AOAC

USDA/FSIS Microbiology Laboratory Guidebook

3rd Edition/1998

CHAPTER 3. EXAMINAT EXAMINATION ION OF FRESH, REFRIGERATED AND FROZEN PREPARED PREPARED  MEAT,  MEAT , POULTR POULTRY Y AND AND PASTEU PASTEURIZE RIZED D EGG EGG PRODUC PRODUCTS TS Charles P. Lattuada, Larry H. Dillard and Bonnie E. Rose 3.1

Introduction

The laboratory methods contained in this section of the Guidebook are used to detect and, when desired, quantitate selected  microorg  micr oorganis anisms ms in samp samples les coll collecte ected d in fede federall rally y insp inspecte ected d meat meat, ,  poultry  poul try and egg proc processi essing ng esta establi blishme shments nts. . They gene generall rally y foll follow ow the Compendium of Methods for the Microbiological Examination of Foods and AOAC International's Official Methods of Analysis. The  methods  meth ods pres presente ented d in this sect section ion may be used to anal analyze yze samp samples les of: a.  b.

fresh, fres h, fr froz ozen en, , smo smoke ked, d,  poultry  poul try prod products ucts; ;

cure cu red d

or or

dehyd deh ydra rate ted d

meat mea t

and and

prepared prep ared/rea /ready-t dy-to-ea o-eat t products prod ucts such as pot pies, pies , luncheon meats, dinners, battered or breaded meat and  poultry  poul try prod products ucts; ;

c.

refr re frig iger erat ated ed me meat at or po poul ultr try y sa sala lads ds; ;

d.

dehy de hydr drat ated ed sou soups ps an and d sauc sauces es amount of meat or poultry;

e.

meat me at sna snack cks, s, hor hors s d'oe d'oeuv uvre res, s, piz pizza za and and spe speci cial alty ty ite items ms; ;

f.

variou vari ous s ingr ingred edie ient nts s inco incorp rpor orat ated ed wit with h meat meat and and pou poult ltry ry  products  prod ucts suc such h as spic spices, es, vege vegetabl tables, es, brea breading ding mate material rial, ,  milk powd powder, er, drie dried d egg, egg, vege vegetabl table e protei proteins; ns;

cont co ntai aini ning ng

g.

pasteurized egg products;

h.

enviro envi ronm nmen enta tal l sam sampl ples es fr from om ar area eas s in in above are processed or manufactured.

the th e

requis requ isit ite e

which whi ch an any y

of of

the the

The quantity and types of mesophilic microorganisms present in or on any of these products offer a means of evaluating the degree of sanitation used during the process. If the results obtained for coliforms, and are Escherichia coli, Staphylococcus aureus unusually high, they might result in some type of official follow-up action. Any such follow-up analysis will use the appropriate Final Action Method found in the latest edition of Official Methods of Analysis of AOAC International or any of its supplements. Pertinent sections in the 16th Edition are:

3-1

USDA/FSIS Microbiology Laboratory Guidebook

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3rd Edition/1998

 Aerobic Plat  Aerobic Plate e Count Count (APC (APC): ): 966.2 966.23 3 Coliform Group and E. coli: 966.24 S. aureus: 987.09

3.11 Comp Compari arison son Wit With h the the AOAC AOAC Meth Method od The procedures in the following sections of this Chapter are either the same as those published by the AOAC or generally follow an AOAC  method.  meth od. The foll followin owing g is a listin listing g of devi deviati ations: ons: a.

The Th e pr proc oced edur ure e for for d det eter ermi mini ning ng nu numb mber ers s of co coli lifo form rm an and d E  . differ from the AOAC procedure as follows:

coli

i.

Use Us e a si sing ngle le tu tube be of la laur urel el su sulf lfat ate e try trypt ptos ose e bro broth th (LST) per dilution, rather than three tubes per dilution. ii. ii . Inc Incub ubate ate in inocu oculat lated ed LST LST and and EC EC brot broths hs for for 24 24 ± 2 h. h. iii. Cons Consider ider the the presen presence ce of gas gas in LST LST and EC broth broths s as positive for coliform and E. coli respectively, with no further testing required.  b.

