AOAC

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CHAPTER 3.

CHAPTER 3. EXAMINATEXAMINATION OF FRESH, REFRIGERATED AND FROZEN PREPION OF FRESH, REFRIGERATED AND FROZEN PREPAREDARED MEAT

MEAT, PO, POULTRULTRY ANY AND PAD PASTEUSTEURIZERIZED EGD EGG PRG PRODUCODUCTSTS

Charles P. Lattuada, Larry H. Dillard and Charles P. Lattuada, Larry H. Dillard and Bonnie E. Rose

Bonnie E. Rose

3

3..11 IInnttrroodduuccttiioonn

The laboratory methods contained in this section of the Guidebook The laboratory methods contained in this section of the Guidebook are used to detect and, when desired, quantitate selected are used to detect and, when desired, quantitate selected micr

microorgoorganisanisms ms in in sampsamples les collcollecteected d in in fedefederallrally y inspinspecteected d meatmeat,, poul

poultry try and and egg egg procprocessiessing ng estaestabliblishmeshmentsnts. . They They genegenerallrally y follfollowow the Compendium of Methods for the Microbiological Examination of the Compendium of Methods for the Microbiological Examination of Foods

Foods and and AOAC AOAC International's International's Official Official Methods Methods of of Analysis. Analysis. TheThe meth

methods ods prespresenteented d in in this this sectsection ion may may be be used used to to analanalyze yze sampsamplesles of:

of:

a.

a. frfresesh, h, frfrozozenen, s, smomokeked, d, cucurered od or dr dehehydydrarateted md meaeat at andnd poul

poultry try prodproductsucts;; b.

b. preppreparedared/rea/ready-tdy-to-eao-eat t prodproducts ucts such such as as pot pot piespies,, luncheon meats, dinners, battered or breaded meat and luncheon meats, dinners, battered or breaded meat and poul

poultry try prodproductsucts;; c.

c. rerefrfrigigereratated ed memeat at or or popoulultrtry y sasalaladsds;; d.

d. dedehyhydrdratated sed sououps ps anand sad saucuces es cocontntaiainining ng ththe ree reququisisititee amount of meat or poultry;

amount of meat or poultry; e.

e. memeat sat snanackcks, hs, horors d's d'oeoeuvuvreres, ps, pizizza aza and snd spepecicialalty ity itetemsms;; f.

f. vavaririouous ins ingrgrededieientnts ins incocorprpororatated wed witith meh meat aat and pnd pououltltryry prod

products ucts sucsuch h as as spicspices, es, vegevegetabltables, es, breabreading ding matematerialrial,, milk

milk powdpowder, er, driedried ed egg, gg, vegevegetabltable pre proteioteins;ns; g

g.. ppaasstteeuurriizzeed d eeggg g pprroodduuccttss;; h.

h. enenviviroronmnmenentatal sl samamplples es frfrom om arareaeas is in wn whihich ch anany oy of tf thehe above are processed or manufactured.

above are processed or manufactured.

The quantity and types of mesophilic microorganisms present in or The quantity and types of mesophilic microorganisms present in or on any of these products offer a means of evaluating the degree of on any of these products offer a means of evaluating the degree of sanitation

sanitation used used during during the the process. process. If If the the results results obtained obtained forfor coliforms,

coliforms, Escherichia Escherichia colicoli, and, and Staphylococcus Staphylococcus aureusaureus areare unusually high, they might result in some type of official unusually high, they might result in some type of official follow-up

follow-up action. action. Any Any such such follow-up follow-up analysis analysis will will use use thethe appropriate Final Action Method found in the latest edition of appropriate Final Action Method found in the latest edition of Official Methods of Analysis of AOAC International or any of its Official Methods of Analysis of AOAC International or any of its supplements.

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♦ ♦ ♦

♦ Aero Aerobic bic PlatPlate Coe Count unt (APC(APC): 9): 966.266.233 ♦

♦ ♦

♦ Coliform Group andColiform Group and E. coliE. coli: 966.24: 966.24

♦ ♦ ♦

♦ _{S. aureus}_{S. aureus}: 987.09: 987.09 3.11

3.11 CompCompariarison son WitWith thh the Ae AOAC OAC MethMethodod

The procedures in the following sections of this Chapter are either The procedures in the following sections of this Chapter are either the same as those published by the AOAC or generally follow an AOAC the same as those published by the AOAC or generally follow an AOAC meth

method. od. The The follfollowinowing is g is a lia listinsting of g of devideviatiations:ons: a.

a. ThThe pre prococededurure foe for dr deteterermiminining ng nunumbmberers of s of cocolilifoform rm anandd E E .. coli

coli differ from the AOAC procedure as follows:differ from the AOAC procedure as follows: i.

i. UsUse a e a sisingngle le tutube be of of lalaururel el susulflfatate te tryryptptosose be brorothth (LST) per dilution, rather than three tubes per (LST) per dilution, rather than three tubes per dilution.

dilution. ii

ii.. IncIncububate ate ininocuoculatlated Led LST aST and End EC brC brotoths fhs for 2or 24 ± 2 h4 ± 2 h.. iii.

iii. ConsConsider tider the prhe presenesence of gce of gas in LSas in LST and EC bT and EC brothrothss as positive for coliform and

as positive for coliform and E. coliE. coli respectively,respectively, with no further testing required.

with no further testing required. b.

