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J. Soc. CosmeticChemists,18, 191-198 (Mar. 4, 1967)

The Applicationof Microbiologyto CosmeticTestingSTANLEY W. OLSON, M.S.,*

PresentedSeptember0-1, 166, Seminar, New York City

Synopsis Sanitation and preservation hold the key to the control of microbial contaminationin cosmetic products. Ultraviolet radiation can be used industrially to control the build-upof micro flora in the stored deionized water that is utilized in the manufacture of cosmetics.A titration technique has been developed for measuring the relative antimicrobial activity oftest preservative systems in products. The technique employs varying dosages of selectedtest microbes which are inoculated into the test systems. The method has been found pre-dictive in that preservative systems inactivating high dosages of test microorganisms areeffective under practical conditions.

INTRODUCTIONCosmeticsneed not be completely free from nonpathogenicbac-teria and fungi, but the residualorganismspresentin any product at thetime producedmust be prevented from multiplying during the product's

shelfand uselife by an effectivepreservative (1).While the desired objective of a microbiological program in theproduction of cosmeticsis to develop "sterile" products, the desiredob-jective is not always readily attainable. "Sterile," as used in this con-text, means free from living microflora which can be detected by routinesterility tests. Ideally, cosmeticsshould be self-sterilizing against allmicrobesencounteredduringproduction,packaging,andusage: Whencompletesterility is not feasible,the cosmeticsmust be free of viablehuman pathogensand inhibitory againstresidualnonpathogens.Actively viable microorganismscan be deleterious to both the es-thetics and to the functional characteristics of cosmetic products.* Microbiology Section of The Toni Company, St. Paul, Minnesota.

191


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192 JOURNAL OF THE SOCIEFY OF COSMETIC CHEMISTS

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Figure 1. Hair conditioninglotion. Thesample on the left is sterile. Pseudo-monads are propagating in the other three

samples

Figure $. Hair styling gel. The sampleon the left is sterile. Aspergillus mold ispropagating in the sample on the right

Figure 2. Shampoo. Pseudomonadshave attacked the shampoo in both bottlesEffectson color,odor, emulsionstability, foaming,and clarity can bedemonstrated. Somesamplesfor illustration are as follows:Figure 1 showsfour samplesof an experimentalhair conditioninglotion. The sampleon the left is a sterilecontrol. Pseudomonadshavebeenallowedto propagatein the other three samples. The first con-taminatedsampleshowsan emulsionseparationdueto microbialattackon the nonionic emulsifiers. The last two contaminated samplesillus-trate discolorationdue to Pseudomonadpigmentation. Figure2 showstwosamplesof a grosslycontaminatedsodiumlauryl


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MICROBIOLOGY IN COSMETIC TESTING 193

sulfatetype'shampoo.Pseudomonadshaveattackedthe detergent,causingthe product to discolorand separate badly.Figure 3 illustrates what can happen to an inadequately preservedhair styling gel. The control sample on the left representsa clear gel.Mold growth in the sample on the right has caused the gel to becometurbid. Aspergillusmold was isolated from the turbid sample.To avoid problemsof this type, sanitation techniquesand preserva-tive methods need to be selected and employed carefully. They needto be monitored continuously to seek improvements in the systemschosenas they are required.

SANITATIONDuring production, common sourcesof microbial contamination incosmeticproducts are raw materials, equipment, and air.Since water for batch-making can be the major threat to productsterility, control over the sanitary quality of this water will be empha-

sized in this discussion.Under summer temperature storage conditions, demineralized ordeionizedwater can easily support bacterial populations as large as 10'

bacteria/mi. In a few casesas many as 106 baeteria/ml have beenobserved. To prevent gross pollution of the batch water supply, thepropagation of microflora coming from the undeionized water, thedeionizerunits, and the storagetanks must be controlled.Although radiation treatment of stored deionizedwater is not widelypracticed in the cosmeticindustry, it is potentially a valuable means forcontrolling water quality. This paper will stress the application ofradiation to water sanitation and specificallythe usesof ultraviolet (UV)radiation.Effective forms of ionizing radiation include ultraviolet light,cathoderays, and gamma rays. The target theory, hypothesizingelectronrays hitting a microbialcell causevital cell atoms to ionize,'";!hasbeenusedto explainthemicrobiocidaleffectof ionizingradiations(2). In this connection,Hollaender (3) has reported that, when germi-cidal effectiveness of ultraviolet is plotted against wavelength, theresultingcurve resembledthe absorptioncurve for nucleic acids.Mercury vapor sourcesof ultraviolet are classified(3) as either high-pressure (400-60,000 mm Hg) or low-pressure (0.004-0.02 mm Hg)lamps. The peak effectivenessof ultraviolet for microbiocidalactivityhas been shownby Luckiesh (4) to be at a wavelength around 2600 A,falling virtually to zero at 3200 A. Since low pressuremercury vapor