The proc procedur edure e for the enum enumerat eration ion of S. aureus differs from the AOAC procedure in that only one tube, instead of three, per dilution is used to determine the estimated count.

3.12 Gene General ral Guidel Guidelines ines for for Testing Testing Fresh or Prepare Prepared d Foods a.

Do not not com combi bine ne the the com compo pone nent nts s of co comp mpos osit ite e item items s such such as as frozen dinners into a single sample. To the greatest extent possible, examine as separate samples the vegetable or non-meat portion(s) and the meat portion.

 b.

The quantit quantity, y, cond conditio ition n and suit suitabil ability ity of the samp sample le are very important. i.

The qua The quant ntit ity y sho shoul uld d be be suf suffi fici cien ent t to to per perfo form rm th the e analysis and have a reasonable amount in reserve for repeat testing. ii. ii . The con condit dition ion of rece receipt ipt sho should uld be in in keepi keeping ng with with good microbiological practices for the analysis(es) requested. iii. The sample sample shoul should d be, to the the greates greatest t extent extent  possible  poss ible, , repr represen esentati tative ve of the whol whole e of the original product at the time the sample was taken. iv. iv . Whe When n appro appropri priate ate and and if if possib possible le, , sample samples s shoul should d be received at the laboratory in their original unopened package(s) (intact sample). 3.13 Test Tests s Cover Covered ed in This Sect Section ion

3-2

USDA/FSIS Microbiology Laboratory Guidebook

a.  b. c. 3.2

Aerobic plate count Coliform Coli form and E. coli quantitative estimates S. aureus

Equipment and Materials a.  b. c. d. e. f. g. h. i. j. k. l.  m. n. o.  p.

3.21

3rd Edition/1998

Balance, capacity ≥2 kg, sensitivity ± 0.1 g Blender Blen der and ster sterile ile blen blender der jars Stomacher™ and sterile stomacher bags o Incubators at 35 ± 1.0°C, and 20 ± 1.0 C Water bath at 45.5 ± 0.05°C Water bath at 37 ± 1.0°C Manu Ma nual al or or Auto Automa mati tic c colo colony ny cou count nter er and and tal tally ly reg regis iste ter r Ster St eril ile, e, dis dispo posa sabl ble/ e/re reus usab able le dis dishe hes, s, pan pans s or tra trays ys for for sample cutting Ster St eril ile e force for ceps ps, , spoon spo on, , knife kni fe, , sciss sci ssor ors s and ot and othe her r sterile sampling equipment Sterile 1, 1, 5 and 10 10 ml ml pi pipettes Ster eri ile 100 x 15 mm pe pet tri di dis shes Transfer loop, 3 mm  Microsco Micr oscope pe and and clean clean slid slides es Refrigerated centrifuge Refrigerator pH meter meter

Media a.  b. c. d. e. f. g.

Plate Plat e coun count t agar agar (PC (PCA) A) in in cont contai aine ners rs sui suita tabl ble e for for maki making ng  pour plat plates es Laurel Laur el sulf sulfate ate tryp tryptose tose (LS (LST) T) bro broth th with ferm ferment entatio ation n tubes EC br broth wi wit th fe ferm rme enta tat tio ion n tu tubes Surfa Su rface ce dr dried ied Bai Bairdrd-Pa Parke rker r pl plate ates s (eg (egg g tel tellur lurite ite gl glyci ycine ne  pyruvate  pyru vate agar agar, , ETGPA ETGPA) ) Brai ain n he heart inf nfu usi sio on (B (BHI HI) ) br broth Tryp Tr ypti tica case se so soy y bro broth th wi with th 10 10% % sod sodiu ium m chl chlor orid ide e and and 1% sodium pyruvate (PTSBS) Toluidine blue DNA agar

