testes de microbiologia- bam - fda

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http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm073!".htm

BAM: Aerobic Plate CountJanuary 2001

Bacteriological Analytical ManualChapter 3

Aerobic Plate CountAuthors: Larry Maturin and James T. Peeler

For additional information, contact John Bryce.

Chapter Contents• Conventional Plate Count Method

• !iral Plate Method

• "eferences

The aero#ic !late count $%PC& is intended to indicate the level of microor'anism in a !roduct. (etailed !rocedures for determinin'the %PC of foods have #een develo!ed #y the %ssociation of )fficial %nalytical Chemists $%)%C& $*& and the %merican Pu#lic+ealth %ssociation $%P+%& $1&. The conventional !late count method for eaminin' fro-en, chilled, !recooed, or !re!ared foods,outlined #elo/, conforms to %)%C Official Methods of Analysis, sec. .2*, /ith one !rocedural chan'e $.2*C&. The suita#lecolony countin' ran'e $10& is 2320. The automated s!iral !late count method for the eamination of foods and cosmetics $&,outlined #elo/, conforms to %)%C Official Methods of Analysis, sec. 44.24. For !rocedural details of the standard !late count, seeref. 2.5uidelines for calculatin' and re!ortin' !late counts have #een chan'ed to conform /ith the antici!ated chan'es in the 1thedition of Standard Methods for the Examination of Dairy Products $2& and the International Dairy Federation $6(F& !rocedures $&.

Conventional Plate Count Method %. Equipment and materials1. 7or area, level ta#le /ith am!le surface in room that is clean, /ell3li'hted $100 foot3candles at /orin' surface&

and /ell3ventilated, and reasona#ly free of dust and drafts. The micro#ial density of air in /orin' area, measured in fallout !our!lates taen durin' !latin', should not eceed 1 colonies8!late durin' 1 min e!osure.

2. tora'e s!ace, free of dust and insects and ade9uate for !rotection of e9ui!ment and su!!lies*. Petri dishes, 'lass or !lastic $at least 1 0 mm&:. Pi!ets /ith !i!et aids $no mouth !i!ettin'& or !i!ettors, 1, , and 10 ml, 'raduated in 0.1 ml units

. (ilution #ottles, o- $10 ml&, #orosilicate3resistant 'lass, /ith ru##er sto!!ers or !lastic scre/ ca!s. Pi!et and !etri dish containers, ade9uate for !rotection4. Circulatin' /ater #ath, for tem!erin' a'ar, thermostatically controlled to : ; 1


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resultin' in a lo/ count. "e!ort counts less than 2 or more than 20 colonies as estimated aero#ic !late counts $%PC&. se thefollo/in' 'uideD

1. Aormal !lates $2320&. elect s!reader3free !late$s&. Count all colony formin' units $CF&, includin' those of!in!oint si-e, on selected !late$s&. "ecord dilution$s& used and total num#er of colonies counted.

2. Plates /ith more than 20 colonies. 7hen num#er of CF !er !late eceeds 20, for all dilutions, record thecounts as too numerous to count $TATC& for all #ut the !late closest to 20, and count CF in those !ortions of !late that arere!resentative of colony distri#ution. ee ref. 2 for detailed 'uidelines. Mar calculated %PC /ith %PC to denote that it /asestimated from counts outside 2320 !er !late ran'e $see (3*&.

*. !readers. !readin' colonies are usually of * distinct ty!esD 1& a chain of colonies, not too distinctly se!arated,that a!!ears to #e caused #y disinte'ration of a #acterial clum!> 2& one that develo!s in film of /ater #et/een a'ar and #ottom of

dish> and *& one that forms in film of /ater at ed'e or on surface of a'ar. 6f !lates !re!ared from sam!le have ecessive s!reader'ro/th so that $a& area covered #y s!readers, includin' total area of re!ressed 'ro/th, eceeds 0E of !late area, or $#& area ofre!ressed 'ro/th eceeds 2E of !late area, re!ort !lates as s!readers. 7hen it is necessary to count !lates containin' s!readersnot eliminated #y $a& or $#& a#ove, count each of the * distinct s!reader ty!es as one source. For the first ty!e, if only one chaineists, count it as a sin'le colony. 6f one or more chains a!!ear to ori'inate from se!arate sources, count each source as onecolony. (o not count each individual 'ro/th in such chains as a se!arate colony. Ty!es 2 and * usually result in distinct coloniesand are counted as such. Com#ine the s!reader count and the colony count to com!ute the %PC.

:. Plates /ith no CF. 7hen !lates from all dilutions have no colonies, re!ort %PC as less than 1 times thecorres!ondin' lo/est dilution used. Mar calculated %PC /ith asteris to denote that it /as estimated from counts outside the 2320 !er !late ran'e. 7hen !late$s& from a sam!le are no/n to #e contaminated or other/ise unsatisfactory, record the result$s& asla#oratory accident $L%&.

