgerY of the Faculty of Medicine, Columbia University.
458
E#ect
of Eugenol
and Oil of Cloves
459
teria. This investigator futher noted that bacteria in general were less sensitive than molds to these spices and that individual species of both groups of microorganisms varied in t,heir sensitivity. Thus, he found that B. subtilis did not grow upon an agar medium which contained oil of cloves in a dilution of 1300, while S. macrescens was only slightly sensitive to its presence. Fabian, Kehl, and Little,* 1939, investigated the role of spices in pickled food spoilage and observed that ground cinnamon and cloves were the only spices that exhibited antibacterial action in low dilution. Oil of cloves was found to restrain the growth of B. subtilis in a dilution of 1:lOO and the Staphylococcus aureus in 1:800. Castell,g 1944, also noted that in low dilutions some of the spices, especially cloves and cassia, had inhibitory qualities. He doubted, however, if such dilutions could be used in practical application. Bullock and Lightbrown,lO 1942, advised the addition of clove oil or cinnamon oil to infusions of quassia to prevent spoilage due to bacterial cont.amination. The effect of essential oils upon pathogenic yeasts and molds has also been investigated. Myers and Thienes, I*, I2 1925, isolated from patients suffering with a dermatitis, a yeastlike microorganism. In comparison to 1 per cent phenol, which required sixty minutes to destroy this microorganism, they found that thymol killed in one minute, cinnamon oil in thirty minutes, and clove oil in ninety minutes. Kingery and Adkisson,13 1928, tested a variety of pathogenic fungi against various essential oils and found the most effective in fungi&da1 activity were thymol, oil of cinnamon, and oil of cloves. Oil of cloves was found to possess a phenol coefficient of 8.0 by Rideal, Rideal and Sciver,14 1928. Cinnamon, cassia, senfol, lemon grass, and cloves were found by Collier and Nitta,ls 1930, to be the most effective of 106 essential oils tested. Wide variation in the dilution of the oil which inhibited growth was noted with different bacterial species. Clove oil restrained the growth of a hemolytic streptococcus in a dilution of 1:1,600, a hemolytic staphylococcus in 1:800, a mixture of five gonococcal strains in 1:1,200, B. coli in 1:800, and the Vibrio Nasik in 1:600. More recently, Hettche and Rosenthal,‘6 1936, tested twenty-four essent,ial oils and observed that the oils of sandalwood, hop, cloves, and cassia possessed the most potent antibacterial properties. A review of the literature pertaining to the antiseptic, anesthetic, and therapeutic properties of some of the essential oils was made by Petrie,17 1938. This author tried various combinations of essential oils as sterilizing and palliative agents in tooth cavities. For shallow cavities he advocated mixtures of oils which vaporize rapidly, such as peppermint, lemon, eucalyptus, wintergreen, and aniseed in 90 per cent alcohol. Deep cavities, he claimed, should first be painted with oil of citronella and then a pledget of cotton saturated with an alcoholic solution of oils of lemon, thymol, cloves, and cassia should be sealed in the cavity. Recently, Shaw, Sprawson, and May, Is 1945, stated that zinc oxide with Bartels,lg 1946, found that low-unit eugenol destroyed penicillin completely. dilutions lost most or all of their inhibitory activity toward Staphylococus aureus when mixed with eugenol zinc oxide cement, but that high-unit concentrations, though affected, still possessed definit,e growth-restraining action.
460
Henry
A. Bade&s
This review of the literature indicates bacteriology, mycology, medicine, dentistry, certain essential oils to possess antibacterial component, eugenol, is one of the more active
that investigators in the fields of and food preservation have found properties. Oil of cloves and its of these oils.
EXPERIMENTAL
The following methods were employed in testing the antibacterial qualities of oil of cloves and eugenol: 1. Agar cup plate. Melted infusion agar was cooled to 45’ C. and seeded with Staphylococcus aureus or B. coli. Plates were poured and when the agar was solid, small holes were bored in the center of each plate with either a lipless test tube or a cork borer. Oil of cloves or eugenol was placed in these agar depressions. A clear zone surrounding the test substance was indicative of restraint of microbic growth. 2. Infusion agar, 100 ml. amounts, was adjusted to pH 6.0, pH 7.0, and pH 8.0, respectively. Oil of cloves or eugenol was added to each lot to give a concentration of 0.5 per cent. Plates were poured and after solidification, onehalf of the medium was removed and replaced with plain infusion agar of the same pH reaction. The surface of the medium was inoculated in parallel streaks with B. subtilis, Staphylococcus aureus, B. coli and B. pyocyaneus. Each streak of inoculum was begun on the plain infusion agar portion and carried over onto the agar containing the clove oil or eugenol. Antibacterial properties were indicated by the restraint of growth on the clove agar and the plain infusion agar neighboring it. (See Table I and Figs. 1 and 2.) Tasrx
I.