3.2 .22 2 Re Rea age gen nts a. Butt tte erfi fie eld ld' 's ph phos osp phat ate e di dilu lue ent  b. Gram stai stain n reagen reagents ts c. Desi De sicc ccat ated ed rab rabbi bit t plas plasma ma (co (coag agul ulas ase) e) ED EDTA  TA  d. Tris Buffer e. Ammonium sulfate [(NH4)2SO4], reagent grade f. Triton X-100 g. 3M tr trich chl lor oro oace cet tic aci cid d so solu lut tion h. 1N HCl solution 3.3 3. 3 Pr Prep epar arat atio ion n and and Dilu Diluti tion on of of Samp Sample les s 3-3

USDA/FSIS Microbiology Laboratory Guidebook

3rd Edition/1998

See Section 1.3 1.5 (Sterilization of Instruments, Disinfection of Containers, and Cutting and Weighing Samples) 3.31 3. 31 Fo Food od Homo Homoge gena nate tes s a.

Using Usin g steri ste rile le spoo sp oons ns, , force for ceps ps, , sciss sci ssor ors, s, etc. et c., , aseptically weigh 50 ± 0.1 g of the sample into a sterile blender jar or stomacher bag.

 b.

If the samp sample le is froz frozen, en, rem remove ove port portions ions, , whe wheneve never r  possible  poss ible, , without without thawing thawing the larger larger sample sample and and weigh 50 ± 0.1 g of the sample into a sterile blender jar or stomacher bag. It is well known that freeze/thaw cycles are damaging to bacteria. This is particularly important when a re-examination of the product may be necessary. Otherwise, partially thaw the sample at 2-5° C for about 18 h, or by placing the sample in a watertight container and immersing it in cold water for 1-2 h.

c.

Add Ad d 450 450 ml st ster eril ile e Butt Butter erfi fiel eld' d's s phos phosph phat ate e dilu diluen ent t and and stomach for 2 minutes, or blend at high speed for two  minutes.  minu tes. The tota total l volu volume me in the blen blender der jar must completely cover the blades. This becomes the 1:10 dilution.

d.

Perm Pe rmit it th the e foa foam m to to set settl tle; e; th then en pi pipe pet t 10 10 ml ml of of the the  blended 1:10 dilu  blended dilution tion into a 90 ml dilu dilution tion blan blank k to  make the 1:10 1:100 0 dilu dilution tion. . Repeat Repe at thi this s proc procedur edure e to -3 -4  prepare  prep are seri serial al dilu dilution tions s of 10 , 10 , etc. Shake all dilutions 25 times in a one foot arc. Use a separate 10  ml pipe pipette tte to prep prepare are each dil dilutio ution. n. Pipettes Pipe ttes must deliver accurately the required volumes. Do not deliver less than 10% of a pipette's volume. For example, to deliver one ml, do not use a pipette of more than 10 ml volume.

e.

The anal The analys yst t sho shoul uld d str striv ive e to mi mini nimi mize ze the the tim time e from from whe when n the sample is stomached or blended until all the dilutions have been placed in or on the appropriate  medium;  medi um; ide ideally ally this time sho should uld not exce exceed ed 15 minu minutes tes whenever possible.

f.

If th the e sam sampl ple e con consi sist sts s of of les less s tha than n 50 50 g, g, wei weigh gh ab abou out t half the sample, and add the amount of diluent required to make a 1:10 dilution (nine times the weight of the  portion  port ion of sampl sample e used) used) and proc proceed eed as above above. .

g.

Hold re Hold rese serv rves es of ea each ch sa samp mple le at or be belo low w -15 -15°C (5°F), unless the product is stored normally at ambient 3-4

USDA/FSIS Microbiology Laboratory Guidebook

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temperature or unless a specific protocol specifies otherwise. Samples should be held until a determination is made that a repeat test is not necessary or for the length of time designated by the testing protocol. 3.32 3. 32 Wh Whol ole e Bir Bird d Rin Rinse se a.

Since Sinc e the there re ar are e dif diffe fere renc nces es be betw twee een n sam sampl ple e typ types es an and d sizes (eg. chicken vs. turkey carcasses), be sure to check the specific program protocol before using this  procedur  proc edure. e.

 b.