(. Computing and recording counts #see refs $ %&

To avoid creatin' a fictitious im!ression of !recision and accuracy /hen com!utin' %PC, re!ort only the f irst t/o si'nificant di'its."ound off to t/o si'nificant fi'ures only at the time of conversion to PC. For mil sam!les, /hen !lates for all dilutions have nocolonies, re!ort %PC as less than 2 colonies estimated count. "ound #y raisin' the second di'it to the net hi'hest num#er /henthe third di'it is , 4, =, or and use -eros for each successive di'it to/ard the ri'ht from the second di'it. "ound do/n /hen thethird di'it is 1, 2, *, or :. 7hen the third di'it is , round u! /hen the second di'it is odd and round do/n /hen the second di'it iseven.

E'amples

Calculated Count APC

12,700 13,000

12,400 12,000

15,500 16,000

14,500 14,000

1. Plates /ith 2320 CF.

a. Calculate the %PC as follo/sD

/hereDA Aum#er of colonies !er ml or ' of !roductG C um of all colonies on all !lates countedn1 Aum#er of !lates in first dilution countedn2 Aum#er of !lates in second dilution countedd (ilution from /hich the first counts /ere o#tained

E'ample

1:100 1:1000

232, 244 33, 28

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*480.022 2:,:0H 2:,000

#. 7hen counts of du!licate !lates fall /ithin and /ithout the 2320 colony ran'e, use only those countsthat fall /ithin this ran'e.

2. %ll !lates /ith fe/er than 2 CF. 7hen !lates from #oth dilutions yield fe/er than 2 CF each, record actual!late count #ut record the count as less than 2 I 18d /hen d is the dilution factor for the dilution from /hich the first counts /ereo#tained.

E'ample

Colonies

1:100 1:1000 EAPC/ml (g)

18 2 6,500,000 EAPC(b)

TNTC 6,490 >5,900,000 EAPC

a Based on !late area of cm2# %PC, estimated %PCc

Based on !late area of cm2

.piral Plate MethodThe s!iral !late count $PLC& method for microor'anisms in mil, foods, and cosmetics is an official method of the %P+% $2& andthe %)%C $*&. 6n this method, a mechanical !later inoculates a rotatin' a'ar !late /ith li9uid sam!le. The sam!le volume dis!enseddecreases as the dis!ensin' stylus moves from the center to the ed'e of the rotatin' !late. The micro#ial concentration isdetermined #y countin' the colonies on a !art of the !etri dish /here they are easily counta#le and dividin' this count #y thea!!ro!riate volume. )ne inoculation determines micro#ial densities #et/een 00 and 00,000 microor'anisms8ml. %dditionaldilutions may #e made for sus!ected hi'h micro#ial concentrations.

%. Equipment and materials1. !iral !later $!iral ystems 6nstruments, 6nc., 4=*0 )ld 5eor'eto/n "oad, Bethesda, M( 20=1:&2. !iral colony counter $!iral ystems& /ith s!ecial 'rid for relatin' de!osited sam!le volumes to s!ecific !ortions

of !etri dishes*. acuum tra! for dis!osal of li9uids $23: liter vacuum #ottle to act as vacuum reservoir and vacuum source of 030 cm +'&

:. (is!osa#le micro #eaers, ml. Petri dishes, !lastic or 'lass, 10 1 mm or 100 1 mm

http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063346.htm#r2-standardhttp://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063346.htm#r2-standardhttp://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063346.htm#r3-officialhttp://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063346.htm#r2-standardhttp://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063346.htm#r3-official


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. Plate count a'ar $standard methods& $M12:&4. Calculator $o!tional&, ine!ensive electronic hand calculator is recommended=. Polyethylene #a's for storin' !re!ared !lates. Commercial sodium hy!ochlorite solution, a#out E Aa)Cl $#leach&10. terile dilution /ater 11. yrin'e, /ith Luer ti! for o#structions in stylus> ca!acity not critical12. 7or area, stora'e s!ace, refri'erator, thermometers, tally, incu#ator, as descri#ed for Conventional Plate Count

Method, a#ove.1*. odium hy!ochlorite solution $.2E&. %vaila#le commercially.B. Preparation of agar plates/

%utomatic dis!enser /ith sterile delivery system is recommended to !re!are a'ar !lates. %'ar volume dis!ensed into !lates isre!roduci#le and contamination rate is lo/ com!ared to hand3!ourin' of a'ar in o!en la#oratory. 7hen !ossi#le, use laminar airflo/ hood alon' /ith automated dis!enser. Pour same 9uantity of a'ar into all !lates so that same hei'ht of a'ar /ill #e !resentedto s!iral !later stylus ti! to maintain contact an'le. %'ar !lates should #e level durin' coolin'.

