17.9.27 (f) Water bath.—48–50°C. AOAC Official Method 2000.06 (g) Water baths.—Circulating, thermostatically controlled, in Foods 43 ± 0.2°C, and 42 ± 0.2°C. with a Low Microbial Load, Detection (h) Sterile spoons.—Or other appropriate instruments for trans- First Action 2000 ferring food. (i) Sterile glassware.—Culture dishes, 15 × 100 mm; glass or (Applicable for the detection of Salmonella in foods with a low mi- plastic sterile pipets, 1 mL, with 0.01 mL graduations; and 5 and crobial load.) 10 mL, with 0.1 mL graduations. Caution: Potassium cyanide (KCN) broth is extremely poison- (j) Inoculating needle and inoculating loop.—Ca3mmidor µ ous. Dehydrated selenite cystine (SC) broth is very 10 L, nichrome, platinum-iridium, chromel wire, or sterile plastic. toxic if inhaled or swallowed. SC broth is very toxic if (k) Sterile test or culture tubes.—16 × 150 mm and swallowed. 20 × 150 mm; serological tubes, 10 × 75 mm or 13 × 100 mm. (l) Test or culture tube racks. See Tables 2000.06A and B for the results of the inter- and (m) Vortex mixer. intralaboratory studies supporting acceptance of the method. (n) Sterile shears, large scissors, scalpel, and forceps. (o) Lamp.—For observing serological reactions. A. Principle (p) Fisher or Bunsen burner. Rappaport-Vassiliadis medium and tetrathionate broth are selec- (q) pH meter. tive enrichment media that allow resuscitated Salmonella to multi- (r) Magnetic stirrer. ply to discernible levels in the presence of competing microflora. The incubated media are streaked onto bismuth sulfite, Hektoen en- C. Reagents and Media teric, and xylose lysine desoxycholate agar plates where, during in- (a) Trypticase (tryptic) soy broth.—967.25A(t) (17.9.01). cubation, discrete Salmonella colonies are formed. Colony growth is (b) pH test paper.—967.25B(1) (17.9.01). identified as Salmonella with biochemical and serological testing. (c) Sodium hydroxide.—1M NaOH, 967.25B(c) (17.9.01). B. Apparatus (d) Hydrochloric acid.—1M HCl, 967.25B(d) (17.9.01). (e) Lactose broth.—967.25A(a) (17.9.01). (a) Blender.—Calibrated, 10 000–12 000 rpm, with sterile (f) Brilliant green dye water.—967.25A(w) (17.9.01). blender jars. (g) Cellulase solution.—1%. 967.25B(p) (17.9.01). Add 1.0 g (b) Glassware.—Sterile, 16 oz. (500 mL) wide-mouth, cellulase to 99 mL H2O in a sterile container. Dissolve. Filter screw-cap jars; sterile 500 mL Erlenmeyer flasks; sterile 250 mL through 0.45 µm membrane into sterile 150 mL bottle. Store at beakers; sterile glass or paper funnels of appropriate size; and, op- 2–5°C for up to 14 days. tionally, containers of appropriate capacity to accommodate com- (h) Rappaport-Vassiliadis (RV) medium.—967.25A(x) posite test samples. (17.9.01). (c) Rods.—Sterile, bent glass or plastic; tongue depressor. (i) Tetrathionate (TT) broth.—967.25A(c) (17.9.01). (d) .—35 ± 2°C. (j) Selenite cystine (SC) broth.—967.25A(b) (17.9.01). (e) Refrigerator.—Incubator or laboratory, 4 ± 1°C. (k) Bismuth sulfite (BS) agar.—967.25A(f) (17.9.01).

Table 2000.06A. Interlaboratory study results for sensitivity ratea for detection of Salmonella by selective enrichment in selenite cystine broth (SC; 35°C), (LT; 35°C), tetrathionate broth (TT; 35°C), TT broth (43°C), and Rappaport-Vassiliadis medium (RV; 42°C) Food Labsb(c) No. test portions MPNd SC (35°C) LT (35°C) TT (35°C) TT (43°C) RV (42°C) Dried egg yolk 15 (0) 75 High 0.979 — 0.979 0.979 1.000 15 (0) 75 Low 1.000 — 1.000 1.000 1.000 Dry active yeast 15 (0) 75 High — 0.860 0.947 0.860 0.877 15 (0) 75 Low — 0.559 0.882 0.824 0.853 Ground black pepper 13 (0) 65 High 0.983 — 0.983 1.000 1.000 13 (0) 65 Low 1.000 — 1.000 1.000 1.000 Guar gum 15 (0) 75 High 0.906 — 0.887 0.831 0.622 15 (0) 75 Low 0.824 — 0.765 0.588 0.471 Instant nonfat dry milk 15 (0) 75 High 1.000 — 1.000 1.000 1.000 15 (0) 75 Low 0 — 0 1.000 0 a Sensitivity rate is the ratio of the number of test positives to the number of known positives. Known positive is defined as a positive test portion from any one or more of the 4 selective enrichments. b(c) Where b = the number of collaborating laboratories and (c) = the number of collaborating laboratories removed as outliers. d MPN = most probable number.