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194 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

lamps exhibit a high output of radiation at 2537 A, this type of lamp isvery efficient and is most commonlyusedindustrially; about 90% of theemittance from these lamps is microbiocidal.Studies conductedby Koller (5) showedthat the killing power ofUV is virtually unaffectedby temperature in the 5-37C range. Whilethe shapeof the ultraviolet effectivenesscurve is generally independentof the type of bacteria, the tendency to spore formation does greatlyinfluence the responsesin specific cases. Thus the spore-forming B.subtills is about 5-10 times as resistant to UV as E. coli. Molds andyeastsare usually 100-1000 times more resistantthan bacteria. For ex-ample, to obtain a 0.0001 survival ratio in water, a UV exposure of24,000 uw-sec/cm would be required for bacterial sporesand 192,000uw-sec/cm for fungi. "Survival ratio" is the fraction of the numberinitially present which survivesUV radiation.Koller (5) also notes that, in order to sterilize water effectively, thewater must have a high transmissionfor UV. In other words, the watermust be free from suspendedmatter which might shieldmicrobesfromradiation. The UV lamps may be installed in reflectors mounted overthe water surface. The tank shouldbe deep enoughto absorbpracticallyall the UV, since radiation absorbed by the walls is wasted. Arrange-ment of the water inlet and outlet shouldassurethorough mixing. Thedegreeof disinfection,the survival ratio, dependsupon the intensity ofthe source,the transmissiondepth, and the rate of water flow.An interesting point, also noted by Koller (5), is helpful to thecosmetic chemist: Those bacteria surviving irradiation are more sus-ceptibleto subsequentcidal treatment, being more easily killed by milddisinfectantsand exhibiting increasedsensitivity to heat.

It may be useful now to describea typical water sanitizingsystememployingultraviolet radiation. Our plant employssucha processthathas been in successfuloperation for a number of years. The deionizedcity water is continuouslyrecirculated from two 5000 gallon storagetanks through an 85 gallon stainlesssteel UV exposuretank at the rateof 180 gallons/min. The water bed in the exposuretank is 25 cm deep,90 cm wide, and 91 cm long. Bafflesare installed in the tank to decreasethe velocity of water flow at the bottom of the tank. This increasesUVexposuretime at the bottom. Mounted about 30 cm above the exposuretanks are seven General Electric (90 cm long) 30-watt UV lamps.These mlaps are spaced 13 cm apart and have specular aluminumreflectors. The lamps are low-pressuremercury lamps having a rated4000 hour life. Based on six lamps being operative, the calculated UV


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5:. .. , .a '.;:.-;?:::' . ;';Figure 4. UV exposure tanks. Storedddonized water is redralated continu-

ously through these knks

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Figure 5. A UV exposure tank with coverremoved. Seven specular aluminum re-fleetors are shown mounted over seven UV

lamps on top of the tank

exposureat the water's surfaceis 26,000 tw-sec/cm" at the water'smiddle it is 18,000, and at the water's bottom it is 17,000. A singlepassof the water through the UV exposuretank is calculated to result in asurvival ratio of 0.0016.

Figure 4 showsthe UV exposuretanks under actual operation. Thetank on the left treats stored deionizedcity water. An identical expo-sure tank on the right is used to treat stored deionized well water.A view of the UV lamps mounted over the recirculating water is shownin Fig. 5.When the UV treatment system for deionized city water was firstplaced in operation, the microbial count at zero time was 38,000 mi-crobes/mi. After / hour operation the count dropped to 3300;after 1/ hours to 390; after 2 hours to 120, and after 18 hours to 81.The system has been in almost continuousoperation sincethat time.

With periodiccleaningand sterilizingof the deionizedwater storagetanks, the countshave been kept at a relatively low level. Employingtight controls,the countscan be held to under 100 microbes/mi. Build-up of UV resistant organismsin the stored deionizedwater has not been aproblem.Periodic sterilization of the physical equipment--tanks, pipes andpumps--is requiredand desirable. Keepinga 2% solutionof hydrogenperoxide in contact with the equipment for a two-hour period has beeneffective. Deionizer beds are treated with formalin as the need arises.Cruickshank et al., (6) found that irrigation of ion exchange beds with0.25% formalin (0.1% formaldehyde)servedas an effectivedisinfectant.