Tm
EFFWC
OF Ckovs
OIL
AND EUGFNK
IN
AGAR
MEDIUMS
OF DIFFERENT
PH
REACTIONS GROWTH MICROORGANISM
REACTlON
FXTGENOL
subtilis subtilis
pH pH pH
6.0 7.0 8.0
B. B. B.
pH pH pH
6.0 7.0 8.0
Staphylococcus Staphylococcus Staphylococcus
pH pH pH
6.0 7.0 8.0
B.
coli
B. B.
coli coli
pH pH pH
6.0 7.0 8.0
tttt tttt tttt
s‘ubtilis aweus aweus aurew
ttt ttt ++-It++ tt tt
INHIBITION AGAR
IN
MM.
/ CLOVES1
tttt ttt ttt tt ttt tt tt tt tt Unaffected Unaffected Unaffected
H. pyocyaneus
Unaffected
Unaffected
Unaffected Unaffected
TJnaffected
*Inhibition of 16 to ++. B. pyoc?lalteus grew or oil of cloves. tTwo manufacturers’
-
ADJACENT
Unaffected
20 mm. designated by + + t +; 11 to 1.7 mm. by +++; 6 to 10 well even upon the portion of medium containing either the brands
of
TO”
1 CLOVESzAGARt
tttt ttt ttt tt tt tt tt tt tt
R. H.
pyocyaneus pyocyaneus
ZONE
AGARt
mm. by eugenol
cloves.
3. Agar in test tubes, cooled to 45O C., was seeded with various microorganisms and poured in plates. Small amounts of eugenol alone or mixed with zinc oxide or zinc oxide alone were placed on the surface of the medium. Zones of growth inhibition about the test materials indicated sensitivity of the microorganisms. (See Table 11,)
Fig. l.-Forty-eight er half plate (control Note, B. pyocyamus other microorganisms
Upper half plate (test portion). infusion agar plus 0.5 per cent eugenol. Lmvhours’ incubation. A, B, wbtilis; B, Staphylococcus aweus; C, B. coli; D, B. pyocyaWUS. portion), plain infusion agar. grows on the test portion. Note also the absence of growth along heavy inoculated streak 1lneS of on test portion and adjacent control portion. (See results, Table I.)
462
Henry
A. Bartels
Eflect TABLE
II.
EFFECT
ON GROWTH
of Eugenol OF
NIOROORGANISMS ZINC OXIDE
OF EUGENOL, GROWTH EUGENOL
MICROORGANISM
Staphylococms aweus Staphylococcus albzls Staphylococcus citreus Serratia mmcesoens (Col. strain) (Feb. strain) Monilia albicnn.~ B. subtilis IC’. typhi E. coli B. Friedliinder B. protezls B. pyocyanms (Col. strain) (New York University strain) (New York State Laboratorv strain) ‘Area measured was from the periphery
463
rind Oil of Cloves EUGENOL-ZINC
INHIBITION
AND
IN MILLIMETERS*
EUGENOLZINC OXIDE
ZINC
OXIDE
65 5
: 5
i 0
(i
5 5 7 5 7 5 3 4
i 0 0 0 0 0 0
: 0
0 0 0 development.
6 9 5 9 : 5
of test
OXIDE,
: 0 material
to region
of colony
4. Infusion agar plates. were inoculated in parallel streaks with four test microorganisms mentioned previously. In the center of each streak was placed either zinc oxide or zinc oxide with eugenol. Interruption of growth along the streak lines in the vicinit,y of the test material indicated inhibitory activity. (See Fig. 3.) 5. Eugenol, 10 per cent, in aqueous suspension, alcoholic solution, or detergent (oxydon) suspension was added in variable amounts to 10 ml. volumes of infusion broth, which previously was inoculated either with Staphylococcus aureus or B. pyocyaneus. RESULTS
The following information was obtained from the preceding experiments : 1. Microorganisms vary in their sensitivity toward eugenol or oil of cloves. E. typhi and Monil& al&cans (Candida albicans) were affected to a greater extent than the other microorganisms tested. B. pyocyaneus (Ps. aewginosa) was the most resistant. Three strains of this bacterium obtained from different sources (Columbia University, New York University, and New York State Laboratory) were tested and all showed similar resistance toward oil of cloves or eugenol. Other studies are being made on this subject. 2. Variation in hydrogen-ion reaction between pH 6.0, pH 7.0, and pH 8.0 had little effect on t,he inhibitory qualities of either oil of cloves or eugenol. 3. Zinc oxide has no apparent antibacterial effect in the dried state on Even when added to infusion broth in 1 per cent amounts, microorganisms. it did not cause destruction of either Staphylococcus aureus or B. pyocyaneus during a three-day period of exposure. 4. Oil of cloves or eugenol when incorporated into a paste with zinc oxide still possesses definite inhibitive properties. 5. Eugenol in alcoholic solution was more effective than when suspended in water or detergent. This no doubt is due to t,he fact that the aqueous and de-
Fig. 3.-Forty-eight hours’ incubation infusion agar. A, coli. Note that zinc oxide does not restrain growth, whereas growth of microorganisms with exception of B. pyocyaneus.
