Aoac Official Method Salmonella
3
17.9.31 AOAC Official Method 2002.10 Salmonella in Fresh Cheese, Dried Egg Products, and Fresh Chilled and Frozen Poultry Culture Method First Action 2002 Final Action 2006 ISO 6579:2002 (Applicable for the detection of Salmonella in fresh cheese, dried egg products, and fresh chilled and frozen poultry.) See Table 2002.10 for the results of the interlaboratory study supporting acceptance of the method. Note: Selective enrichment combination Muller-Kauffmann tetrathionate broth/Rappaport-Vassiliadis soya broth may not be effective for the recovery of S. typhi and S. paratyphi from foods. A. Principle Salmonella are resuscitated under nonselective conditions and then propagated through the use of selective enrichment broths to levels that can be successfully recovered when isolated on selective agars. B. Apparatus (a) Masticator .—Stomacher (masticator), or equivalent, for homogenizing test portions. ( b) Mas ti ca tor bags, ster ile .—Ap pro pri ate ca pac ity to accommodate test portions and masticator used. (c) Top loading balance.—Capacity of 2000 g with readability of 0.1 g. (d) Incubator .—Maintaining 35°–37°C. (e) Water baths.—Maintaining 41.5° ± 1.0°C. (f) Sterile culture tubes with rack.—16 · 150 mm tubes. (g) Syringe with filter .—10 mL sterile plastic syringe with 0.2 mm filter. (h) Pipets.—Sterile glass or plastic pipets, 1 mL with 0.01 mL graduations; 5 and 10 mL with 0.1 mL graduations. (i) Vortex mixer .—For mixing tube contents. (j) Sterile inoculating loops.—Ca 3 mm id or 10 mL, nichrome, platinum-iridium, or sterile plastic. C. Media and Reagents (a) Buffered peptone water (BPW).—Suspend 10 g peptone, 5.0 g NaCl, 9.0 g Na 2 HPO 4 ×12H 2 O, and 1.5 g KH 2 PO 4 in 1 L water, and mix thoroughly. Dispense 225 mL aliquots in 500 mL containers. Autoclave for 15 min at 121°C. Final pH should be 7.0 ± 0.2. (b) Rappaport-Vassiliadis soya peptone broth (RVS).—Medium may be made from individual ingredients or from ISO-compliant com mer cial for mu la tion. Pre pare the fol low ing so lu tions: Solution A.—Dissolve 5.0 g soya peptone, 8.0 g NaCl, 1.4 g KH 2 PO 4 , and 0.2 g K 2 HPO 4 in l L water. Heat to ca 70°C to completely dissolve medium. Prepare solution A on the day that complete medium is to be made. Solution B.—Dissolve 400 g MgCl 2 ×6H 2 O in 1 L water. Solution B can be stored at room temperature in a dark bottle up to 1 year. Solution C.—Dissolve 0.4 g malachite green oxalate in 100 mL water. Solution C can be stored at room temperature in the dark for up to 6 months. To prepare complete RVS broth, combine 1000 mL Solution A, 100 mL Solution B, and 10 mL Solution C. Dispense complete medium in 10 mL aliquots into 16 · 150 mm tubes and autoclave 15 min at 115°C. Final pH should be 5.2 ± 0.2. ( c) Mul ler-Kauffmann tetrathionate broth + novobiocin (MKTT+n).—Suspend 4.23 g meat extract, 8.45 g tryptone, 2.54 g NaCl, 38.04 g CaCO 3 , 30.27 g Na 2 S 2 O 3 (anhydrous), 4.75 g ox bile, and 9.5 mg brilliant green in 1 L water. Boil gently for 1 min. Cool below 45°C and store at 5°–8°C. The base solution should be pH 7.0 ± 0.2. Prepare I–KI solution by dissolving 25 g KI in 25 mL water, adding 20 g resublimed I, dissolving, and diluting to 100 mL with sterile water. Prepare novobiocin solution by dissolving 0.04 g novobiocin sodium salt in 5 mL water. Filter-sterilize through 0.2 mm filter. On the day the medium is used, add 20 mL I–KI solution and 5.0 mL novobiocin per 1 L basal broth. Resuspend precipitate by gentle agitation, and aseptically dispense 10 mL portions into 16 · 150 mm sterile test tubes. Do not heat medium after addition of I–KI and novobiocin solutions. (d) Xylose lysine desoxycholate (XLD) agar .