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PRESERVATION

Even with good sanitation practices, cosmeticsmust be preservedto copewith contaminationencounteredduring production,packaging,and normal usage. As other workersin the field of cosmeticpreservationagree, test preservative systemsmust be evaluated in finished productformulations before final selectionof a preferred system is made.A useful technique for measuringthe relative antimicrobial activityof various preservative systemcandidatesis the inoculationof varyingconcentrationsof selectedtest organismsinto the test systems. Thistechniqueis predicatedon the hypothesisthat the higher the concentra-tion of selectedmicrobes a test system can inactivate the greater theefficacy of the system.This titration technique for evaluating the efficacy of preservationsystemsis illustrated in an application to a hair-conditioninglotion.The product was an o/w emulsionhaving a pH between 3.25 and 4.25;nonionic emulsifiers were utilized in the formula at a concentration ofapproximately4%.Illustrative of a few of the preservatives evaluated in this productwere: (a) 0.1% methyl p-hydroxybenzoate(MP), (b) 0.2% MP, and(c) 0.2% benzoicacid. It shouldbe understoodthat these compoundsare but a small sampleof thosenormally employedin screeningpreserva-fives. Among the test organismsinoculatedinto the test systemswas aPseudomonadthat had been isolated from a contaminated experimentalbatch of the product preservedwith 0.1% MP.A pure culture of the test Pseudomonadwas taken from a nutrientagar slant and allowed to propagate for 24 hours at 30-32C in nutrientbroth. The resulting suspendedculture was centrifuged and washedthree times with buffered distilled water (pH 7.2). Serial dilutions ofthe washed culture were prepared in buffered distilled water (pH 7.2).One ml aliquot of each dilution were inoculated into 9 ml aliquots ofeach preserved product.Sterility testing of the inoculatedproduct samplesshowedthat 0.1%MP inactivated levels of the test Pseudomonadup to about 10S/ml ofproduct. The product with 0.2% MP inactivated dosagesup to about107. The system containing 0.2% benzoic acid appeared bactericidalagainstthe highestinoculumtested,i.e., 10O/mlof product.Under practice conditions, Pseudomonad contamination was a re-curring problem with the product containing only 0.1% MP. Theproduct containing 0.2% benzoic acid has consistentlybeen producedin a sterile condition.


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The basic titration techniqueemployed successfullyon the hair con-ditioning lotion has been applied successfullyto the development ofpreservative systemsfor a wide variety of cosmeticproducts. Includedin thesestudieshave been a variety of both o/w and w/o emulsionsaswell as dispersionsand solutions. A broad spectrumof bacteria andfungi has been utilized in these evaluationsof preservative systemsintest products.In addition to Ps. aeruginosa,initial screeningevaluations of testpreservative systemsutilize representativesof the gram positive cocci,gram positive rods, and molds. If contamination has been a problempreviouslywith a product similar to the one under study, pure culturesof these contaminants are also employed in the screeningprogram.Thus, a typical screening study might include a Pseudomonad (Ps.aeruginosa),an Aspergillusmold (Aspergillusniger), a Bacillus (Bacilluscirculans),a Micrococcus(Micrococcuspyogenesv. aureus),and anyorganismsisolatedfrom productssimilar to the one under test.The three most effectivepreservativesystems,judged by the resultsof the screeningtests, are then subjectedto more extensiveinoculationstudies. During the extensivetesting phase,organismssuchas Candidaalbicans, Cephalosporiurnsp., Corynebacteriumpseudodiphthericum,Es-cherichiacoli, Fusarium oxysporum,Penicillium sp., and Streptococcusfaecalis are utilized.

The preferred preservativesystemis selectednot only on the basisof its antimicrobialefficacybut on its productcompatibilityand medicalacceptability as well.SUMMARY

As a protection to both the cosmeticproductsand the cosmeticusers,adequate microbiological controls are an important part of a cosmetictesting program.Contamination of the deionized water utilized in production can beminimized by ultraviolet radiation. UV treatment of the water accom-panied by periodicchemicaldecontaminationof the equipmentcan holdcounts on stored water to under 100 mmrobes/ml.Residual microorganismsfinding their way into the finishedcosmeticproductsare controlledthrough the developmentof effective preserva-tive systems. A titration techniquehas been developedto measuretheantimicrobial activity of preservative systemsin products. This tech-nique employsvarying dosagesof test microbes. The resultsindicate


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that preservative systems inactivating high dosagesof test microbesare adequate under production conditions.

ACKNOWLEDGMENTThe author gratefully acknowledgesthe assistanceof the followingco-workersin the preparation of this paper: Mr. Aaron Efron, Chem-ical Engineer; Mr. Carl Fraction, Microbiologist; and Miss BarbaraWarncoke, Secretary.

(ReceivedSeptember20, 1966)REFERENCES

(1) Wedderburn, D. L., Hygiene in manufacturing plant and its effect on the preservationof emulsions,J. Soc. CosmeticChemists,16,395-403 (June, 1965).(2) Foster, E. M., et al., Dairy Microbiology,Prentice-Hall, Inc., New York, 1957, pp. 97.(3) Hollaender, A., Radiation Biology, Vol. II, McGraw-Hill Book Company, New York, 1955,pp. 55.

(4) Luckiesh, M., Germicidal, Erythemal and Infra-red Energy, D. Van Nostrand Co., NewYork, 1946.

(5) Koller, L. R., Ultraviolet Radiation, 2nd Edition, John Wiley and Sons, Inc., New York,1965, pp. 236-256.(6) Cruickshank, O. A., and Braithwaite, D. O., Sterilization of cation exchange resins,sulfonatedphenol formaldehyde type, Ind. Eng. Chem.,41,472 (1949).

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