The follo/in' method is su''ested for !re!ourin' a'ar !latesD se automatic dis!enser or !our constant amount $a#out 1 ml8100mm !late> 0 ml810 mm !late& of sterile a'ar at 0340


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+. To chec total volume dis!ensed #y s!iral !later, /ei'h amount dis!ensed from stylus ti!. Collect in tared ml !lastic#eaer and /ei'h on analytical #alance $; 0.2 m'&.

6.

J. Fi'ure 1. 10 cm !late, area $*#&

.

L. E'amination and reporting of spiral plate counts/

Countin' rule of 20. %fter incu#ation, center s!iral !late over 'rid #y adKustin' holdin' arms on vie/er. Choose any /ed'e and#e'in countin' colonies from outer ed'e of first se'ment to/ard center until 20 colonies have #een counted. Com!lete #y countin'

remainin' colonies in se'ment /here 20th colony occurs. 6n this countin' !rocedure, num#ers such as *#, :c $Fi'. l& refer to arease'ments from outer ed'e of /ed'e to desi'nated arc line. %ny count irre'ularities in sam!le com!osition are controlled #ycountin' the same se'ments in the o!!osite /ed'e and recordin' results. am!le of s!irally inoculated !late $Fi'. l& demonstratesmethod for determinin' micro#ial count. T/o se'ments of each /ed'e /ere counted on o!!osite sides of !late /ith *1 and *0colonies, res!ectively. The sam!le volume contained in the darened se'ments is 0.001 ml. To estimate num#er ofmicroor'anisms, divide count #y volume contained in all se'ments counted. ee eam!le under Fi'. l.

6f 20 CF are not /ithin the : se'ments of the /ed'e, count CF on entire !late. 6f the num#er of colonies eceeds 4 in second,third, or fourth se'ment, /hich also contains the 20th colony, the estimated num#er of microor'anisms /ill 'enerally #e lo/#ecause of coincidence error associated /ith cro/din' of colonies. 6n this case, count each circumferentially adKacent se'ment inall = /ed'es, countin' at least 0 colonies, e.'., if the first 2 se'ments of a /ed'e contain 1 colonies and the third se'mentcontains the 20th and 4th $or more&, count colonies in all circumferentially adKacent first and second se'ments in all = /ed'es.Calculate contained volume in counted se'ments of /ed'es and divide into num#er of colonies.

7hen fe/er than 20 colonies are counted on the total !late, re!ort results as less than 00 estimated PLC !er ml. 6f colony

count eceeds 4 in first se'ment of /ed'e, re!ort results as 'reater than 00,000 estimated PLC !er ml. (o not count s!iral!lates /ith irre'ular distri#ution of colonies caused #y dis!ensin' errors. "e!ort results of such !lates as la#oratory accident $L%&. 6f s!reader covers entire !late, discard !late. 6f s!reader covers half of !late area, count only those colonies that are /ell distri#utedin s!reader3free areas.

Com!ute PLC unless restricted #y detection of inhi#itory su#stances in sam!le, ecessive s!reader 'ro/th, or la#oratoryaccidents. "ound off counts as descri#ed in 63(, a#ove. "e!ort counts as PLC or estimated PLC !er ml.

1eferences1. %merican Pu#lic +ealth %ssociation. 1=:. Com!endium of Methods for the Micro#iolo'ical amination of Foods, 2nd ed.

%P+%, 7ashin'ton, (C2. %merican Pu#lic +ealth %ssociation. 1*. tandard Methods for the amination of (airy Products, 1th ed. %P+%,

7ashin'ton, (C.*. %ssociation of )fficial %nalytical Chemists. 10. )fficial Methods of %nalysis, 1th ed. %)%C, %rlin'ton, %.:. (onnelly, C.B., J.. 5ilchrist, J.T. Peeler, and J.. Cam!#ell. 14. !iral !late count method for the eamination of ra/

and !asteuri-ed mil. Appl. Environ. Microbiol. 3)D21324.. 5ilchrist, J.., C.B. (onnelly, J.T. Peeler, and J.. Cam!#ell. 144. Colla#orative study com!arin' the s!iral !late and

aero#ic !late count methods. . Assoc. Off. Anal. !hem. $+D=043=12.. 6nternational (airy Federation. 1=4. Mil and Mil ProductsD numeration of Microor'anismsNColony Count at *


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4. Jarvis, B., .+. Lach, and J.M. 7ood. 144. valuation of the s!iral !late maer for the enumeration of microor'anisms infoods. . Appl. "acteriol. 23D1:314.