—See 967.25A(d) (see 17.9.01). (e) Second selective agar .—The second agar used is at the discretion of the analyst. The agar used should be complementary to XLD and appropriate for isolation of lactose-positive strains of Salmonella, S. typhi, and S. paratyphi. (f) Nutrient agar .—Suspend 3.0 g meat extract, 5.0 g peptone, and 15 g agar in 1 L water, and mix thoroughly. Heat to boiling to dissolve completely. Autoclave at 121°C for 15 min. Cool in water bath, and pour 20 mL portions into 15 · 100 mm Petri dishes. Let agar cool and dry before use. Final pH should be 7.0 ± 0.2. (g) Triple sugar iron agar (TSI).—Suspend 3.0 g meat extract, 3.0 g yeast extract, 20.0 g enzymatic digest of casein, 5.0 g NaCl, 10.0 g lactose, 10.0 g sucrose, 1.0 g glucose, 0.3 g iron(III) citrate, 0.3 g Na 2 S 2 O 3 , 0.024 g phenol red, and 13 g agar in 1 L water, and mix thoroughly. Heat to boiling to dissolve completely. Dispense medium into 16 · 150 mm tubes, 1/3 full, and cap or plug to maintain aerobic conditions during use. Autoclave tubes for 15 min at 121°C. Before the medium solidifies, place tubes in a slanted position to form deep butts (ca 3 cm) and adequate slants (ca 5 cm) on solidification. The final pH should be 7.4 ± 0.2. (h) Urea agar .—Suspend 1.0 g peptone, 1.0 g glucose, 5.0 g NaCl, 2.0 g KH 2 PO 4 , 0.012 g phenol red, and 15 g agar in 1 L water. Heat to boiling to dissolve completely. Autoclave for 15 min at 121°C. Cool to 50°–55°C. Prepare urea solution by dissolving 400 g urea in water and dilute to a final volume of 1 L. Sterilize by filtration through 0.2 mm filter. To prepare complete medium, aseptically add 50 mL urea solution to 950 mL cooled urea agar base. Mix thoroughly. Note: Do not heat the complete medium. Dispense complete medium in 10 mL quantities into sterile tubes. Let tubes set in sloping position. The final pH should be 6.8 ± 0.2. ( i ) L -lysine decarboxylation me dium (Falkow) .—See 967.25A(m)(2) (see 17.9.01). (j) Bromocresol purple solution.—0.2%. Dissolve 0.2 g in sterile water, and dilute to 100 mL. (k) Sterile physiological saline solution.—See 940.36B(c) (see 17.1.02). (l) Salmonella polyvalent somatic (O) antiserum.—Antiserum A-I and Vi. (m) Salmonella polyvalent flagellar (H) antiserum.—Poly A–Z. ª 2006 AOAC INTERNATIONAL
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Transcript of Aoac Official Method Salmonella
17.9.31AOAC Of fi cial Method 2002.10Sal mo nella in Fresh Cheese,
Dried Egg Prod ucts,and Fresh Chilled and Frozen Poul try
Culture Method
First Ac tion 2002Final Action 2006
ISO 6579:2002
(Ap pli ca ble for the de tec tion of Sal mo nella in fresh cheese, driedegg prod ucts, and fresh chilled and frozen poul try.)
See Ta ble 2002.10 for the re sults of the interlaboratory studysup port ing ac cep tance of the method.
Note: Se lec tive en rich ment com bi na tion Mul ler-Kauffmanntetrathionate broth/Rappaport-Vassiliadis soya broth may not beef fec tive for the re cov ery of S. typhi and S. paratyphi from foods.
A. Prin ci ple
Sal mo nella are re sus ci tated un der nonselective con di tions andthen prop a gated through the use of se lec tive en rich ment broths tolev els that can be suc cess fully re cov ered when iso lated on se lec tiveagars.
B. Ap pa ra tus
(a) Mas ti ca tor.—Stomacher (mas ti ca tor), or equiv a lent, forho mog e niz ing test por tions.
(b) Mas ti ca tor bags, ster ile.—Ap pro pri ate ca pac ity toac com mo date test por tions and mas ti ca tor used.
(c) Top load ing bal ance.—Ca pac ity of 2000 g with readability of 0.1 g.
(d) In cu ba tor.—Main taining 35°–37°C.