Aseptically Aseptica lly tra transfe nsfer r the car carcass cass to a sterile sterile Stom Stomache acher r 3500 bag (or equivalent), draining as much excess fluid as possible during the transfer.  Note: Larger (24 x 30 Note: 30-36 36 in.) bags will have to be used with turkeys.

c.

Add Add 400 400 ml (chi (c hick cken ens) s) or 600 60 0 ml ml (turk (tu rkey eys) s) of Butterfield's Phosphate Diluent (BPD) to the carcass in the bag. Pour approximately one half the volume into the interior cavity of the bird and the other half over the skin. Note: If Salmonella is the ONLY target analyte, Buffered Peptone Water (BPW) may be substituted for the BPD.

d.

Rins Ri nse e the the bi bird rd, , ins insid ide e and and ou out, t, wi with th a roc rocki king ng mo moti tion on for 1 min at a rate of approximately 35 forward and back swings per minute. This is done by grasping the carcass in the bag with one hand and the closed top of the bag with the other. Rock with a reciprocal motion in an 1824 inch arc, assuring that all surfaces (interior and exterior) are rinsed.

e.

Asepti Asep tica call lly y rem remov ove e the the car carca cass ss fro from m the the bag bag, , drai draini ning ng excess rinsed liquid into the bag, dispose of the carcass, and culture the bird rinse liquid according to  protocol  prot ocol dire directio ctions. ns.

3.33 Egg Products o

a.

Liquid eg eggs mu must be be he held at at 4. 4.4°C (40 F) or below for valid analysis.

 b.

Frozen sam Frozen samples ples must be tha thawed wed as rapi rapidly dly as poss possible ible in a water bath at 45°C.

c.

Expo Ex pose sed d or or lea leaki king ng sa samp mple les s sho shoul uld d not not be an anal alyz yzed ed. .

d.

Mix Mi x

the sa the samp mple le

with wi th

a 3-5

steri ste rile le sp spoo oon, n,

spat sp atul ula, a,

or by

USDA/FSIS Microbiology Laboratory Guidebook

3rd Edition/1998

shaking.

3.4

e.

Asep As epti tica call lly y wei weigh gh a min minim imum um of 10 100 0 g of of egg egg sa samp mple le in into to a sterile blender jar or sealable bag containing 900 ml of the appropriate enrichment or buffer. If a specific  protocol  prot ocol requires requires a samp sample le size greater greater than 100 g, the 1:10 ratio must be maintained in the same enrichment or  buffer.  buff er.

f.

Mix the Mix the 1:10 1:10 sam sampl ple e enri enrich chme ment nt/b /buf uffe fer r well well by by shak shakin ing, g, stomaching, or blending.

g.

Dried Drie d egg egg sa samp mple les s sho shoul uld d be be reh rehyd ydra rate ted d slo slowl wly y by by gradually adding the enrichment/diluent to the sample. This is done by adding a small portion of liquid to the sample and mixing aseptically to obtain a homogeneous suspension. Repeat this procedure three times and then add the remainder of the liquid. Mix until a lump-free suspension is obtained.

h.

Incuba Incu bate te or tr tran ansf sfer er to th the e app appro ropr pria iate te en enri rich chme ment nt  medium  medi um and incu incubate bate acco accordin rding g to the prot protocol ocol(s) (s) bein being g used.

Aerobic Plate Count (APC) a.

Pour Po ur Pl Plat ates es (R (Ref efer eren ence ce AO AOAC AC 96 966. 6.23 23 C) i.

Using Usin g the the di dilu luti tion ons s pre prepa pare red d in in sec secti tion on 3. 3.3, 3, pi pipe pet t -1 -2 -3 -4 1 ml from the 10 , 10 , 10 , 10 etc. dilutions into each of four petri dishes, two for each incubation temperature. Plate additional dilutions when expecting higher bacterial levels.

ii. ii .