=. Aiemela, . 1=*. tatistical evaluation of "esults from ?uantitative Micro#iolo'ical aminations. "e!ort Ao. 1, 2nd ed.Aordic Committee in Food %nalysis, !!sala, /eden.

. Tomasie/ic-, (.M., (.. +otchiss, 5.7. "ein#old, ".B. "ead, Jr., and P.%. +artman. 1=0. The most suita#le num#er ofcolonies on !lates for countin'. . Food Prot. 23D2=232=.

10. Oi!es, M."., J.. 5ilchrist, and J.T. Peeler. 1=1. Com!arison of yeast and mold counts #y s!iral, !our, and strea !latemethods. . Assoc. Off. Anal. !hem. $2D1:31:.

+y!ertet ourceD Bacteriolo'ical %nalytical Manual, dition =, "evision %, 1=. Cha!ter *.

BAM: Microbiological Methods for Cosmetics %u'ust 2001

Bacteriological Analytical ManualChapter )3Microbiological Methods for CosmeticsAuthors: %nthony (. +itchins, Tony T. Tran, and James . McCarron

For additional information, contact "e#ecca Bell

1evision istory:

• %u'ust 2001D ectionD 6dentification of Micro#es. "evised Part ( and added reference 2#.

• %u'ust 2001D M4 formulation corrected.

The a#ility of microor'anisms to 'ro/ and re!roduce in cosmetic !roducts has #een no/n for many years. Microor'anisms maycause s!oila'e or chemical chan'es in cosmetic !roducts and inKury to the user $:,,10,1:31,20,21&. Methods for isolation ofmicroor'anisms from cosmetic !roducts are direct colony counts and enrichment culturin'. Products that are not solu#le in /aterare initially treated to render them misci#le #efore isolation !rocedures are conducted. (ilution and !latin' media that !artiallyinactivate !reservative systems commonly found in cosmetic !roducts are used. The isolated microor'anisms are identified #yroutine micro#iolo'ical methods or #y commercial identification its. The scheme for these analyses is summari-ed in Fi'. 1.

%. 9ui!ment and materials1. Pi!ets, sterile, 1, , and 10 ml, 'raduated

2. 5au-e !ads, sterile, : : inch*. terile instrumentsD force!s, scissors, scal!el and #lades, s!atulas, and micros!atulas:. Test tu#es, scre/3ca!, 1* 100, 1 12, and 20 10 mm. (ilution #ottles, scre/3ca!. Balance, sensitivity of 0.01 '

mailto:rebecca.bellmailto:rebecca.bell


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4. Petri dishes, sterile, !lastic, 1 100 mm=. Bent 'lass rods, sterile. 6ncu#ators, *0 ; 2 ML%, modified letheen a'ar> P(%, !otato detrose a'ar> M%, malt etract a'ar>BP, Baird3Parer> J, o'el3Johnson.

C. Media for identification of Enterobacteriaceae1. %ndrade@s car#ohydrate #roth and indicator $M1*& for testin' meta#olism of rhamnose, mannitol, sor#itol,

ara#inose

2. Lysine iron a'ar $M=&*. Malonate #roth $M2& or !henylalanine malonate #roth $(ifco&:. Motility3indole ornithine medium $M&. M"3P #roth $M10:&. immons citrate a'ar $M1*=&4. Tri!le su'ar iron $T6& a'ar $M1:&=. Christensen@s urea a'ar $M:0&. MacConey a'ar $M1&10. Lysine decar#oylase medium for 5ram3ne'ative nonfermentative #acteria $M==&11. Phenylalanine deaminase a'ar $M12*& $see also C63 above&

12. %P6 20, "oche nterotu#e, or other e9uivalent identification its

6ncu#ate all #iochemical tests usin' media in B and C, a#ove, at *3*4


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. 6ndole medium $M:& and indole medium $C(C& $M&. in'@s B medium $M&4. Lysine decar#oylase $L(C& medium for 5ram3ne'ative AF #acteria $M==&=. Motility nitrate medium $M101&. Aitrate #roth, enriched $C(C& $M10&10. in'@s )F #asal medium $M40& for testin' meta#olism of sucrose, lactose, fructose, esculin, ylose, 'lucose

$detrose&, mannitol, salicin, sor#itol, and maltose11. )idase test stri!s12. Christensen@s urea a'ar $M:0&1*. (ecar#oylase #asal medium $for ar'inine decar#oylase& $M::&