(e) Wa ter baths.—Main taining 41.5° ± 1.0°C.
(f) Ster ile cul ture tubes with rack.—16 ´ 150 mm tubes.
(g) Sy ringe with fil ter.—10 mL ster ile plas tic sy ringe with 0.2 mm fil ter.
(h) Pipets.—Ster ile glass or plas tic pipets, 1 mL with 0.01 mLgrad u a tions; 5 and 10 mL with 0.1 mL grad u a tions.
(i) Vor tex mixer.—For mix ing tube con tents.
(j) Ster ile in oc u lat ing loops.—Ca 3 mm id or 10 mL, ni chrome,plat i num-irid ium, or ster ile plas tic.
C. Me dia and Re agents
(a) Buffered peptone wa ter (BPW).—Sus pend 10 g peptone,
5.0 g NaCl, 9.0 g Na2HPO4×12H2O, and 1.5 g KH2PO4 in 1 L wa ter,and mix thor oughly. Dis pense 225 mL aliquots in 500 mL
con tain ers. Au to clave for 15 min at 121°C. Fi nal pH should be 7.0 ±0.2.
(b) Rappaport-Vassiliadis soya peptone broth (RVS).—Me diummay be made from in di vid ual in gre di ents or from ISO-com pli antcom mer cial for mu la tion. Pre pare the fol low ing so lu tions:So lu tion A.—Dis solve 5.0 g soya peptone, 8.0 g NaCl, 1.4 g
KH2PO4, and 0.2 g K2HPO4 in l L wa ter. Heat to ca 70°C tocom pletely dis solve me dium. Pre pare so lu tion A on the day thatcom plete me dium is to be made. So lu tion B.—Dis solve 400 g
MgCl2×6H2O in 1 L wa ter. So lu tion B can be stored at roomtem per a ture in a dark bot tle up to 1 year. So lu tion C.—Dis solve 0.4 g mal a chite green ox a late in 100 mL wa ter. So lu tion C can be stored atroom tem per a ture in the dark for up to 6 months. To pre parecom plete RVS broth, com bine 1000 mL So lu tion A, 100 mLSo lu tion B, and 10 mL So lu tion C. Dis pense com plete me dium in
10 mL aliquots into 16 ´ 150 mm tubes and au to clave 15 min at
115°C. Fi nal pH should be 5.2 ± 0.2.
(c) Mul ler-Kauffmann tetrathionate broth + novobiocin(MKTT+n).—Sus pend 4.23 g meat ex tract, 8.45 g tryptone, 2.54 gNaCl, 38.04 g CaCO3, 30.27 g Na2S2O3 (an hy drous), 4.75 g ox bile,and 9.5 mg bril liant green in 1 L wa ter. Boil gently for 1 min. Cool
be low 45°C and store at 5°–8°C. The base so lu tion should be pH 7.0± 0.2. Pre pare I–KI so lu tion by dis solv ing 25 g KI in 25 mL wa ter,add ing 20 g resublimed I, dis solv ing, and di lut ing to 100 mL withster ile wa ter. Pre pare novobiocin so lu tion by dis solv ing 0.04 gnovobiocin so dium salt in 5 mL wa ter. Fil ter-ster il ize through
0.2 mm fil ter. On the day the me dium is used, add 20 mL I–KIso lu tion and 5.0 mL novobiocin per 1 L basal broth. Re sus pendpre cip i tate by gen tle ag i ta tion, and asep ti cally dis pense 10 mL
por tions into 16 ´ 150 mm ster ile test tubes. Do not heat me diumaf ter ad di tion of I–KI and novobiocin so lu tions.
(d) Xylose lysine desoxycholate (XLD) agar.—See 967.25A(d)(see 17.9.01).
(e) Sec ond se lec tive agar.—The sec ond agar used is at thedis cre tion of the an a lyst. The agar used should be com ple men tary toXLD and ap pro pri ate for iso la tion of lac tose-pos i tive strains ofSal mo nella, S. typhi, and S. paratyphi.
(f) Nu tri ent agar.—Sus pend 3.0 g meat ex tract, 5.0 g peptone,and 15 g agar in 1 L wa ter, and mix thor oughly. Heat to boil ing to
dis solve com pletely. Au to clave at 121°C for 15 min. Cool in wa ter
bath, and pour 20 mL por tions into 15 ´ 100 mm Petri dishes. Letagar cool and dry be fore use. Fi nal pH should be 7.0 ± 0.2.