© 2002 AOAC INTERNATIONAL Table 2000.06B. Intralaboratory study results for specificity ratea for detection of Salmonella by selective enrichment in selenite cystine broth (SC; 35°C), lauryl tryptose broth (LT; 35°C), tetrathionate broth (TT; 35°C), TT broth (43°C), and Rappaport-Vassiliadis medium (RV; 42°C) Food Labsb(c) “Known negative”d SC (35°C) LT (35°C) TT (35°C) TT (43°C) RV (42°C) Dried egg yolk 15 (0) 75 1.00 — 1.00 1.00 1.00 Dry active yeast 15 (0) 75 — 1.00 1.00 1.00 1.00 Ground black pepper 13 (0) 65 1.00 — 1.00 1.00 1.00 Guar gum 15 (0) 75 1.00 — 1.00 1.00 1.00 Instant nonfat dry milk 15 (0) 75 1.00 — 1.00 1.00 1.00 a Specificity rate is the ratio of the number of test negatives to the number of “known negatives.” “Known negative” is defined as an uninoculated control. b(c) Where b = the number of collaborating laboratories and (c) = the number of collaborating laboratories removed as outliers. d Total number of expected negative values; all test samples were uninoculated controls.

(l) Hektoen enteric (HE) agar.—967.25A(e) (17.9.01). (e) Dry active yeast.—Aseptically weigh 25 g test portion into (m) Xylose lysine desoxycholate (XLD) agar.—967.25A(d) 500 mL wide-mouth jar, B(b), or other appropriate container. Add (17.9.01). 225 mL Trypticase soy broth, C(a), and mix well to form a smooth suspension. Cap securely and let stand at room temperature (n) Triple sugar iron agar (TSI).—967.25A(g) (17.9.01). (20–25°C) for 1 h. Mix well by swirling and determine pH with test (o) (LIA).—967.25A(m)(1) (17.9.01). paper, C(b). Adjust pH, if necessary, to 6.8 ± 0.2 with 1 M NaOH, D. Preparation of Test Suspensions C(c), or HCl, C(d); cap securely, and thoroughly mix contents be- fore determining final pH. Incubate for 24 ± 2hat35± 2°C. (a) Black pepper.—Aseptically weigh 25 g test portion into 500 mL wide-mouth jar, B(b), or other appropriate container. Add E. 225 mL Trypticase soy broth, C(a), and mix well. Cap securely and (a) Growth in selective broth.—Gently shake incubated test sus- let stand at room temperature (20–25°C) for 1 h. Mix well by shak- pension, D(a), (b), (c), or (e), and transfer 0.1 mL to 10 mL RV me- ing and determine pH with test paper, C(b). Adjust pH, if necessary, dium, C(h), and an additional 1 mL to 10 mL TT broth, C(i). Gently ± to 6.8 0.2 with 1M NaOH, C(c), or HCl, C(d), cap securely, and mix shake incubated test sample mixture, D(d), and transfer 1 mL to contents thoroughly before determining final pH. Incubate for 10 mL SC broth, C(j), and an additional 1 mL to 10 mL TT broth, ± ± ° 24 2hat35 2 C. C(i). Incubate RV medium 24 ± 2hat42± 0.2°C in a circulating (b) Dried egg yolk.—Aseptically weigh 25 g test portion into water bath, B(g). Incubate selenite cystine broth 24 ± 2hat 500 mL wide-mouth jar, B(b), or other appropriate container. Add 35 ± 2°C, B(d). Incubate tetrathionate broth 24 ± 2hat35± 2°C. ca 15 mL lactose broth, C(e). Stir with glass rod, B(c), spoon, B(h), For low microbial load foods, other than guar gum, mix with Vor- or tongue depressor B(c) to a smooth suspension. Add 3 additional µ portions of lactose broth, 10, 10, and 190 mL, for a total 225 mL. Stir tex mixer, B(m), and streak 3 mm (10 L) loopful of incubated RV after each addition until test portion is suspended without lumps. medium on selective enrichment plates of BS agar, C(k), HE agar, µ Cap securely and let stand at room temperature (20–25°C) for 1 h. C(l), and XLD agar, C(m). Repeat with 3 mm (10 L) loopful incu- Mix well by shaking and determine pH with test paper. Adjust pH, if bated TT broth. Incubate plates 24 ± 2hat35± 2°C. BS plates necessary, to 6.8 ± 0.2 with 1M NaOH, C(c), or HCl, C(d); cap se- should also be examined after 48 ± 2 h of incubation. curely, and thoroughly mix contents before determining final pH. For guar gum, mix with Vortex mixer, and streak 3 mm (10 µL) Incubate for 24 ± 2hat35± 2°C. loopful incubated SC broth on selective enrichment plates of BS (c) Instant nonfat dry milk.