1:. east etract $& a'ar $M1=1&1. Pseudomonas a'ars F $M12= and P $M12& $(ifco&1. Cetrimide a'ar $PseudoselTM, BBL> (ifco&, or e9uivalent $M*4&14. 5lycerol, sterile $(ifco&, or e9uivalent1=. %P6, AFT, or other e9uivalent commercial identification system1. oser@s citrate #roth $M42&. )ther media and rea'ents1. %9ueous solution of 40E ethanol and 1E +Cl $v8v& or :E iodine in 40E ethanol solution or 2E 'lutaraldehyde

solution2. T/een =0 $Polysor# =0&*. thanol, E $v8v&:. Lyo!hili-ed ra##it coa'ulase !lasma /ith (T%. *E $v8v& %9ueous solution of hydro'en !eroide. 5ram stain $"*2& and endos!ore stain $"*2a&4. Cooed meat medium $M:2&

F. +andlin' of cosmetic sam!les for micro#iolo'ical analysis

%naly-e sam!les as soon as !ossi#le after their arrival. 6f necessary, store sam!les at room tem!erature. (o not incu#ate,refri'erate, or free-e sam!les #efore or after analysis. 6ns!ect sam!les carefully #efore o!enin' and note any irre'ularities ofsam!le container. (isinfect surface of sam!le container /ith a9ueous miture of 40E ethanol $v8v& and 1E +Cl $v8v& or otherdisinfectant $see 3l& #efore o!enin' and removin' contents. se laminar flo/ hood if !ossi#le. (ry surface /ith sterile 'au-e#efore o!enin'. se re!resentative !ortion of contents for micro#ial analysis, e.'., 1 ' $ml& !ortion of sam!le.

For !roducts /ei'hin' less than 1 ' $ml&, analy-e entire contents. 6f only one sam!le unit is availa#le and multi!le analyses arere9uested $i.e., micro#ial, toicolo'ical, and chemical&, tae su#sam!le for micro#iolo'ical eamination #efore those for otheranalyses. 6n this situation, amount of su#sam!le used for micro#ial analysis /ill de!end on other analyses to #e !erformed. Foream!le, if total sam!le content is ml, use 1 or 2 ml !ortion for micro#ial analyses.

5. Preliminary sam!le !re!aration

%mounts of sam!le and diluent 'iven here can #e adKusted accordin' to amount of sam!le availa#le. 6f sam!le has manysu#sam!les, amount of test material can #e increased and /orload streamlined #y com!ositin'. %nalysts should use their #est Kud'ment as to /hen and ho/ much material to com!osite.

1. 7iquids. (ecimally dilute 1 ml li9uid directly into ml modified letheen #roth $MLB& in 20 10 mm scre/3ca!test tu#e for the 1031 dilution.

2. .olids and po(ders. %se!tically remove and /ei'h 1 ' sam!le into 20 10 mm scre/3ca! test tu#e containin'1 ml sterile T/een =0. (is!erse !roduct in T/een =0 /ith sterile s!atula. %dd = ml sterile MLB and mi thorou'hly. This /ill #e the1031 dilution.

*. Cream and oil6based products. %se!tically remove and /ei'h 1 ' sam!le into 20 10 mm scre/3ca! tu#econtainin' 1 ml sterile T/een =0 !lus five to seven 3mm 'lass #eads $or ten to f ifteen *3mm 'lass #eads&. Mi total contents /ithorte mier. %dKust total volume to 10 ml /ith sterile MLB $= ml& for the 10 31 dilution.

:. Aerosols of po(ders soaps liquids and other materials. (econtaminate no--le of s!ray can as much as!ossi#le #y s/a##in' /ith 'au-e !ad moistened /ith 40E $v8v& a9ueous ethanol. !el some !roduct to flush out no--le> thens!ray a!!ro!riate amount into tared dilution #ottle, e.'., 1 ' of !roduct into ml sterile MLB. Thorou'hly mi !roduct and #roth, and

re/ei'h. This /ill #e a 1031dilution if eactly 1 ' of sam!le /as o#tained.. Anhydrous materials. Treat as in 532 or 53*, as a!!ro!riate.

+. Micro#iolo'ical evaluations

Aot all analyses descri#ed #elo/ /ill necessarily #e !erformed> ho/ever, the aero#ic !late count, enrichment culture, and thefun'al count should #e !erformed routinely.

1. Aerobic plate count #APC&. se s!read !late techni9ue to facilitate reco'nition of different colony ty!es and, ifnecessary, for differential count. %lso use Baird3Parer $BP& or o'el3Johnson $J& a'ar if Staphylococcus s!!. are sus!ected.Pre!are and la#el du!licate sets of !etri dishes containin' modified letheen a'ar $ML%& and BP a'ar for sam!les of 1031 to 103 dilutions. %dd either or 10 ml of !re!ared cosmetic !re!aration $see 5, a#ove& to : or 0 ml, res!ectively, of MLB, for 1032 dilution.