b. The The procproceduredure e for for the the enumenumerateration ion ofof S. aureusS. aureus differsdiffers from the AOAC procedure in that only one tube, instead from the AOAC procedure in that only one tube, instead of three, per dilution is used to determine the of three, per dilution is used to determine the estimated count.

estimated count. 3.12

3.12 GeneGeneral Guiral Guideldelines foines for Testinr Testing Fresh or Preg Fresh or Preparepared Foodsd Foods a.

a. Do nDo not cot comombibine tne the che comompoponenentnts of s of cocompmposositite ite itemems sus such ach ass frozen

frozen dinners dinners into into a a single single sample. sample. To To the the greatestgreatest extent possible, examine as separate samples the extent possible, examine as separate samples the vegetable or non-meat portion(s) and the meat portion. vegetable or non-meat portion(s) and the meat portion. b.

b. The The quanquantittity, y, condconditioition n and and suitsuitabilability ity of of the the sampsamplele are very important.

are very important. i.

i. ThThe qe quauantntitity sy shohoululd bd be se sufuffificicienent tt to po pererfoform rm ththee analysis and have a reasonable amount in reserve analysis and have a reasonable amount in reserve for repeat testing.

for repeat testing. ii

ii.. The The conconditdition ion of rof receeceipt ipt shoshould uld be ibe in ken keepieping wng withith good microbiological practices for the analysis(es) good microbiological practices for the analysis(es) requested.

requested. iii.

iii. The saThe sample smple shoulhould be, to td be, to the grehe greatesatest extet extentnt poss

possibleible, , reprrepresenesentatitative ve of of the the wholwhole e of of thethe original product at the time the sample was taken. original product at the time the sample was taken. iv

iv.. WheWhen apn appropropripriate aate and ind if posf possibsiblele, sam, sampleples shos shoululd bed be received at the laboratory in their original received at the laboratory in their original unopened package(s) (intact sample).

unopened package(s) (intact sample). 3.13

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a

a.. AAeerroobbiic c ppllaatte e ccoouunntt b.

b. ColiColiform form andand E. coliE. coli quantitative estimatesquantitative estimates c.

c. S. aureusS. aureus 3.2

3.2 Equipment Equipment and and MaterialsMaterials a

a.. BBaallaannccee, , ccaappaacciittyy ≥≥≥≥2 kg, sensitivity ± 0.1 g2 kg, sensitivity ± 0.1 g b.

b. BlenBlender der and and stersterile ile blenblender der jarsjars c

c.. SSttoommaacchheerr™™™™ and sterile stomacher bagsand sterile stomacher bags d d.. IInnccuubbaattoorrs s aat t 335 5 ± ± 11..00°°°°C, and 20 ± 1.0C, and 20 ± 1.0ooCC e e.. WWaatteer r bbaatth h aat t 4455..5 5 ± ± 00..0055°°°°CC f f.. WWaatteer r bbaatth h aat t 337 7 ± ± 11..00°°°°CC g.

g. MaManunual oal or Aur Autotomamatitic coc cololony cny couountnter aer and tnd talally rly regegisisteterr h.

h. StStererilile, de, disispoposasablble/e/rereususabable dle disishehes, ps, panans or ts or trarays fys foror sample cutting

sample cutting i.

i. StStererilile fe fororcecepsps, s, spopoonon, k, kninifefe, s, scicissssorors as and nd ototheherr sterile sampling equipment

sterile sampling equipment j

j.. SStteerriille 1e 1, 5 , 5 aannd 1d 10 m0 ml pl piippeetttteess k.

k. SSttereriille e 1100 00 x x 115 5 mmm m pepettri ri didisshheses l

l.. TTrraannssffeer r lloooopp, , 3 3 mmm m m.

m. MicrMicroscooscope ape and cnd clean lean slidslideses n n.. RReeffrriiggeerraatteed d cceennttrriiffuuggee o o.. RReeffrriiggeerraattoorr p. p. pH mpH metereter 3.21 Media 3.21 Media a.

a. PlPlatate coe coununt agt agar (ar (PCPCA) iA) in con contntaiaineners srs suiuitatablble foe for mar makikingng pour

pour platplateses b.

b. LaurLaurel el sulfsulfate ate tryptryptose tose (LS(LST) T) brobroth th with with fermfermententatioationn tubes

tubes c.

c. EEC bC brrooth th wiwitth fh feermrmeenntatattioion tn tuubbeses d.

d. SuSurfarface ce drdried ied BaiBaird-rd-PaParkerker r plplateates s (eg(egg g teltellurlurite ite glglyciycinene pyru

pyruvate vate agaragar, ET, ETGPAGPA)) e.

e. BBrraiain hn heeaart rt iinfnfuusisioon (n (BBHIHI) b) brroothth f.

f. TrTrypyptiticacase se sosoy by broroth th wiwith th 1010% s% sododiuium cm chlhlororidide ae and nd 1%1% sodium pyruvate (PTSBS)

sodium pyruvate (PTSBS) g

g.. TToolluuiiddiinne e bblluue e DDNNA A aaggaarr 3

3.2.222 ReReaagegennttss a.

a. BBuutttteerrfifieeldld''s ps phhosospphhatate de diilulueenntt b.

b. Gram Gram staistain ren reagenagentsts c.

c. DeDesisiccccatated red rababbibit plt plasasma (ma (cocoagagululasase) e) EDEDTA TA d

d.. TTrriis s BBuuffffeerr e

e.. AAmmmmoonniiuum m ssuullffaatte e [[((NNHH44))22SOSO44], reagent grade], reagent grade

f

f.. TTrriittoon n XX--110000 g.

g. 33M tM trriichchllororooaacecettic ic aacicid sd sooluluttiionon h

h.. 11N N HHCCl l ssoolluuttiioonn 3.

