17.9.02 AOAC Offi Of ficial cial Method 967.26
Salm Sal m o n e l l a in in Processed Pro cessed Foods Detec De tection tion First Action Action 1967 Final Fi nal Action Action 1974
A. Prep a ra tion of Test Suspensions
( a ) Dr ie d wh ol e eg g, dr ie d eg g yo lk , an d dr ie d eg g white. —As white. —As ep eptically tically open labo lab o ra ratory tory sam ple con tainer and asepti asep tically cally weigh 25 g test portion por tion into sterile, sterile, empty, wide-mouth, screw-cap pt (500 mL) jar. Add ca 15 mL sterile ster ile lactose lac tose broth, 967.25A((a) ( see 17.9.01). 967.25A see 17.9.01). Stir with sterile ster ile glass rod, sterile ster ile spoon, or sterile ster ile tongue depres pressor sor to smooth sus pen pension. sion. Add 3 addi ditional tional portions por tions lactose lactose broth (10, 10, and 190 mL) for to tal of 225 mL. Stir after af ter each addi addition tion until until test portion is sus pended with out lumps. Cap jar se curely and let stand at room tem per peraature 60 min. Mix well by shak shaking, ing, and deter termine mine pH with test pa per, 967.25B 967.25B((l) ( see see 17.9.01). Ad just just pH, if nec neces essary, sary, to 6.8 ± 0.2 with sterile ster ile 1M NaOH or HCl, 967.25B 967.25B((c) or ( d) ( see see 17.9.01), 17.9.01), cap ping jar securely and mixing mix ing well before deter de termin mining ing final final pH. Loosen jar cap ca 14 turn and incu incu bate 24 ± 2 h at 35°C. (b) Dry whole milk. —Aseptically —Aseptically weigh 25 g test portion into sterile, ster ile, wide-mouth screw-cap 500 mL (1 pt) jar. Add 225 mL sterile sterile H2O and mix well. Cap jar securely se curely and let stand 60 min at room tem per peraature. Mix well by swirling swirl ing and deter determine mine pH with test pa per, pa per, 967.25B((l) ( see 17.9.01). 967.25B see 17.9.01). Adjust pH, if neces nec essary, sary, to 6.8 ± 0.2 with sterile ster ile 1M NaOH or HCl, 967.25B 967.25B((c) or (d) ( see see 17.9.01). Add 0.45 mL 1% aqueous aqueous brilliant brilliant green dye solu solution, tion, 967.25B 967.25B((n) ( see see 17.9.01), and mix well. Loosen jar cap ca ¼ turn and incu in cu bate 24 ± 2 h at 35°C. (c) Dried active active yeast. —Aseptically —Aseptically weigh 25 g test portion into sterile, ster ile, empty, wide-mouth, screw-cap pt (500 mL) jar. Add 225 mL sterile ster ile Trypticase (tryptic) soy broth, 967.25A 967.25A((t) ( see 17.9.01), see 17.9.01), and let yeast form smooth sus pension. Cap se securely curely and let stand 60 min at room tem per peraature. Deter Determine mine pH with test pa per, 967.25B 967.25B((l) ( see see 17.9.01). Ad just pH, if nec essary, essary, to 6.8 ± 0.2 with sterile ster ile 1M NaOH or HCl, 967.25B 967.25B((c) or (d) ( see see 17.9.01), 17.9.01), cap ping jar securely and mixing mix ing well before be fore deter determining mining final final pH. (If pH is ad justed before before yeast is evenly sus pended, final pH will be less than de desired.) sired.) Incu In cu bate 24 ± 2 h at 35°C, with jar cap loos ened ¼ turn. (d) On Onion ion powder powder and garlic garlic powder powder .—Autoclave 225 mL portions Trypticase (tryptic) soy broth, 967.25A 967.25A((t) ( see see 17.9.01), with added K 2SO3 (5 g/L) for 0.5% final K 2SO3 concentration, in 225 mL portions in 500 mL flasks, for 15 min at 121°C. Aseptically determine the volume and adjust, if n ecessary ecessary,, to 225 mL. Aseptically weigh 25 g test portion into sterile, wide-mouth, screw-cap 500 mL (1 pint) jar. Add the 225 mL sterile Trypticase (tryptic) soy broth with K 2SO3 to the test portion and mix thoroughly with a sterile glass rod or spoon. Let stand 60 min and determine pH with test paper, 967.25B 967.25B((l) ( see see 17.9.01). Adjust pH, if necessary, to 6.8 ± 0.2 with sterile 1M NaOH or 1 M HCl, 967.25B 967.25B((c) or (d) ( see see 17.9.01). Incubate 24 ± 2 h at 35 °C, with jar cap loosened ¼ turn. (e) Milk choc olate and ca sein. sein. —Asep —Aseptically tically weigh 25 g test port ion into ster sterile ile blen der jar. Add 225 mL ster ile re consti stituted tuted NFDM, 967.25A 967.25A((v) ( see 17.9.01), see 17.9.01), to choc olate products, and add 225 mL lactose broth, 967.25A 967.25A((a) ( see 17.9.01), to casein prod ucts. Blend each test port ion/b roth mix mixture ture 2 min at high speed and decant blended ho homog mogeenate into sterile ster ile 500 mL jar. Cap jar securely se curely and let stand 60 min at room tem per tem peraature. Mix