Use sep separa arate te ster steril ile e pipe pipette ttes s for for each each dil diluti ution. on.

iii. Add molte molten n Plate Plate Count Count Agar cool cooled ed in a water water bath bath to 45 ± 1°C. Uniformly mix the agar and the inoculum by gently swirling or tilting each plate, taking care not to generate bubbles. iv. iv .

Allow the Allow the aga agar r to har harden den and the then n place place one ser series ies of duplicate plates in a 35 ± 1°C incubator for 48 h. Incubate the other series at 20 ± 1°C for four or five days.

v.

Use a co Use colo lony ny co coun unte ter r an and d cou count nt co colo loni nies es on th the e duplicate plates in a suitable range (30-300 colonies per plate). If plates do not contain 3-6

USDA/FSIS Microbiology Laboratory Guidebook

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30-300 colonies, record the dilution counted and the number of colonies found. Average the counts obtained from duplicate plates, multiply by the dilution factor and report this number as the aerobic plate count per gram or milliliter at the incubation temperature used.  b.

Alternat Alte rnate e Method Methods s - AOAC i.

Aerobi Aero bic c Pl Plat ate e Cou Count nt in Fo Food ods: s: Hy Hydr drop opho hobi bic c Gri Grid d *  Membrane  Memb rane Fil Filter ter Met Method hod (AOAC 986.32) ii. ii . Dry Reh Rehydr ydrata atable ble Fil Film m (Pet (Petrif rifilm ilm Aer Aerobi obic c Plate Plate™ ) *  Method  Meth od (AOAC 990.12) * iii. ii i. Spi Spira ral l Plat Plate e Meth Method od (AOAC 977.27) *Since these methods are available commercially,  manufact  manu facturer urer's 's direc direction tions s should should be follo followed wed. . 3.5

the

Coliform Group and Escherichia coli a.

Esti Es tima mate ted d Cou Count nt Pr Proc oced edur ure e (Re (Refe fere renc nce e AOA AOAC C 966 966.2 .24) 4) i.

ii. ii .

Using Usin g the the di dilu luti tion ons s pre prepa pare red d in in sec secti tion on 3. 3.3, 3, pi pipe pet t -1 -2 -3 1 ml from the 10 , 10 , 10 etc. dilutions into LST  broth,  brot h, one tube tube per dilution dilution. . Inoc Inoculat ulate e additiona additional l dilutions when expecting higher bacterial levels. The highest dilution of sample must be sufficiently high to yield a negative end point. Use sep separa arate te ster steril ile e pipe pipette ttes s for for each each dil diluti ution. on.

iii. Incubate Incubate the tubes tubes of LST LST broth broth at 35°C for 24 ± 2 h.

 b.

iv. iv .

Examine Exami ne each each tub tube e for for gas gas forma formatio tion n as evid evidenc enced ed by by displacement of fluid in the inverted tubes or by effervescence when tubes are shaken gently.

v.

Cons Co nsid ider er an any y tub tube e of of LST LST br brot oth h dis displ play ayin ing g gas gas as coliform positive, and report the number of coliform per gram in accordance with the highest dilution with gas. When a "skip" occurs, report by using the missing estimate (for example: If the -1 -2 -4 10 , 10 , and 10 dilutions produce gas but the -3 10 dilution tube is non-gassing, report "1,000 coliforms per gram.")

Fecal Coli Fecal Coliform form (E. coli) (Reference AOAC 966.24) i.

Estimated

Count

Procedure

Use Us e a 3 mm mm cal calib ibra rate ted d loo loop p to to tra trans nsfe fer r one one lo loop opfu ful l 3-7

USDA/FSIS Microbiology Laboratory Guidebook

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

to

a

Incuba Incu bate te th the e EC EC tub tubes es in a 45. 45.5 5 ± 0.0 0.05 5°C covered water bath for 24 ± 2 h. Submerge the EC tubes in the bath so that the water level is above the level of medium in the tubes.

iii. Record Record every every tube tube producin producing g gas, as evide evidence nced d by displacement of liquid in the inverted tube or by effervescence when tubes are shaken gently. iv.

c.