(ilute sam!les decimally in MLB $89ED save dilutions for enrichment ste!& to o#tain com!lete dilution series from 10 31 to 103.$Be'in /ith 1032 if all the 1031 dilution is used u!.& Thorou'hly mi dilutions and !i!et 0.1 ml of each dilution onto surfaces of solidmedia in !rela#eled !etri dishes. !read inoculum over entire surface /ith #ent 'lass rod that /as first sterili-ed #y di!!in' in Eethanol and 9uicly flamed to remove the ethanol. Let medium a#sor# inoculum #efore invertin' and incu#atin' !lates for := h at*0 ; 2


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Count all colonies in !lates containin' 2320 colonies, and record results !er dilution counted. %vera'e the colony countso#tained, and multi!ly the avera'e #y 10 and then #y the a!!ro!riate dilution factor $10 31 3 103&. "e!ort results as %PC8' $ml&sam!le. 6f !lates do not contain 2320 colonies, record dilution counted and note num#er of colonies found.

For BP !lates, count /ell3distri#uted colonies that are conve, shiny #lac, either /ith or /ithout a clear -one surroundin' thecolony. $89ED Coa'ulase3!ositive colonies !roduce clearin', #ut coa'ulase3ne'ative colonies may or may not !roduce clearin'. 6fcoa'ulase3ne'ative colonies clear, their irre'ularity re!ortedly distin'uishes them from coa'ulase3!ositive colonies.& elect !lateshavin' more than 20 colonies /hen those at a 'reater dilution do not contain colonial ty!es descri#ed a#ove. Plates from minimaldilutions havin' fe/er than 2 colonies may also #e used if necessary. From each BP !late demonstratin' 'ro/th, !ic one or morety!ical colonies to confirm their coa'ulase reaction. Transfer colonies to a'ar slants of any suita#le maintenance medium, e.'.,try!ticase $try!tic& soy a'ar $T%&, #rain heart infusion $B+6& a'ar. 6ncu#ate slants until 'ro/th is evident.

Calculate num#er of Staphylococcus aureus or'anisms !resent #y first determinin' the fraction of colonies tested that arecoa'ulase !ositive. Multi!ly this fraction #y avera'e num#er of Staphylococcus colonies counted on the BP !lates. Multi!ly thenum#er o#tained #y the a!!ro!riate dilution factor and re!ort as num#er of S. aureus8' $ml& sam!le.

6f no colonies are o#tained on ML% or BP media, o#serve already !re!ared MLB dilutions /hile enrichin' them at *0 ; 2


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Further characteri-ation of 5ram3!ositive rods is 'enerally unnecessary. Consult refs. 4 and 12 if further characteri-ation of theseor'anisms is re9uired.

2. "ram6positive cocci. trea ML% !late from %PC media $ML% or BP&, incu#ate 1=32: h at * ; 2 leave 1 tu#e loosely ca!!ed /ith no

overlay&, and enriched a'ar slant for use in coa'ulase test."e!ort or'anism as S. aureus if it is coa'ulase3!ositive and8or /ill ferment mannitol> S. epidermidisif it is fermentative as /ell asoidative on )F detrose, is coa'ulase3ne'ative, and /ill not ferment mannitol> or Micrococcus s!ecies if it is oidative only on )Fdetrose.

*. "ram6negative rods. 6noculate T6 a'ar slant, MacConey a'ar !late, cetrimide a'ar, and ML% !late /ith all5ram3ne'ative rods. 6ncu#ate 1=32: h at * ; 2 alaline& S +2 indicatean Enterobacteriaceae isolate. 8, 8AC $AC no chan'e& or AC8AC reactions indicate nonfermentative $AF& 5ram3ne'ative#acilli. 6f T6 reactions are mased #y hydro'en sulfide !roduction, inoculate lactose and 'lucose car#ohydrate #roths and incu#ate1=32: h at * ; 2 they should

#e /hite. The stri!s are sta#le indefinitely.

se !latinum loo! to smear mass of cells on !ortion of stri!. $Aichrome /ire 'ives false3!ositive reactions.& "ead at 10s, no longer . Positive is indicated #y a dee! !ur!le color> ne'ative is indicated #y the a#sence of color or /hen a !ur!le colora!!ears after 10 s.Pseudomonas s!!. are oidase3!ositive.


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#. Biochemical tests

From each !ositive !resum!tive T6 a'ar slant inoculate du!licate a'ar slants, oser@s citrate #roth, malonate #roth,decar#oylase #asal medium containin' ar'inine, motility nitrate a'ar, nutrient 'elatin $C(C&, Pseudomonas a'ar F, andPseudomonas a'ar P.