well by shaking, shaking, and deter determine pH with test pa per, 967.25B 967.25B((l) ( see 17.9.01). essary, sary, to 6.8 ± 0 .2 with sterile sterile 1M see 17.9.01). Ad just pH, if neces NaOH or HCl, 967.25B 967.25B((c) or (d) ( see see 17.9.01), cap ping jar securely se curely and mixing mixing well be fore de ter termin mining ing final fi nal pH. To choc olate-reconstituted olate-reconstituted NFDM test sam ple sam ple s, add 0. 0.45 45 mL 1% aque ous bril brilliant liant green dye, 967.25B 967.25B((n) ( see see 17.9.01), and mix well. Loosen jar caps ¼ turn and in cu bate jar 24 ± 2 h at 35°C. ( f ) In In sta sta nt no non n fat dr dryy mil k (N (NFDM FDM ). ). —A —Asep sep tically open labo lab ora ratory tory sam ple container container and asepti asep tically cally weigh 25 g test portion into sterile sterile beaker (250 mL) or other ap pro pri priate ate container. container. Cover with sterile sterile foil cover or sterile ster ile cap to prevent pre vent contam contamiina nation. tion. Using sterile ster ile glass or pa per (made with tape to with withstand stand autoclaving) funnel, fun nel, p our 25 g analyt lytiical unit gently and slowly over surface surface of 225 mL brilliant brilliant green H2O, 967.25A 967.25A((w) ( see 17.9.01), see 17.9.01), contained contained in sterile ster ile 500 mL Erlenmeyer or other ap pro priate container. Let container con tainer with test portion–pre-enrichment broth stand undis undisturbed turbed 60 ± 5 min. Incu Incu bate loosely capped con tainer, without without mixing mixing or pH ad just justment, ment, for 24 ± 2 h at 35°C. B. Iso la tion
(a) Growth in selec selective tive broth. —Gently —Gently shake in incu cu bated test portion mix mixture, ture, A, and transfer 1 mL to 10 mL sele selenite cystine broth, 967.25A 967.25A((b)( )(1 (2) ( see 17.9.01), and addi additional tional 1 mL to 1) or (2 see 17.9.01), 10 mL tetrathionate broth, 967.25A 967.25A((c) ( see see 17.9.01). Incu bate 24 ± 2 h at 35°C. (For dried ac tive yeast, substi sub stitute tute lauryl sulfate tryptose broth, 967.25A 967.25A((u) ( see 17.9.01), see 17.9.01), for sele selenite cystine broth, 967.25A((b)( 967.25A )(1 1) or (2 (2) ( see 17.9.01). see 17.9.01). Mix on a Vortex Vortex mixer, and streak 3 mm loopful of incu incu bated selenite cy stine broth on selective media me dia plates of XLD, 967.25A 967.25A((d) ( see ( see 17.9.01), 17.9.01), HE, 967.25A 967.25A((e) ( see 17.9.01), see 17.9.01), and BS, 967.25A 967.25A((f ) ( see 17.9.01). see 17.9.01). Re peat with 3 mm loopful of incu incu bated tetrathionate broth. Incu bate plates 24 ± 2 h at 35°C. ( b) Ap pea rance of typi typ ical Salmonella colo col onies. —(1 —( 1) On XLD . —Pin —Pin k col colo onies with or without with out black centers. cen ters. Many Salmo Sal monella nella may may have large, glossy black cen ters or may ap pear as almost al most com pletely black col colo onies. Atypi Atypically, a few Sal Salmo monella nella cultures cul tures produce produce yellow yellow colo colonies with or without black cen centers. ters. (2) On HE. —Blue-green —Blue-green to blue col colo onies with or without without black centers. cen ters. Many Sal Salmo monella nella colo colonies may have large glossy black centers cen ters or may ap pear as almost almost com pletely black colo colonies. (3 (3) On BS. —Brown, —Brown, gray, or black, some sometimes times with metal me tallic lic sheen. Surrounding Sur rounding medium medium is usually usually brown at first, turn ing black with increas in creasing ing incu incu ba bation tion time. Some strains produce pro duce green colo colonies with little little or no dark en ening ing of surround surrounding ing medium. medium. Exam Examine ine XLD and HE plates for typi typ ical or sus pi picious cious Sal Salmo monella nella col colo onies after after 24 ± 2 h incu ba bation tion at 35°C. BS plates should be ex am amined ined for typiical or sus pi typ picious cious Sal Salmo monella nella col colo onies after after 24 ± 2 h and 48 ± 2 h incu in cu ba bation tion at 35°C. C. Treat ment of Ty p i cal or Sus p i cious Col onies