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See Section 1.3 - 1.5 (Sterilization of Instruments, See Section 1.3 - 1.5 (Sterilization of Instruments, Disinfection of Containers, and Cutting and Weighing Samples) Disinfection of Containers, and Cutting and Weighing Samples) 3.

3.3131 FoFood Hood Homomogegenanatetess a.

a. UsUsining g ssteteririle le spspoooonsns, , ffororcecepsps, , sscicissssorors, s, etetc.c.,, aseptically weigh 50 ± 0.1 g of the sample into a aseptically weigh 50 ± 0.1 g of the sample into a sterile blender jar or stomacher bag.

sterile blender jar or stomacher bag. b.

b. If If the the sampsample le is is frozfrozen, en, remremove ove portportionsions, , whewheneveneverr poss

possibleible, witho, without thawut thawing the laing the larger sarger sample anmple and weigh 50d weigh 50 ± 0.1 g of the sample into a sterile blender jar or ± 0.1 g of the sample into a sterile blender jar or stomacher

stomacher bag. bag. It It is is well well known known that that freeze/thaw freeze/thaw cyclescycles are

are damaging damaging to to bacteria. bacteria. This This is is particularlyparticularly important when a re-examination of the product may be important when a re-examination of the product may be necessary.

necessary. Otherwise, partially Otherwise, partially thaw thaw the the sample sample at at 2-52-5°°°°CC

for about 18 h, or by placing the sample in a watertight for about 18 h, or by placing the sample in a watertight container and immersing it in cold water for 1-2 h.

container and immersing it in cold water for 1-2 h. c.

c. AdAdd 45d 450 ml 0 ml ststererilile Bue Buttttererfifieleld'd's phs phososphphatate die diluluenent ant andd stomach for 2 minutes, or blend at high speed for two stomach for 2 minutes, or blend at high speed for two minu

minutes. tes. The The totatotal l voluvolume me in in the the blenblender der jar jar mustmust completely

completely cover cover the the blades. blades. This This becomes becomes the the 1:101:10 dilution.

dilution. d.

d. PePermrmit it ththe fe foaoam tm to so setettltle; e; ththen en pipipepet 1t 10 m0 ml ol of tf thehe blen

blended ded 1:10 1:10 diludilution tion into into a a 90 90 ml ml diludilution tion blanblank k toto make

make the the 1:101:100 0 diludilutiontion. . RepeRepeat at thithis s procproceduredure e toto prep

prepare are seriserial al diludilutiontions s of of 1010-3-3, , 1010-4-4, , etc. etc. Shake Shake allall dilutions 25

dilutions 25 times in times in a one a one foot arc. foot arc. Use a Use a separate 10separate 10 ml

ml pipepipette tte to to prepprepare are each each dildilutioution. n. PipePipettes ttes mustmust deliver

deliver accurately accurately the the required required volumes. volumes. Do Do not not deliverdeliver less

less than than 10% 10% of of a a pipette's pipette's volume. volume. For For example, example, toto deliver one ml, do not use a pipette of more than 10 ml deliver one ml, do not use a pipette of more than 10 ml volume.

volume. e.

e. ThThe ane analalysyst st shohoululd sd strtrivive to e to mimininimimize tze the the timime fre from wom whehenn the sample is stomached or blended until all the the sample is stomached or blended until all the dilutions have been placed in or on the appropriate dilutions have been placed in or on the appropriate medi

medium; um; ideideally ally this this time time shoshould uld not not exceexceed ed 15 15 minuminutestes whenever possible.

whenever possible. f.

f. If If ththe se samamplple ce cononsisiststs os of lf lesess ts thahan 5n 50 g0 g, w, weieigh gh ababououtt half the sample, and add the amount of diluent required half the sample, and add the amount of diluent required to make a 1:10 dilution (nine times the weight of the to make a 1:10 dilution (nine times the weight of the port

portion ion of sof samplample use used) ed) and and procproceed eed as aas abovebove..

g.

g. HoHold ld rereseservrves es of of eaeach ch sasampmple le at at or or bebelolow -w -1515°°°°C C (5(5°°°°F),F), unless the product is stored normally at ambient unless the product is stored normally at ambient

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temperature or unless a specific protocol specifies temperature or unless a specific protocol specifies otherwise.

otherwise. Samples Samples should should be be held held until until a a determinationdetermination is made that a repeat test is not necessary or for the is made that a repeat test is not necessary or for the length of time designated by the testing protocol.

length of time designated by the testing protocol. 3.