(g) Tri ple sugar iron agar (TSI).—Sus pend 3.0 g meat ex tract,3.0 g yeast ex tract, 20.0 g en zy matic di gest of ca sein, 5.0 g NaCl,10.0 g lac tose, 10.0 g su crose, 1.0 g glu cose, 0.3 g iron(III) ci trate,0.3 g Na2S2O3, 0.024 g phe nol red, and 13 g agar in 1 L wa ter, andmix thor oughly. Heat to boil ing to dis solve com pletely. Dis pense
me dium into 16 ́ 150 mm tubes, 1/3 full, and cap or plug to main tain
aer o bic con di tions dur ing use. Au to clave tubes for 15 min at 121°C.Be fore the me dium so lid i fies, place tubes in a slanted po si tion toform deep butts (ca 3 cm) and ad e quate slants (ca 5 cm) onso lid i fi ca tion. The fi nal pH should be 7.4 ± 0.2.
(h) Urea agar.—Sus pend 1.0 g peptone, 1.0 g glu cose, 5.0 gNaCl, 2.0 g KH2PO4, 0.012 g phe nol red, and 15 g agar in 1 L wa ter.Heat to boil ing to dis solve com pletely. Au to clave for 15 min at
121°C. Cool to 50°–55°C. Pre pare urea so lu tion by dis solv ing 400 gurea in wa ter and di lute to a fi nal vol ume of 1 L. Ster il ize by
fil tra tion through 0.2 mm fil ter. To pre pare com plete me dium,asep ti cally add 50 mL urea so lu tion to 950 mL cooled urea agar base. Mix thor oughly. Note: Do not heat the com plete me dium. Dis pensecom plete me dium in 10 mL quan ti ties into ster ile tubes. Let tubes set in slop ing po si tion. The fi nal pH should be 6.8 ± 0.2.
(i) L-lysine decarboxylat ion me dium (Falkow) .—See967.25A(m)(2) (see 17.9.01).
(j) Bromocresol pur ple so lu tion.—0.2%. Dis solve 0.2 g in ster ile wa ter, and di lute to 100 mL.
(k) Ster ile phys i o log i cal sa line so lu tion.—See 940.36B(c) (see17.1.02).
(l) Sal mo nella polyvalent so matic (O) an ti serum.—An ti serumA-I and Vi.
(m) Sal mo nella polyvalent flagellar (H) an ti serum.—Poly A–Z.
ã 2006 AOAC IN TER NA TIONAL
ã 2006 AOAC IN TER NA TIONAL
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D. Prep a ra tion of Test Sus pen sions
(a) Fresh cheese.—Asep tically weigh 25 g test por tion into
stomacher bag. Add 225 mL prewarmed (35°C) BPW, C(a), andho mog e nize in stomacher for 1–3 min. In cu bate the test sus pen sion
for 16–20 h at 35°–37°C.(b) Dried egg prod ucts.—Asep tically weigh 25 g test por tion
into stomacher bag. Add 225 mL BPW, C(a), and ho mog e nize instomacher for 1–3 min. In cu bate test sus pen sion for 16–20 h at
35°–37°C.(c) Poul try prod ucts.—Asep tically weigh 25 g test por tion into
stomacher bag. Add 225 mL BPW, C(a), and ho mog e nize instomacher for 1–3 min. In cu bate test sus pen sion for 16–20 h at
35°–37°C.
E. Iso la tion
(a) Growth in se lec tive broth.—Gently shake in cu bated testsus pen sion, D, and trans fer 0.1 mL into 10 mL RVS me dium, C(b),and an ad di tional 1.0 mL into 10 mL MKTT+n broth, C(c). In cu bate
RVS me dium at 41.5° ± 1°C for 24 ± 3 h. In cu bate MKTT+n broth at
35°–37°C for 24 ± 3 h. Mix on a Vor tex mixer all se lec tive tubes.Streak loopful of in cu bated RVS me dium onto se lec tive en rich mentplates of XLD agar, C(d), and the sec ond agar se lected (see C(e) forde tails). Re peat iso la tion with 3 mm loopful of MKTT+n test broth.