;E agar slants. 6noculate du!licate a'ar slants. 6ncu#ate one slant at *


12/16

#.t& P#ssib' P $ aeruginosa

%alonate utili&ation

Neatie P#bab' *#t P $ aeruginosa

P#sitie P#ssib' P $ aeruginosa

'itrate reduction

Neatie, *# as P#bab' *#t P $ aeruginosa


%otilit"

Neatie P#bab' *#t P $ aeruginosa


lagellation

P#a "aea P#ssib' P $ aeruginosa

A*' #t&e "aeati#* N#t P $ aeruginosa

Pseudomonas agar

N# "/#es+e*t !i%e*t N#t P $ aeruginosa

/#es+e*t .ates#/be !i%e*t (!'#edi*e) P $ aeruginosa

Pseudomonas agar P

N# !i%e*t N#t P $ aeruginosa

" !i%e*t is !ese*t, +#*"i% as !#+'a*i*e P $ aeruginosa

C.

(. 6nter!retation

Cosmetic !roducts are not e!ected to #e ase!tic> ho/ever, they must #e com!letely free of hi'h3virulence micro#ial !atho'ens,and the total num#er of aero#ic microor'anisms !er 'ram must #e lo/. ince there are no /idely acce!ta#le standards fornum#ers, tem!orary 'uidelines are used instead. For eye3area !roducts, counts should not #e 'reater than 00 colony formin'units $CF&8'> for non3eye3area !roducts, counts should not #e 'reater than 1000 CF8'. The !resence of !atho'ens /ould #e!articularly im!ortant in evaluatin' as unacce!ta#le a cosmetic /ith a mar'inally acce!ta#le count, e.'., :00 CF8' for an eye3area!roduct. Patho'ens or o!!ortunistic !atho'ens /hose incidence /ould #e of !articular concern, es!ecially in eye3area cosmetic!roducts, include S. aureus, Streptococcus pyo#enes,P . aeru#inosa and other s!ecies, and %lebsiella pneumoniae. omemicro#es normally re'arded as non!atho'enic may #e o!!ortunistically !atho'enic, e.'., in /ounds.

. Cosmetic preservative efficacy. The a#ove 'uidelines for inter!retation of results a!!ly to cosmetic !roducts #efore thetime of use. Cosmetics contain antimicro#ial !reservatives and thus are e!ected to /ithstand a certain amount of a#use #y users.Formerly, there /ere no validated tests for cosmetic !reservative efficacy $&, althou'h the test for !harmaceutical !reservativeefficacy in the .. Pharmaco!eia $2& or the cosmetic test in the technical 'uidelines of the Cosmetic, Toiletry, and Fra'rance %ssociation $CTF%& $1& /ere used. "ecently, the CTF% test has #een %)%C validated $2#& for use /ith li9uid cosmetics. % test for

solid cosmetic !reservative efficacy has #een !ro!osed $1=&. Cosmetics in reusa#le test its, such as those in retail stores, can #emicro#iolo'ically evaluated semi9uantitatively #y a sterile s/a# test $14&.

1eferences


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1. %nonymous. 1=. Preservation testin' of a9ueous li9uid and semi3li9uid eye cosmetics. InD CTF% Technical 5uidelines. TheCosmetic, Toiletry and Fra'rance %ssociation, 6nc., 7ashin'ton, (C.

2. %nonymous. 10. %ntimicro#ial !reservatives 3 effectiveness. InD nited tates Pharmaco!eia, 22nd "evision, !. 1:4=. ..Pharmaco!eial Convention, "ocville, M(.

2#. %)%C 6AT"A%T6)A%L. 2000. )fficial Methods of %nalysis, 14th ed., Method =.10. %)%C 6AT"A%T6)A%L, 5aithers#ur',M(.

*. Brenner, (.J., J.J. Farmer, F.7. +icman, M.%. %s#ury, and %.5. tei'er/alt. 144. Taonomic and Aomenclature Chan'esin Enterobacteriaceae. Centers for (isease Control, %tlanta, 5%.

:. (e Aavarre, M.5. 1:1. The Chemistry and Manufacture of Cosmetics. an Aostrand, Ae/ or.

. (unnin'an, %.P. 1=. Micro#iolo'ical control of cosmetics. Dru# !osmet. Ind. 4+)D:*3:, 1231=.

. /in', 7.+. 1=. d/ards and /in'@s 6dentification of Enterobacteriaceae, :th ed. lsevier, Ae/ or.

4. 5erhardt, P., ".5.. Murray, ".A. Costilo/, .7. Aester, 7.%. 7ood, A.". rie', and 5.". Philli!s. 1=1. Manual of Methods for5eneral Bacteriolo'y. %merican ociety for Micro#iolo'y, 7ashin'ton, (C.

=. +eim#roo, M.., 7.L.L. 7an', and 5. Cam!#ell. 1=. tainin' #acterial fla'ella easily. . !lin. Microbiol. )>D212321.

. +itchins, %.(. 1*. Cosmetic !reservation and safetyD F(% tatus. . Assoc. Food Dru# Officials *>D:23:.