3.3232 WhWholole Be Birird Rd Rininsese a.

a. SiSincnce te thehere re arare de dififfefererencnces es bebetwtweeeen sn samamplple te typypes es anandd sizes (eg. chicken vs. turkey carcasses), be sure to sizes (eg. chicken vs. turkey carcasses), be sure to check the specific program protocol before using this check the specific program protocol before using this proc

proceduredure.e. b.

b. AsepAsepticatically lly tratransfensfer r the the carcarcass cass to to a a stersterile ile StomStomacheacherr 3500 bag (or equivalent), draining as much excess fluid 3500 bag (or equivalent), draining as much excess fluid as possible during the transfer.

as possible during the transfer. Note

Note: Larger (24 x : Larger (24 x 30-30-36 in.) bags will have to 36 in.) bags will have to be usedbe used with turkeys.

with turkeys. c.

c. AdAdd 4d 400 00 ml ml (c(chihickckenens) s) or or 60600 m0 ml (l (tuturkrkeyeys) s) ofof Butterfield's Phosphate Diluent (BPD) to the carcass in Butterfield's Phosphate Diluent (BPD) to the carcass in the

the bag. bag. Pour Pour approximately approximately one one half half the the volume volume intointo the interior cavity of the bird and the other half over the interior cavity of the bird and the other half over the

the skin. skin. Note: Note: IfIf SalmonellaSalmonella is the ONLY targetis the ONLY target analyte, Buffered Peptone Water (BPW) may be substituted analyte, Buffered Peptone Water (BPW) may be substituted for the BPD.

for the BPD. d.

d. RiRinsnse te the he bibirdrd, i, insnsidide ae and nd ouout, t, wiwith th a ra rocockiking ng momotitionon for 1 min at a rate of approximately 35 forward and back for 1 min at a rate of approximately 35 forward and back swings

swings per per minute. minute. This This is is done done by by grasping the grasping the carcasscarcass in the bag with one hand and the closed top of the bag in the bag with one hand and the closed top of the bag with

with the other. the other. Rock with Rock with a a reciprocal motion reciprocal motion in in an an 18- 18-24 inch arc, assuring that all surfaces (interior and 24 inch arc, assuring that all surfaces (interior and exterior) are rinsed.

exterior) are rinsed. e.

e. AsAsepeptiticacalllly ry rememovove the the ce cararcacass fss frorom tm the bhe bagag, dr, draiaininingng excess rinsed liquid into the bag, dispose of the excess rinsed liquid into the bag, dispose of the carcass, and culture the bird rinse liquid according to carcass, and culture the bird rinse liquid according to prot

protocol ocol diredirectioctions.ns. 3.33 Egg Products

3.33 Egg Products a

a.. LLiiqquuiid ed eggggs ms muusst bt be he heelld ad at 4t 4..44°°°°C (40C (40ooF) or below forF) or below for valid analysis.

valid analysis. b.

b. FrozFrozen en samsamples must be ples must be thathawed as wed as rapirapidly as dly as posspossible inible in a water bath at 45

a water bath at 45°°°°C.C.

c.

c. ExExpoposesed od or lr leaeakiking ng sasampmpleles ss shohoululd nd not ot be be ananalalyzyzeded.. d.

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shaking. shaking. e.

e. AsAsepeptiticacalllly wy weieigh gh a ma mininimimum um of of 10100 g o0 g of ef egg gg sasampmple le inintoto a sterile blender jar or sealable bag containing 900 ml a sterile blender jar or sealable bag containing 900 ml of

of the the appropriate enrichment appropriate enrichment or or buffer. buffer. If If a a specificspecific prot

protocol requiocol requires a res a sampsample le size greatsize greater er than 100 than 100 g, g, thethe 1:10 ratio must be maintained in the same enrichment or 1:10 ratio must be maintained in the same enrichment or buff

buffer.er. f.

f. MiMix thx the 1:e 1:10 s10 samamplple ene enririchchmementnt/b/bufuffefer wer well bll by shy shakakining,g, stomaching, or blending.

stomaching, or blending. g.

g. DrDrieied ed egg gg sasampmpleles ss shohoululd bd be re rehehydydrarateted sd slolowlwly by byy gradually adding the enrichment/diluent to the sample. gradually adding the enrichment/diluent to the sample. This is done by adding a small portion of liquid to the This is done by adding a small portion of liquid to the sample and mixing aseptically to obtain a homogeneous sample and mixing aseptically to obtain a homogeneous suspension.

suspension. Repeat Repeat this this procedure procedure three three times times and and thenthen add

add the the remainder of remainder of the the liquid. liquid. Mix Mix until until a a lump-freelump-free suspension is obtained.

suspension is obtained. h.

h. InIncucubabate te or or trtranansfsfer er to to ththe ae apppproroprpriaiate te enenririchchmementnt medi

medium um and and incuincubate bate accoaccordinrding g to to the the protprotocolocol(s) (s) beinbeingg used.

used. 3.4

3.4 Aerobic Aerobic Plate Plate Count Count (APC)(APC) a.

a. PoPour ur PlPlatates es (R(Refefererenence ce AOAOAC AC 96966.6.23 23 C)C) i.

i. UsUsining tg the he didilulutitionons ps prerepaparered id in sn secectition on 3.3.3, 3, pipipepett 1 ml from the 10

1 ml from the 10-1-1, , 1010-2-2, , 1010-3-3, , 1010-4-4 etc. dilutionsetc. dilutions into each of four petri dishes, two for each into each of four petri dishes, two for each incubation

incubation temperature. temperature. Plate Plate additional additional dilutionsdilutions when expecting higher bacterial levels.

when expecting higher bacterial levels. ii

ii.. Use Use sepseparaarate ste sterterilile pie pipepettettes fos for ear each dch dililutiution.on. iii.

iii. Add moAdd moltelten Platn Plate Coune Count Agar ct Agar coolooled in a wated in a water bater bathh to 45 ± 1

to 45 ± 1°°°°C. C. Uniformly Uniformly mix mix the the agar agar and and thethe inoculum by gently swirling or tilting each plate, inoculum by gently swirling or tilting each plate, taking care not to generate bubbles.