In cu bate plates 24 ± 3 h at 35°–37°C. If growth is slight or if no
typ i cal col o nies of Sal mo nella are pres ent, reincubate at 35°–37°Cfor ad di tional 24 h. Re-ex am ine plates for typ i cal col o nies ofSal mo nella.
(b) Ap pear ance of typ i cal Sal mo nella col o nies on XLD.—Pinkcol o nies with or with out black cen ters. Many Sal mo nella may havelarge, glossy black cen ters or may ap pear as al most com pletelyblack col o nies. Atyp i cally, a few Sal mo nella cul tures pro duceyel low col o nies with or with out black cen ters.
F. Treat ment of Col onies
(a) In oc u la tion of TSI.—Pick with a ster ile nee dle 2 or moretyp i cal or sus pi cious col o nies, if pres ent, from each XLD plate andsec ond agar plate. In oc u late TSI slant, C(g), by streak ing agar slant
and then stab bing the butt. Store picked se lec tive plates at 5°–8°C.
In cu bate slants at 35°–37°C for 24 ± 2 h. Cap tubes loosely tomain tain aer o bic con di tions while in cu bat ing slants to pre ventex ces sive H2S pro duc tion. Sal mo nella cul tures typ i cally haveal ka line (red) slant and acid (yel low) butt, with or with out H2S(black en ing of agar) in TSI.
(b) In oc u la tion of L-lysine decarboxylation me dium.—Using aster ile nee dle, trans fer a por tion of TSI cul ture to L-lysinedecarboxylation me dium, C(i). Close tube caps tightly af ter
in oc u la tion and in cu bate at 35°–37°C for 24 ± 2 h. Sal mo nella spp.give pur ple color of al ka line re ac tion through out broth (fi nal color is slightly darker than orig i nal pur ple color of me dium). Some timestubes that are yel low af ter 8–12 h of in cu ba tion change to pur plelater. Neg a tive test is per ma nently yel low through out broth. Ifme dium ap pears to be dis col ored (nei ther pur ple nor yel low), add afew drops of 0.2% bromocresol pur ple dye, C(j), and re read there ac tion.
(c) Se lec tion for iden ti fi ca tion.—Re tain all pre sump tive pos i tiveSal mo nella cul tures on TSI (al ka line slant and acid butt) agar forbio chem i cal and serological test whether or not cor re spond ing lysinedecarboxylation re ac tion is pos i tive (al ka line) or neg a tive (acid). Donot ex clude a TSI cul ture that ap pears to be non-Sal mo nella if there ac tion in L-lysine decarboxylation broth is typ i cal for Sal mo nella.Treat these cul tures as pre sump tive pos i tive and sub mit them tofur ther ex am i na tion. Lysine decarboxylation me dium is use ful inde tec tion of S. Arizonae and atyp i cal Sal mo nella strains that uti lizelac tose and/or su crose. Dis card only ap par ent non-Sal mo nella TSIcul tures (acid slant and acid butt) if cor re spond ing lysinedecarboxylation broth is not typ i cal (acid) for Sal mo nella. Testre tained TSI cul tures as di rected in F(d) to de ter mine if they areSal mo nella spp., 967.27D(e)(1) (see 17.9.03), or S. Arizonaeor gan isms, 967.27D(e)(2) (see 17.9.03). If TSI slants fail to givetyp i cal Sal mo nella re ac tions, pick ad di tional sus pi cious col o niesfrom se lec tive me dium plate not giv ing any pre sump tive pos i tivecul tures, and in oc u late TSI and lysine decarboxylation broth as inF(a) and (b).
(d) Iden ti fi ca tion.—Ap ply bio chem i cal and serologicaliden ti fi ca tion tests to 3 pre sump tive pos i tive TSI cul tures pickedfrom se lec tive agar plates streaked from RVS me dium and to 3pre sump tive pos i tive TSI cul tures picked from se lec tive agar platesstreaked from MKTT+n broth as di rected in 967.27 (see 17.9.03)and 967.28 (see 17.9.07). Ex am ine min i mum of 6 TSI and 6 lysinedecarboxylation broth cul tures for each 25 g test por tion tested. AnyAOAC-ap proved Sal mo nella bio chem i cal iden ti fi ca tion test kitmay be used in stead of per form ing the in di vid ual bio chem i cal testspre sented in this method.
Ref er ence: J. AOAC Int. 86, 275(2003).
ã 2006 AOAC IN TER NA TIONAL