B.
pyocyaneus; B, zinc oxide-eugenol
B.
~02~: paste,
C, Staphylococcus large or small
aureus~ amount.
D,, B. mhlbits
E@ect of Eugenol and Oil of CEov.es tergent suspensions either separate out in the form of globules or sediment at the bottom of the tubes of broth, and so act only in a restricted area of the broth, allowing growth to occur in the upper part of the tube. 6. Oil of cloves and eugenol were comparable in their antibacterial properties. Peck’s observation that eugenol was without effect was due possibly to use of an inferior brand of eugenol or to the presence of a resistant microorganism in specimens of saliva which he employed as inoculum. Our results with B. pyocyaneus indicate this latter possibility. SUMMARY
Bacterial species vary in their individual sensitivity to either oil of cloves or eugenol. B. pyocyaneus (Ps. aeruginosa) was markedly resistant. Variation in hydrogen-ion concentration had no appreciable effect on the activity of clove oil or eugenol. Incorporated in a paste with zinc oxide, eugenol, or oil of cloves still retains definite antibacterial properties. REFERENCES 1. McCulloch. E. C.: Disinfection and Sterilization. Philadeluhia. 1936. Lea & Febiger,v pp. 16, 482, and 510 Cloves-Oil of Cloves-Eugenol; Their Medico-Dental History, Dental 2. Molnar, E. J.: Items Interest 64: 521, 662, 745, 876, 971, 1942. 3. Topley, W. W, and Wilson, G. S.: The Principles of Bacteriology and Immunity, ed. 2, Baltimore, 1936, William Wood & Company, pp. 117. 4. Peck, A. H.: The Essential Oils and Some Other Agents; Their Antiseptic Value, Also Their Irritating and Nonirritating Properties, Dent. Rev. 12: 593, 1898. 5. MaWhinney, E. : Antiseptics and Disinfectants, Trans. Illinois State Dent. Sot., pp. 125, 1900. 6. Hoffmann, C., and Evans, A. C.: The Use of Spices as Preservatives, J. Ind. & Eng. Chem. 3: 835, 1911. F. M.: The Inhibiting Action of Certain Suices on the Growth of Micro7. Bachmann. organisms, J. Ind. & Eng. Chem. 8: 620, 1916. I F. W., Kehl, C. F., and Little, N. W.: 8. Fabian, The Role of Spices in Pickled Food Spoilage, Food Research 4: 269, 1939. Thermophilic Bacteria in Foods and in Various Ingredients Entering 9. Castell, C. H.: Into the Manufacture of Food, Food Research 9: 410, 1944. K., and Lightbrown, J. W.: 10. Bullock, The Preservation of Concentrated Infusions, Quart. J. Pharm. & Pharmacol. 15: 51, 1942. H. B., and Thienes, C. H.: The Fungicidal Activity of Certain Volatile Oils, 11. Myers, J. A. M. A. 84: 1985, 1925. An Unappreciated Fungicidal Activity of Certain Volatile Oils, J. A. 12. Myers, H. B.: M. A. 89: 1834. 1927. A.: Certain Volatile Oils and Stearoptens as Fungi13. Kingery, L. B., and Adkisson, cides, Arch. Dermat. & Syph. 17: 499, 1928. 14. Rideal, S., Rideal, E. K., and Sciver, A.: Investigations Into the Germicidal Powers and Capillary Activities of Certain Essential Oils, Perfum. Ess. Oil Rec. 19: 285, 1928. 15. Collier, W. A., and Nitta, Y.: The Effect of Ethereal Oils Upon Different Species of Bacteria, Ztschr. f. Hyg. u. Infektionskr. 111: 301, 1930. H. O., and Rosenthal, P.: Investigation of the Bactericidal and Bacterio16. Hettche, static Activity of Volatile Oils, Arch. f. Hyg. 115: 302, 1936. 17. Petrie, J. M.: The Essential Oils in the Treatment and Sterilization of Live Dentine, Dent. Record 58: 250, 1938. 18. Shaw, F. E., Sprawson, E., and May, H. B.: Use of Penicillin in Dental Pulp Canal Therapy, Brit. M. J. 1: 551, 1945. The Effect of Zinc Oxide Eugenol Cement on Penicillin, Ann. J. 19. Bartels, H. A.: ORTHODONTICS AND ORAL SURG. 32: 344,1946. 20. Ruehle, G. L., and Brewer, C. M.: United States Food and Drug Administration Methods of Testing Antiseptics and Disinfectants, Circular 198, 1931, U. S. Department of Agriculture, Washington, D. C. L
‘