taking care not to generate bubbles. iv

iv.. AllAllow tow the ahe agagar to r to harharden den and and thethen pln place ace one one serseriesies of duplicate plates in a 35 ± 1

of duplicate plates in a 35 ± 1°°°°C incubator forC incubator for 48

48 h. h. Incubate Incubate the the other other series series at at 20 20 ± ± 11°°°°C forC for four or five days.

four or five days.

v.

v. UsUse e a a cocololony ny cocoununteter r anand cd couount nt cocololoninies es on on ththee duplicate plates in a suitable range (30-300 duplicate plates in a suitable range (30-300 colonies

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30-300 colonies, record the dilution counted and 30-300 colonies, record the dilution counted and the

the number number of of colonies colonies found. found. Average Average the the countscounts obtained from duplicate plates, multiply by the obtained from duplicate plates, multiply by the dilution factor and report this number as the dilution factor and report this number as the aerobic plate count per gram or milliliter at the aerobic plate count per gram or milliliter at the incubation temperature used.

incubation temperature used. b.

b. AlteAlternatrnate Mee Methodthods - s - AOACAOAC i.

i. AeAerorobibic c PlPlatate Ce Couount nt in in FoFoodods: s: HyHydrdropophohobibic Gc Griridd Memb

Membrane rane FilFilter ter MetMethodhod** (AOAC 986.32)(AOAC 986.32) ii

ii.. Dry Dry RehRehydrydrataatable ble FilFilm (Pm (Petetrifrifilm ilm AerAerobiobic Plc Plateate™™™™)) Meth

Methodod** (AOAC 990.12)(AOAC 990.12) ii

iii.i. SpiSpiraral Pl Platlate Me Methethodod** (AOAC 977.27)(AOAC 977.27)

*Since these methods are available commercially, the *Since these methods are available commercially, the manu

manufactfacturerurer's d's direcirectiontions shs should ould be fbe folloollowedwed.. 3.5

3.5 Coliform Coliform Group Group andand Escherichia coliEscherichia coli a.

a. EsEstitimamateted Cd Couount nt PrPrococededurure (e (ReRefefererencnce Ae AOAOAC 9C 96666.2.24)4) i.

1 ml from the 10-1-1, 10, 10-2-2, 10, 10-3-3 etc. dilutions into LSTetc. dilutions into LST brot

broth, one tubh, one tube per dilue per dilutiontion. . InocInoculatulate addite additionaionall dilutions when expecting higher bacterial levels. dilutions when expecting higher bacterial levels. The highest dilution of sample must be sufficiently The highest dilution of sample must be sufficiently high to yield a negative end point.

high to yield a negative end point. ii

iii. IncuIncubate bate the tthe tubes ubes of LSof LST broT broth at th at 3535°°°°C forC for 24 ± 2 h.

24 ± 2 h. iv

iv.. ExaExamimine ene each ach tubtube foe for gar gas fos formarmatiotion as en as evidvidencenced bed byy displacement of fluid in the inverted tubes or by displacement of fluid in the inverted tubes or by effervescence when tubes are shaken gently.

effervescence when tubes are shaken gently. v.

v. CoConsnsidider er anany ty tubube oe of Lf LST ST brbrototh dh disisplplayayining gg gas as asas coliform positive, and report the number of coliform positive, and report the number of coliform per gram in accordance with the highest coliform per gram in accordance with the highest dilution with

dilution with gas. gas. When When a a "skip" "skip" occurs, occurs, report report byby using

using the the missing missing estimate estimate (for (for example: example: If If thethe 10

10-1-1, , 1010-2-2, and 10, and 10-4-4 dilutions produce gas but thedilutions produce gas but the 10

10-3-3 dilution tube is non-gassing, report "1,000dilution tube is non-gassing, report "1,000 coliforms per gram.")

coliforms per gram.") b.

b. FecaFecal l ColiColiform form ((E. coliE. coli) Estimated Count Procedure) Estimated Count Procedure (Reference AOAC 966.24)

(Reference AOAC 966.24) i.

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from every gas-positive LST broth tube to a from every gas-positive LST broth tube to a correspondingly marked tube of EC broth.

correspondingly marked tube of EC broth. ii

ii.. InIncucubabate te ththe Ee EC tC tububes es in in a 4a 45.5.5 ± 05 ± 0.0.055°°°°C coveredC covered water bath

water bath for for 24 ± 24 ± 2 2 h. h. Submerge the Submerge the EC EC tubes intubes in the bath so that the water level is above the level the bath so that the water level is above the level of medium in the tubes.

of medium in the tubes. iii.

iii. RecoRecord everd every tubry tube prode producinucing gas, as eg gas, as evidevidencenced byd by displacement of liquid in the inverted tube or by displacement of liquid in the inverted tube or by effervescence when tubes are shaken gently.

effervescence when tubes are shaken gently. i

ivv.. RRepepoorrt tt thhe e nunummbber er ooff E. coliE. coli per gram in accordanceper gram in accordance with

with the the highest highest dilution dilution displaying displaying gas. gas. When When aa "skip" occurs, report by using the missing estimate "skip" occurs, report by using the missing estimate (for

(for example: example: If If the the 1010-1-1, , 1010-2-2, and 10, and 10-4-4 dilutionsdilutions prod

produce uce gas gas but but the the 1010-3-3 dilution dilution tube tube isis non-gassing, report "1,000

non-gassing, report "1,000 E. coliE. coli per gram.")per gram.") c

c.. AAlltteerrnnaatte Me Meetthhoodds - s - AAOOAACC i

i.. CCoolliiffoorrm m aanndd Escherichia coliEscherichia coli Counts in Foods:Counts in Foods: Hydrophobic Grid Membrane Filter/MUG Method