10. 6'le/si, B. 1=. Pro#in' Pseudomonas aeru#inosa, an o!!ortunistic !atho'en. ASM &e's **D*0*3*04.

11. in', .). 1: $revised 142&. The 6dentification of nusual Patho'enic 5ram3Ae'ative Bacteria. Centers for (isease Control, %tlanta, 5%.

12. rie', A."., and J.5. +olt. 1=:. Ber'ey@s Manual of ystematic Bacteriolo'y. 7illiams 7ilins, Baltimore.

1*. Balo/s, %., 7.J. +ausler, Jr., .L. +errmann, +.(. 6sen#er', and +.J. hadomy. 11. Manual of Clinical Micro#iolo'y, th ed. %merican ociety for Micro#iolo'y, 7ashin'ton, (C.

1:. Madden, J.M. 1=:. Micro#iolo'ical methods for cosmetics, !!. 4*30*. InD Cosmetic and (ru' PreservationD Princi!les andPractice. J.J. a#ara $ed&. Marcel (eer, Ae/ or and Basel.

1. Morse, L.J., +.L. 7illiams, F.P. 5renn, Jr., .. ldrid'e, and J.". "otta. 14. e!ticemia dueto%lebsiella pneumoniae ori'inatin' from a hand3cream dis!enser. &. En#l. . Med. )>>D:423:4*.

1. mart, "., and (.F. !ooner. 142. Micro#iolo'ical s!oila'e in !harmaceuticals and cosmetics. . Soc. !osmet. !hem. )3D42134*4.

14. Tran, T.T., and %.(. +itchins. 1:. Micro#iolo'ical survey of shared3use cosmetic test its availa#le to the !u#lic. . Ind.Microbiol. 43D*=3*1.

1=. Tran, T.T., %.(. +itchins, and .7. Collier. 10. (irect contact mem#rane method for evaluatin' !reservative efficacy in solidcosmetics. Int. . !osmet. Sci. 4)D1431=*.

1. 7eaver, ".., (.5. +ollis, 7.%. Clar, and P. "iley. 1=*. "evised Ta#les for the 6dentification of nusual Patho'enic 5ram3Ae'ative Bacteria $.). in'&. Centers for (isease Control, %tlanta, 5%.

20. 7ilson, L.%., and (.5. %hearn. 144. Pseudomonas3induced corneal ulcers associated /ith contaminated eye mascaras. Am.. Ophthalmol. %2D112311.

21. 7ilson, L.%., %.J. Jilian, and (.5. %hearn. 14. The survival and 'ro/th of microor'anisms in mascara durin' use. Am. .Ophthalmol. >?D301.

+y!ertet ourceD Bacteriolo'ical %nalytical Manual, =th dition, "evision %, 1=. Cha!ter 2*.


14/16

BAM Media M?4: MacCon!ey Agar January 2001

Bacteriological Analytical ManualM?4MacCon!ey Agar Proteose !e!toneor !oly!e!tone

* '

Pe!tone or 'elysate 14 '

Lactose 10 '

Bile salts Ao. *or #ile salts miture

1. '

AaCl '

Aeutral red 0.0* '

Crystal violet 0.001 '

%'ar 1*. '

(istilled /ater 1 liter

us!end in'redients and heat /ith a'itation to dissolve. Boil 132 min. %utoclave 1 min at 121


15/16

BAM Media M3>: Cetrimide Agar January 2001

Bacteriological Analytical ManualM3>Cetrimide Agar Pancreatic di'est of 'elatin 20.0 '

Ma'nesium chloride 1.: '

Potassium sulfate 10.0 '

%'ar 1*. '

Cetrimide

$cetyl trimethyl ammonium #romide&0.* '

us!end :.* ' of in'redients or commercial !o/der $PseudoselTM, BBL> Becton3(icinson& in 1 liter of !urified /ater. %dd 10 ml'lycerol. Mi /ell. +eat to #oilin', a'itatin' fre9uently> maintain #oilin' for 1 min to dissolve !o/der.

Autoclave at 44%C for 4* min/

Final !+, 4.2 ; 0.2. Bacto cetrimide a'ar #ase !lus 'lycerol $(ifco& is a similar medium.


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BAM Media M?>: Mannitol .alt Agar January 2001

Bacteriological Analytical ManualM?>

Mannitol .alt Agar Beef etract 1 '

Poly!e!tone 10 '

AaCl 4 '

Mannitol 10 '

Phenol red 0.02 '

%'ar 1 '

(istilled /ater 1 liter

+eat /ith a'itation to dissolve a'ar and #oil 1 min. (is!ense 20 ml !ortions into 1 100 mm !etri dishes. %utoclave 1 min at121

testes de microbiologia- bam - fda

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