Rep epo ort th the nu num mber of E. coli per gram in accordance with the highest dilution displaying gas. When a "skip" occurs, report by using the missing estimate -1 -2 -4 (for example: If the 10 , 10 , and 10 dilutions -3  produce  prod uce gas but the 10 dilution tube is non-gassing, report "1,000 E. coli per gram.")

Alternate Me Methods - AOAC i. ii.

Coliform and Escherichia coli Counts in Foods: * Hydrophobic Grid Membrane Filter/MUG Method Coliform and Escherichia coli Counts in Foods: Dry * Rehydratable Film 

*Since these methods are available commercially,  manufacturer  manufact urers's s's dire directio ctions ns s shoul hould d be foll followe owed. d. 3.6

the

Staphylococcus aureus

a.

Esti Es tima mate ted d Cou Count nt Pr Proc oced edur ure e (Re (Refe fere renc nce e AOA AOAC C 987 987.0 .09) 9) i.

ii. ii .

Using Usin g the the di dilu luti tion ons s pre prepa pare red d in in sec secti tion on 3. 3.3, 3, pi pipe pet t -1 -2 -3 1 ml from the 10 , 10 , 10 etc. dilutions into tubes containing 10 ml of Trypticase (tryptic) Soy Broth with 10% sodium chloride and 1% sodium   pyruvate  pyru vate (PTSBS (PTSBS), ), one tube tube per dilution dilution. . Inoc Inoculat ulate e additional dilutions when expecting higher  bacteria  bact erial l leve levels. ls. The high highest est dilu dilution tion of samp sample le  must be suff sufficie icientl ntly y high to yiel yield d a nega negative tive end  point.  poin t. Use sep separa arate te ster steril ile e pipe pipette ttes s for for each each dil diluti ution. on.

iii. Incubate Incubate the PTSB PTSBS S tubes tubes at 35 35°C for 48 h. iv. iv . Usi Using ng a 3 mm cal calibr ibrate ated d loop loop, , tran transfe sfer r a loop loopful ful from each growth-positive tube as well as from the tube of the next highest dilution to previously  prepared  prep ared plates plates of Baird-Parke Baird-Parker r agar agar. . Streak Stre ak in a  manner  mann er to to produc produce e wellwell-isol isolated ated colo colonies nies. . 3-8

USDA/FSIS Microbiology Laboratory Guidebook

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3rd Edition/1998

Incu In cuba bate te th the e Bai Baird rd-P -Par arke ker r pla plate tes s at at 35 35°C for 48 h. Typical S. aureus colonies appear as circular, convex, smooth, grey-black to jet-black colonies on uncrowded plates and frequently have an off-white  margin  marg in surr surround ounded ed by a zone of prec precipit ipitatio ation n (turbidity) followed by a clear zone. The colonies usually have a buttery to gummy consistency.

vii. Test two two or more more isolate isolates, s, from from each useab useable le plate plate  meeting  meet ing the above abov e descript desc ription ion (3.6,vi) (3.6 ,vi), , for coagulase as in Section 3.6 (c).  b.

Direct Dire ct Plati Plating ng i.

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

ii. ii .

Pipet Pip et 0.1 ml fro from m each each dil dilut ution ion on pre previ vious ously ly  prepared and drie  prepared dried d Bair Baird-Pa d-Parker rker agar plat plates. es. Use separate accurate pipettes for each dilution.

iii ii i

Distribu Distr ibute te the the ino inocul culum um even evenly ly ove over r the the surfa surface ce of of the plates using separate, sterile, fire polished,  bent-gla  bent -glass ss rods ("ho ("hockey ckey stic sticks") ks") for each plat plate. e.  Mark plat plates es acco accordi rding ng to to the the diluti dilution on used. used.

iv. iv . v.