Hydrophobic Grid Membrane Filter/MUG Method** i

iii.. CCoolliiffoorrm m aanndd Escherichia coliEscherichia coli Counts in Foods: DryCounts in Foods: Dry Rehydratable Film

Rehydratable Film **

*Since these methods are available commercially, the *Since these methods are available commercially, the manu

manufactfacturerurers's s's diredirectioctions sns shoulhould be d be follfolloweowed.d. 3.6

3.6 Staphylococcus aureusStaphylococcus aureus a.

a. EsEstitimamateted Cd Couount nt PrPrococededurure (e (ReRefefererencnce Ae AOAOAC 9C 98787.0.09)9) i.

1 ml from the 10-1-1, , 1010-2-2, , 1010-3-3 etc. dilutions intoetc. dilutions into tubes containing 10 ml of Trypticase (tryptic) Soy tubes containing 10 ml of Trypticase (tryptic) Soy Broth with 10% sodium chloride and 1% sodium Broth with 10% sodium chloride and 1% sodium pyru

pyruvate (Pvate (PTSBSTSBS), one tub), one tube per dilue per dilutiontion. . InocInoculatulatee additional dilutions when expecting higher additional dilutions when expecting higher bact

bacteriaerial l levelevels. ls. The The highhighest est diludilution tion of of sampsamplele must

must be be suffsufficieicientlntly y high high to to yielyield d a a neganegative tive endend poin

point.t. ii

iii. IncuIncubate bate the the PTSBPTSBS tuS tubes bes at 3at 355°°°°C for 48 h.C for 48 h.

iv

iv.. UsiUsing a ng a 3 mm 3 mm calcalibribrateated lod loopop, tr, tranansfesfer a lr a loopoopfulful from each growth-positive tube as well as from the from each growth-positive tube as well as from the tube of the next highest dilution to previously tube of the next highest dilution to previously prep

prepared plateared plates s of Baird-Pof Baird-Parkearker r agaragar. . StreStreak ak in in aa mann

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v.

v. InIncucubabate te ththe Be Baiairdrd-P-Pararkeker pr plalatetes as at 3t 355°°°°C for 48 h.C for 48 h. v

vii.. TTyyppiiccaall S. aureusS. aureus colonies appear as circular,colonies appear as circular, convex, smooth, grey-black to jet-black colonies on convex, smooth, grey-black to jet-black colonies on uncrowded plates and frequently have an off-white uncrowded plates and frequently have an off-white marg

margin in surrsurroundounded ed by by a a zone zone of of precprecipitipitatioationn (turbidity)

(turbidity) followed followed by by a a clear clear zone. zone. The The coloniescolonies usually have a buttery to gummy consistency.

usually have a buttery to gummy consistency. vii.

vii. Test tTest two or mowo or more isore isolatelates, fros, from each um each useabseable plale platete meet

meeting ing the the abovabove e descriptdescription ion (3.6(3.6,vi),vi), , forfor coagulase as in Section 3.6 (c).

coagulase as in Section 3.6 (c). b.

b. DireDirect Pct Platilatingng i

i.. IIff S. aureusS. aureus counts of 100 cfu per gram or more arecounts of 100 cfu per gram or more are expected, direct plating can be done using expected, direct plating can be done using Baird-Parker agar.

Baird-Parker agar. ii

ii.. PipPipet et 0.1 0.1 ml fml frorom eam each ch dildilutution ion on on prepreviviousouslyly prep

prepared ared and and driedried d BairBaird-Pad-Parker rker agar agar platplates. es. UseUse separate accurate pipettes for each dilution.

separate accurate pipettes for each dilution. ii

iiii DisDistrtribuibute tte the ihe inonoculculum eum evenvenly oly ovever thr the sue surfarface oce off the plates using separate, sterile, fire polished, the plates using separate, sterile, fire polished, bent

bent-gla-glass ss rods rods ("ho("hockey ckey sticsticks") ks") for for each each platplate.e. Mark

Mark platplates es accoaccordirding tng to tho the die dilutilution uon used.sed. iv

iv.. InInvevert rt plplatates es anand id incncububatate ae at 3t 355°°°°C for 48 h.C for 48 h.

v.

v. SeSelelect ct plplatates es cocontntaiainining ng apapprproxoximimatatelely 2y 20 o0 or mr mororee well-isolated typical

well-isolated typical S. aureusS. aureus colonies. colonies. CountCount plat

plates es contcontainiaining ng 20-220-200 00 colocoloniesnies. . TypiTypicalcal colonies are circular, convex, smooth, grey-black colonies are circular, convex, smooth, grey-black to jet-black and frequently have an off-white to jet-black and frequently have an off-white marg

margin in surrsurroundounded ed by by a a zone zone of of precprecipitipitatioationn (turbidity)

(turbidity) followed followed by by a a clear clear zone. zone. The The coloniescolonies usually have a buttery to gummy consistency.

usually have a buttery to gummy consistency. vi

vi.. SelSelecect 10 cot 10 colonlonies ies frofrom thom those cse counounted ated and innd inococulaulatete each into separate 13 x 100 millimeter tubes each into separate 13 x 100 millimeter tubes containing 0.2 ml of BHI broth for coagulase containing 0.2 ml of BHI broth for coagulase testing.

