Microbiological Methods for Cosmetics.docx
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Microbiological Methods for Cosmetics.docx
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MicrobiologicalMethodsforCosmetics
BAM:
MicrobiologicalMethodsforCosmetics
August2001
BacteriologicalAnalyticalManual
Chapter23
MicrobiologicalMethodsforCosmetics
Authors:
AnthonyD.Hitchins,TonyT.Tran,andJamesE.McCarron
Foradditionalinformation,contact RebeccaBell
RevisionHistory:
∙August2001:
Section:
IdentificationofMicrobes.RevisedPartDandaddedreference2b.
∙August2001:
M79formulationcorrected.
Theabilityofmicroorganismstogrowandreproduceincosmeticproductshasbeenknownformanyyears.Microorganismsmaycausespoilageorchemicalchangesincosmeticproductsandinjurytotheuser(4,5,10,14-16,20,21).Methodsforisolationofmicroorganismsfromcosmeticproductsaredirectcolonycountsandenrichmentculturing.Productsthatarenotsolubleinwaterareinitiallytreatedtorenderthemmisciblebeforeisolationproceduresareconducted.Dilutionandplatingmediathatpartiallyinactivatepreservativesystemscommonlyfoundincosmeticproductsareused.Theisolatedmicroorganismsareidentifiedbyroutinemicrobiologicalmethodsorbycommercialidentificationkits.TheschemefortheseanalysesissummarizedinFig.1.
A.Equipmentandmaterials
1.Pipets,sterile,1,5,and10ml,graduated
2.Gauzepads,sterile,4x4inch
3.Sterileinstruments:
forceps,scissors,scalpelandblades,spatulas,andmicrospatulas
4.Testtubes,screw-cap,13x100,16x125,and20x150mm
5.Dilutionbottles,screw-cap
6.Balance,sensitivityof0.01g
7.Petridishes,sterile,plastic,15x100mm
8.Bentglassrods,sterile
9.Incubators,30±2°Cand35±2°C
10.Anaerobicatmospheregeneratingenvelopes,indicatorstrips,andjars(BBLorOxoid),oranaerobicincubator,35±2°C,oranaerobicglovebox,35±2°C.
11.CandlejarsorCO2 incubator,35±2°C.
12.LaminarflowhoodwithHEPAfilter,ifavailable
13.Vitekorequivalentautomatedcomputerizedidentificationsystem
B.Media forenumerationandidentificationofGram-positivebacteriaandfungi
1.Anaerobeagar(M11)
2.Bileesculinagar(M18)
3.Brainheartinfusion(BHI)agarandbroth(M24)
4.Maltextractagar(MEA)(M93)
5.Potatodextroseagar(PDA)(M127)
6.Mannitolsaltagar(M97)
7.Modifiedletheenagar(MLA)(M78)andbroth(MLB)(M79)
8.Oxidative-fermentative(OF)testmedium(M117)
9.Sabouraud'sdextrosebroth(M133)
10.Bloodagarbase(M20a)
11.Starchagar(M143)
12.Trypticase(tryptic)soyagar(TSA)(M152)andbroth(TSB)(M154)
13.Baird-Parker(BP)agar(M17)
14.Catalasetest(R12)
15.Vogel-Johnson(VJ)agar(optional)(M176)
16.Commercialbacterialidentificationkit(APIorequivalent)
Fig.1:
SchemeforEnumeration,Isolation,andIdentificationofCosmeticMicrobes
∙Samplepreparation.
∙DilutepreparedsamplesinMLB.
∙Spreadduplicate0.lmlsampleson
(a)
(b)
(c)
(d)
MLA
48h,30°C
PDA(orMEA)
withchlortetracycline
7days,30°C
BP(orVJ)agar
48h,35°C
(optional)
Anaerobicagar
MLA
2-4days,35°C
∙EnrichMLBdilutionsfor7days,at30°C.PurifygrowthonlyifnocoloniesonMLA.
∙CountcoloniesandsubculturedifferentcolonytypesonMLAandMacConkeyagar(andBPorVJagarsifusedinc,above).Forfungalisolates, see text.
∙DetermineGramreaction,cellshape,andcatalaseproductionofpurifiedisolates.
∙Proceedwithidentificationofbacterialisolatesasdescribedintext,oruseidentificationkits.
Fig.l.Abbreviations:
MLB,modifiedletheenbroth;MLA,modifiedletheenagar;PDA,potatodextroseagar;MEA,maltextractagar;BP,Baird-Parker;VJ,Vogel-Johnson.
C.Media foridentificationof Enterobacteriaceae
1.Andrade'scarbohydratebrothandindicator(M13)fortestingmetabolismofrhamnose,mannitol,sorbitol,arabinose
2.Lysineironagar(M89)
3.Malonatebroth(M92)orphenylalaninemalonatebroth(Difco)
4.Motility-indoleornithinemedium(M99)
5.MR-VPbroth(M104)
6.Simmonscitrateagar(M138)
7.Triplesugariron(TSI)agar(M149)
8.Christensen'sureaagar(M40)
9.MacConkeyagar(M91)
10.LysinedecarboxylasemediumforGram-negativenonfermentativebacteria(M88)
11.Phenylalaninedeaminaseagar(M123)(seealsoC-3,above)
12.API20E,RocheEnterotube,orotherequivalentidentificationkits
IncubateallbiochemicaltestsusingmediainBandC,above,at35-37°Cfor18-24h,exceptmalonatebroth(48h)andMR-VPbroth(48horlonger).
D.Media and reagents foridentificationofGram-negativenonfermentative(NF)bacilli
1.Acetamidemedium(M2)
2.Clark'sflagellarstain(R14)
3.Esculinagar,modified(CDC)(M53)
4.Nutrientgelatin(CDC)(M115)
5.Indolemedium(M64)andindolemedium(CDC)(M65)
6.King'sBmedium(M69)
7.Lysinedecarboxylase(LDC)mediumforGram-negativeNFbacteria(M88)
8.Motilitynitratemedium(M101)
9.Nitratebroth,enriched(CDC)(M109)
10.King'sOFbasalmedium(M70)fortestin
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