Inve In vert rt pl plat ates es an and d inc incub ubat ate e at at 35 35°C for 48 h. Select Sele ct pl plat ates es co cont ntai aini ning ng ap appr prox oxim imat atel ely y 20 20 or or mor more e well-isolated typical S. aureus colonies. Count  plates  plat es containi cont aining ng 20-200 20-2 00 colonies colo nies. . Typical Typi cal colonies are circular, convex, smooth, grey-black to jet-black and frequently have an off-white  margin  marg in surr surround ounded ed by a zone of prec precipit ipitatio ation n (turbidity) followed by a clear zone. The colonies usually have a buttery to gummy consistency.

vi. vi .

Select Selec t 10 colon colonies ies fro from m those those coun counted ted and and inoc inocula ulate te each into separate 13 x 100 millimeter tubes containing 0.2 ml of BHI broth for coagulase testing. Test for coagulase as in 3.6 (c). vii. Calc Calculat ulate e the total numbe number r of colonies colonies represen represented ted  by coag coagulas ulase e posi positive tive cultures cultures and mult multiply iply by the appropriate sample dilution factor to record the number of coagulase positive staphylococci per gram. c.

Coagulase Test for Staphylococcus aureus 3-9

USDA/FSIS Microbiology Laboratory Guidebook

3rd Edition/1998

i.

Use an Use an ino inocu cula lati ting ng ne need edle le to ob obta tain in a sma small ll am amou ount nt of growth from each suspect colony and place it into 13 X 100 mm tubes containing 0.2 ml of BHI Broth.

ii. ii .

A know known n coagu coagulas lase e posi positiv tive e and and a kno known wn neg negati ative ve culture should be inoculated into BHI broth at the same time as the samples.

iii. ii i. Inc Incub ubate ate ea each ch tube tube at 35 35°C for 18-24 h. iv. iv .

Add Add 0.5 0.5 ml of of rabbit rabb it plasm pla sma a with with EDTA ED TA, , reconstituted according to the manufacturer's directions, to the BHI cultures.

v.

Mix tho Mix thoro roug ughl hly y and and pl plac ace e the the tu tube bes s in in a 3535-37 37°C. water bath.

vi. vi .

Examine Exami ne thes these e tube tubes s each each hour hour, , from from one one thro through ugh six hours, for clot formation. Any degree of clotting should be interpreted as a positive reaction.

3.61 Special Sampling Procedure for Fermented Sausage Products a.

Introduction During the early stages of sausage fermentation, staphylococci can grow extensively if the starter culture is not added or fermentation fails with no concomitant production of lactic acid and drop in pH. Failure can be caused by poor quality starter cultures or the improper use of starter cultures or "back inoculation". S. aureus growth is aerobic and usually confined to the outer 1/8 inch of the sausage. Enterotoxin may be formed as a result of this growth. Coagulase-positive staphylococcal counts on large sticks of salami have been noted to vary widely. On large sticks, some areas may have very few staphylococci while 6 other areas may have levels in excess of 10 /g. Whenever  possible  poss ible, , obta obtain in 1-2 poun pounds ds of the suspect suspect saus sausage. age. In order to obtain a representative sample, portions should  be taken taken from several several differe different nt areas areas and composit composited ed for testing.

 b.

Procedur Proc edure e i.

If th the e sau sausa sage ge is mo mold ldy, y, wipe wipe th the e mol mold d off off th the e sausage casing with a piece of sterile tissue paper and proceed. 3-10

USDA/FSIS Microbiology Laboratory Guidebook

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3rd Edition/1998

To coll collect ect a samp sample, le, us use e a steri sterile, le, sha sharp rp knif knife e and and cut several thick slices from the sausage near the ends as well as in the middle. Aseptically trim  and save the outer 1/8 to 1/4 inch portion of the sausage and label it "shell portion". Even if the amount of sample is limited, do not cut deeper than 1/4 inch.

iii. Work Working ing asept aseptical ically, ly, blen blend d 25-50 25-50 g of the shell shell  portion  port ion for ente enteroto rotoxin xin test testing; ing; the same blen blended ded sample can be used to test for viable coagulase-positive as described in S. aureus section 3.6. iv. iv .