testing. Test Test for for coagulase coagulase as as in in 3.6 3.6 (c).(c). vii.

vii. CalcCalculatulate the total nue the total numbember of colonir of colonies repres represenesentedted by

by coagcoagulasulase e posipositive culturtive cultures es and and multmultiply by iply by thethe appropriate sample dilution factor to record the appropriate sample dilution factor to record the number of coagulase positive staphylococci per number of coagulase positive staphylococci per gram.

gram. c

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i.

i. UsUse ae an in inonocuculalatiting ng neneededle le to to obobtatain in a sa smamall ll amamouountnt of growth from each suspect colony and place it of growth from each suspect colony and place it into 13 X 100 mm tubes containing 0.2 ml of BHI into 13 X 100 mm tubes containing 0.2 ml of BHI Broth.

Broth. ii

ii.. A knA knowown con coaguagulaslase poe posisitivtive ane and a kd a knonown nwn negegatiativeve culture should be inoculated into BHI broth at the culture should be inoculated into BHI broth at the same time as the samples.

same time as the samples. ii

iii.i. IncIncububate ate eaeach tch tube ube at 3at 355°°°°C for 18-24 h.C for 18-24 h. iv

iv.. AdAdd d 00.5 .5 ml ml oof f rabbrabbit it pplalasmsma a wiwith th EDEDTATA,, reconstituted according to the manufacturer's reconstituted according to the manufacturer's directions, to the BHI cultures.

directions, to the BHI cultures. v.

v. MiMix tx thohororougughlhly ay and nd plplacace te the he tutubebes is in a 3n a 35-5-3737°°°°C.C. water bath.

water bath. vi

vi.. ExaExamimine tne theshese tue tubebes eas each hch hourour, fr, from oom one tne throhrough ugh sixsix hours,

hours, for for clot clot formation. formation. Any Any degree degree of of clottingclotting should be interpreted as a positive reaction.

should be interpreted as a positive reaction. 3.61

3.61 Special Special Sampling Sampling Procedure Procedure for for Fermented Fermented Sausage Sausage ProductsProducts a

a.. IInnttrroodduuccttiioonn

During the early stages of sausage fermentation, During the early stages of sausage fermentation, staphylococci can grow extensively if the starter staphylococci can grow extensively if the starter culture is not added or fermentation fails with no culture is not added or fermentation fails with no concomitant production of lactic acid and drop in pH. concomitant production of lactic acid and drop in pH. Failure can be caused by poor quality starter cultures Failure can be caused by poor quality starter cultures or the improper use of starter cultures or "back or the improper use of starter cultures or "back inoculation".

inoculation". S. aureusS. aureus growth is aerobic and usuallygrowth is aerobic and usually confined to the outer 1/8 inch of the sausage. confined to the outer 1/8 inch of the sausage. Enterotoxin may be formed as a result of this growth. Enterotoxin may be formed as a result of this growth.

Coagulase-positive staphylococcal counts on large sticks Coagulase-positive staphylococcal counts on large sticks of

of salami salami have have been been noted noted to to vary vary widely. widely. On On largelarge sticks, some areas may have very few staphylococci while sticks, some areas may have very few staphylococci while other areas may have levels in excess of 10

other areas may have levels in excess of 1066/g. Whenever/g. Whenever poss

possibleible, , obtaobtain 1-2 in 1-2 pounpounds ds of of the suspecthe suspect t saussausage. age. InIn order to obtain a representative sample, portions should order to obtain a representative sample, portions should be take

be taken from seven from several difral differeferent areant areas and comps and compositosited fored for testing.

testing. b.

b. ProcProcedureduree i.

i. If If ththe se sauausasage ge is is momoldldy, y, wiwipe pe ththe me molold od off ff ththee sausage casing with a piece of sterile tissue paper sausage casing with a piece of sterile tissue paper and proceed.

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ii

ii.. To cTo collollect ect a saa sampmple, le, ususe a ste a sterierile, le, shasharp krp knifnife ane andd cut several thick slices from the sausage near the cut several thick slices from the sausage near the ends

ends as as well well as as in in the the middle. middle. Aseptically Aseptically trim trim and save the outer 1/8 to 1/4 inch portion of the and save the outer 1/8 to 1/4 inch portion of the sausage

sausage and and label label it it "shell "shell portion". portion". Even Even if if thethe amount of sample is limited, do not cut deeper than amount of sample is limited, do not cut deeper than 1/4 inch.

1/4 inch. iii.

iii. WorkWorking aing aseptsepticalically, bly, blenlend 25-d 25-50 g of the sh50 g of the shellell port

portion ion for for enteenterotorotoxin xin testtesting; ing; the the same same blenblendedded sample can be used to test for viable sample can be used to test for viable coagulase-positive

coagulase-positive S. S. aureusaureus as described inas described in section 3.6.

section 3.6. iv

iv.. AnaAnalylyze tze the he samsamplple by e by eiteither her of of ththe foe follllowiowingng proc

proceduredures.es. 3.62

3.62 The (The (PresumptPresumptive) ive) StaphyloStaphylococcal coccal EnterotoEnterotoxin xin Reverse Reverse PassivePassive Latex Agglutination Test

Latex Agglutination Test

The procedure for this test is given in (15.20) and usually is The procedure for this test is given in (15.20) and usually is the method of choice.

the method of choice. 3.6

3.633 TheThermormonucnuclealease se AsAssaysay a

a.. IInnttrroodduuccttiioonn

This procedure is based on the detection of a heat This procedure is based on the detection of a heat stable DNase which is produced by most strains of stable DNase which is produced by most strains of S.S. aureus,

aureus, including 98.3% of the enterotoxigenic strains.including 98.3% of the enterotoxigenic strains. This heat stable DNase is produced in detectable amounts This heat stable DNase is produced in detectable amounts under all conditions which permit the growth of under all conditions which permit the growth of S.S. aureus

aureus and and the the production production of of enterotoxin. enterotoxin. The The DNase DNase isis able to survive processing conditions which would able to survive processing conditions which would destroy viable

destroy viable S. aureus.S. aureus.