(a) In Inoc ocu ula lation tion of triple sugar iron (TSI) agar and lysine iron agar (LIA). —Pick —Pick with nee needle dle 2 or more typi typical or suspi picious cious colo col onies, if present, present, from each XLD, HE, and BS plates having growth. Inoc Inocu ulate TSI slant, 967.25A 967.25A((g) ( see 17.9.01), see 17.9.01), with portion portion of each colony col ony by streaking streaking slant and stab bing butt butt.. Af After ter inoc in ocu ulat lating ing TSI with needle, nee dle, do not ob tain more inoculum from colony col ony and do not heat needle, needle, but inoc inoculate ulate LIA, 967.25A 967.25A((m)( )(1 1) ( see 17.9.01), see 17.9.01), as in 967.27C 967.27C((a) ( see 17.9.03). see 17.9.03). Store picked selec selective plates at 5° –8°C or at room temper peraature (ca 26°C). TERNA NATIONAL TIONAL � 2005 AOAC INTER
(b) Pre sump sumptive tive reac re actions. tions. —Incu —In cu bate TSI and LIA slants at 35°C for 24 ± 2 h . Cap tubes loosely to main tain aer o bic condi con ditions tions while incu in cu bat bating ing slants to prevent pre vent exces excessive sive H2S produc pro duction. tion. Sal Salmo monella nella cultures cultures typi typically have alka al kaline line (red) slant and acid (yel low) butt, with or with out H2S (blacken (blackening ing of agar) in TSI. In LIA, Sal Salmo monella nella cul cultures tures typi typically have alka al kaline line (pur ple ) re ac action tion in butt. Consider Con sider only a distinct dis tinct yel low col oration oration in butt of tube as an acidic (neg ative) reac action. tion. Do not elimiinate cultures elim cul tures that pro duce discol color oration ation in butt solely on this basis. ba sis. Most Sal Salmo monella nella cul cultures tures pro duce H 2S in LIA. Retain Re tain all presump pre sumptive tive posi positive Sal Salmo monella nella cul cultures tures on TSI (alka (al kaline line slant and acid butt) agar for biochem biochemiical and serological tests whether or not corre correspond sponding ing LIA reac reaction tion is posi pos itive (alka (al kaline line butt) or negaative (acid butt). Do not ex clude a TSI culture that ap pears neg ap pears to be non-Sal non- Salmo monella nella if if the reac re action tion in LIA is typi typ ical (alka (al kaline line butt) for Sal Salmo monella nella.. Treat these cultures cul tures as presump pre sumptive tive posi positive and submit sub mit them to f urther ex ami amination. LIA is useful use ful in de tection tection of S. Arizonae and Arizonae and atypi atypical Sal Salmo monella nella strains strains that utilize uti lize lactose lac tose and/or sucrose. sucrose. Discard Dis card only ap par parent ent non-Sal non- Salmo monella nella TSI cultures (acid slant and acid butt) if cor re respond sponding ing LIA reac re actions tions are not typi typ i cal (acid butt) for Sal Salmo mo nella nella.. Test retained re tained pr e sump sumptive tive posi pos i tive TSI cultures cul tures as directed di rected in C (c ) to
IN TERNA NATIONAL TIONAL � 2005 AOAC INTER
de termine deter mine if they are Sal Salmo monella nella spp., 967.27D 967.27D((e )( )(1 1 ) ( see 17.9.03), or S. Arizonae or Arizonae organ ganisms, isms, 967.27D 967.27D((e)( )(2 2) ( see 17.9.03). see 17.9.03). If TSI slants fail to give typi typ ical Sal Salmo monella nella re reac actions, tions, pick addi ad ditional tional sus pi picious cious colo colonies from selec selective tive medium medium plate not giving giv ing presump presumptive tive posi positive culture culture and inoc inocu ulate TSI and LIA slants as in ( a). ( c ) Se Selec lection tion for identi iden ti fi fica cation. tion. —Ap ply —Ap ply bio chem chemii cal and serological identi identifi fica cation tion tests to 3 presump pre sumptive tive posi positive TSI cultures cul tures picked from selec se lective tive agar plates streaked from sele sel enite cystine broth and to 3 presump presumptive tive posi positive TSI cultures cultures picked from selec se lective tive agar plates streaked from tetrathionate broth. If 3 presumptive posi positive TSI cultures cultures are not isolated iso lated from one set of selec lective tive agar plates, test other presump pre sumptive tive posi positive TSI cultures, cul tures, if isolated, iso lated, by biochem biochemiical and serological tests. A miniimum of 6 TSI cul min cultures tures are exam amined ined for each 25 g test portion tested. Refer Ref erences: ences: JAOAC JAOAC 50, 753(1967); 51, 870(1968); 52,455(1969); 56, 1027(1973); 59, 731(1976); 61, 401(1978); 62, 499(1979); 64, 893(1981); 64, 899(1981); 65, 356(1982);67, 807(1984); 69, 277(1986). Revised: Re vised: March 1997