Analyze Analy ze the the  procedur  proc edures. es.

sampl sam ple e

by

either eit her

of

the th e

followi foll owing ng

3.62 The (Presumpt (Presumptive) ive) Staphylo Staphylococcal coccal Enteroto Enterotoxin xin Reverse Passive Latex Agglutination Test The procedure for this test is given in (15.20) and usually is the method of choice. 3.63 3.6 3 The Thermo rmonuc nuclea lease se As Assay say a.

Introduction This procedure is based on the detection of a heat stable DNase which is produced by most strains of S. aureus, including 98.3% of the enterotoxigenic strains. This heat stable DNase is produced in detectable amounts under all conditions which permit the growth of S. The DNase is aureus and the production of enterotoxin. able to survive processing conditions which would destroy viable S. aureus. This method can be used to screen large sausages or a large number of samples to identify "hot spots". It has been shown (Tatini, 1981) that the detection of DNase with this procedure is indicative of S. aureus 5  populati  popu lations ons of ≥10 per gram.

 b.

Procedur Proc edure: e: i.

Blend 20 g of shell, 10 g (NH4)2SO4, and 2 ml Triton X-100 in 40 ml of distilled water.

ii. ii .

Adjus Adj ust t the the pH pH of th this is slur slurry ry to to 4.5-4 4.5-4.8 .8 with with 1N HCl.

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iii. Centrifu Centrifuge ge under under refrigera refrigeration tion at 7-10,0 7-10,000 00 RPM for 15 min. iv. iv .

Decant Decan t and and disc discard ard th the e super supernat natant ant and ad add d 0.05 0.05 ml ml cold 3M trichloroacetic acid for each ml of the original slurry, mix and centrifuge a second time as above.

v.

Deca De cant nt an and d di disc scar ard d th the e su supe pern rnat atan ant. t. Re Re-s -sus uspe pend nd th the e  precipitate  precipit ate in 1 ml of Tris buffer, buffer, adjusted adjusted to pH 8.5, and then adjust the volume to 2 ml with Tris  buffer.  buff er.

vi.

Boi oil l the the solu lut tion for ≥15 but ≤90 min, cool and store under refrigeration until needed.

vii. Cut 2 mm diamet diameter er wells wells into into air dried dried Toluid Toluidine ine Blue DNA Agar. viii. vii i. Dispense Dispense the food food extra extract ct into into one one or more more wells wells using a Pasteur pipette. Do not overfill the well. ix. ix .

Incubate Incub ate th these ese pla plates tes, , agar agar sid side e dow down, n, at 37°C for 4 to 24 h.

x.

Any pi Any pink nk ha halo lo, , ext exten endi ding ng 1 mm mm bey beyon ond d the the we well ll is considered positive for thermonuclease.

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3.7

3rd Edition/1998

Sel Se lec ect ted Ref Refe eren enc ces Cunniff, P. (ed.). 1995. Official Methods of Analysis of AOAC International, 16th Edition. AOAC International Inc., Gaithersburg, MD 20877. Emswiler-Rose, B. S., R. W. Johnston, M. E. Harris, and W. H. Lee. 1980. Rapid detection of staphylococcal thermonuclease on casings of naturally contaminated fermented sausages.  Appl.  Appl . Envir Environ. on. Micr Microbio obiol. l. 440:1 440:13-18 3-18. . Lancette,

G.

A.,

and

aureus, p. 533-550.

S. R. Tatini. 1992. Staphylococcus In C. Vanderzant and D. F. Splittstoesser

(ed.), Compendium of Methods for the Microbiological Examination of Foods. Amer. Publ. Hlth. Assoc., Washington, D.C. 20005. Tatini, S. R. 1981. Thermonuclease as an indicator of staphylococcal enterotoxins in food, p. 53-75. In R. L. Ory (ed.), Antinutrients and Natural Toxicants in Foods. Food and  Nutriti  Nutr ition on Pres Press, s, Inc. Inc., , Westpo Westport, rt, CT.

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