This method can be used to screen large sausages or a This method can be used to screen large sausages or a large number of samples to identify "hot spots".

large number of samples to identify "hot spots".

It has been shown (Tatini, 1981) that the detection of It has been shown (Tatini, 1981) that the detection of DNase with this procedure is indicative of

DNase with this procedure is indicative of S. aureusS. aureus popu

populatilations ons ofof ≥≥≥≥101055 per gram.per gram. b.

b. ProcProceduredure:e: i

i.. BBlleennd d 220 0 g g oof f sshheellll, , 110 0 g g ((NNHH44))22SOSO44, and 2 ml Triton, and 2 ml Triton

X-100 in 40 ml of distilled water. X-100 in 40 ml of distilled water. ii

ii.. AdjAdjusust tht the pe pH of H of ththis sis slurlurry try to 4.o 4.5-45-4.8 w.8 with ith 1N1N HCl.

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iii.

iii. CentCentrifurifuge undege under refrir refrigerageration at 7-tion at 7-10,010,000 RPM for00 RPM for 15 min.

15 min. iv

iv.. DecDecanant ant and did discscard ard ththe sue superpernatnatant ant and and adadd 0.d 0.05 m05 mll cold 3M trichloroacetic acid for each ml of the cold 3M trichloroacetic acid for each ml of the original slurry, mix and centrifuge a second time original slurry, mix and centrifuge a second time as above.

as above. v.

v. DeDecacant nt anand d didiscscarard d ththe e susupepernrnatatanant. t. ReRe-s-sususpepend nd ththee prec

precipitipitate in ate in 1 1 ml of ml of Tris buffTris buffer, adjuster, adjusted to ed to pHpH 8.5, and then adjust the volume to 2 ml with Tris 8.5, and then adjust the volume to 2 ml with Tris buff

buffer.er. v

vii.. BBoioil tl the he ssooluluttiion on ffoorr ≥≥≥≥15 but15 but ≤≤≤≤90 min, cool and90 min, cool and store under refrigeration until needed.

store under refrigeration until needed. vii.

vii. Cut 2 mm diCut 2 mm diametameter weler wells intls into air dro air dried Toied Toluidluidineine Blue DNA Agar.

Blue DNA Agar. vii

viii.i. DispDispense ense the fthe food eood extraxtract inct into onto one or moe or more were wellslls using

using a a Pasteur pipette. Pasteur pipette. Do Do not not overfill overfill the the well.well. ix

ix.. IncIncububate ate ththese ese plaplatestes, a, agar gar sidside de dowown, n, at at 3737°°°°C for 4C for 4 to 24 h.

to 24 h. x.

x. AnAny y pipink nk hahalolo, e, extxtenendiding ng 1 m1 mm bm beyeyonond td the he wewell ll isis considered positive for thermonuclease.

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3

3.7.7 SeSellecectteed Rd Refefeerrenencceses Cunniff, P.

Cunniff, P. (ed.). (ed.). 1995. 1995. Official Methods Official Methods of of Analysis of Analysis of AOACAOAC International,

International, 16th 16th Edition. Edition. AOAC AOAC International International Inc.,Inc., Gaithersburg, MD 20877.

Gaithersburg, MD 20877.

Emswiler-Rose, B. S., R. W. Johnston, M. E. Harris, and W. H. Emswiler-Rose, B. S., R. W. Johnston, M. E. Harris, and W. H. Lee.

Lee. 1980. 1980. Rapid Rapid detection detection of of staphylococcal staphylococcal thermonucleasethermonuclease on casings of naturally contaminated fermented sausages. on casings of naturally contaminated fermented sausages. Appl

Appl. E. Envirnviron. on. MicrMicrobioobiol. 4l. 440:140:13-183-18..

Lancette, G. A., and S. R. Tatini. 1992.

Lancette, G. A., and S. R. Tatini. 1992. StaphylococcusStaphylococcus aureus

aureus, p. 533-550., p. 533-550. InIn C. Vanderzant and D. F. SplittstoesserC. Vanderzant and D. F. Splittstoesser (ed.),

(ed.), Compendium Compendium of of Methods Methods for for the the MicrobiologicalMicrobiological Examination

Examination of of Foods. Foods. Amer. Amer. Publ. Publ. Hlth. Hlth. Assoc., Assoc., Washington,Washington, D.C. 20005.

D.C. 20005. Tatini,

Tatini, S. S. R. R. 1981. 1981. Thermonuclease Thermonuclease as as an an indicator indicator ofof staphylococcal enterotoxins in food, p. 53-75.

staphylococcal enterotoxins in food, p. 53-75. InIn R. L. OryR. L. Ory (ed.),

(ed.), Antinutrients and Antinutrients and Natural Natural Toxicants in Toxicants in Foods. Foods. Food Food andand Nutr