—Use funnel, B(b), to add aseptically agar, HE agar, and XLD agar. Repeat with 3 mm (10 µL) loopful in- 25 g test portion, slowly and gently, to 225 mL brilliant green dye cubated TT broth. Incubate plates 24 ± 2hat35± 2°C. BS plates water, C(f), in 500 mL wide-mouth jar, B(b), or other appropriate should also be examined after 48 ± 2 h of incubation. container. Do not mix. Let test portion soak undisturbed for1hat (b) Appearance of typical Salmonella colonies.—(1) On XLD, room temperature (20–25°C). Do not mix or adjust pH. Incubate for C(m).—Pink colonies with or without black centers. Many Salmo- ± ± ° 24 2hat35 2 C. nella colonies may have large, glossy black centers or may appear (d) Guar gum.—Aseptically weigh 25 g test portion into 250 mL almost completely black. Atypically, a few Salmonella cultures pro- beaker, B(b), or other appropriate container. Add 225 mL lactose duce yellow colonies with or without black centers. (2) On HE, broth, C(e), and 2.25 mL 1% cellulase solution, C(g), to 500 mL C(l).—Blue-green to blue colonies with or without black centers. wide-mouth jar, B(b), or other appropriate container. While vigor- Many Salmonella colonies may have large, glossy black centers or ously stirring cellulase/lactose broth with magnetic stirrer, B(r), may appear almost completely black. (3) On BS, C(k).—Brown, pour the 25 g test portion quickly through a glass funnel, B(b), into gray, or black colonies, sometimes with a metallic sheen. Sur- the cellulase/lactose broth. Cap securely and let stand at room tem- rounding medium is usually brown at first, turning black with in- perature (20–25°C) for 1 h. Incubate the loosely capped container creasing incubation time. Some strains produce green colonies with without pH adjustment for 24 ± 2hat35± 2°C. little or no darkening of surrounding medium.

© 2002 AOAC INTERNATIONAL Examine XLD and HE plates for Salmonella colonies after sumptive positive and examine them further. LIA is useful in detec- 24 ± 2 h of incubation at 35 ± 2°C. BS plates should be examined for tion of S. arizonae and atypical Salmonella strains that use lactose Salmonella colonies after 24 ± 2 h and 48 ± 2 h of incubation at 35 ± and/or sucrose. Discard only apparent non-Salmonella TSI cultures 2°C. (acid slant and acid butt) if corresponding LIA reactions are not typi- cal (acid butt) for Salmonella. Test retained presumptive positive F. Treatment of Colonies TSI culture as directed in 967.26C(c) (17.9.02) to determine if they are Salmonella spp. 967.27D(e)(1) (17.9.03) or S. arizonae organ- (a) Inoculation of TSI agar, C(n), and LIA, C(o).—Pick with nee- isms 967.27D(e)(2) (17.9.03). dle 2 or more colonies, if present, from each XLD, C(m), HE, C(l), If TSI slants fail to give typical Salmonella reactions, pick addi- and BS, C(k) plate having growth. Inoculate TSI slant with portion tional suspicious colonies from selective medium plate not giving pre- of each colony by slant and stabbing butt. After inoculat- sumptive positive culture, and inoculate TSI and LIA slants as in (a). ing TSI with needle, B(j), do not obtain more inoculum from colony (c) Selection for identification.—Apply biochemical (any ap- and do not heat needle, but inoculate LIA. Store picked selective proved biochemical test kit sanctioned for use with Salmonella and ° plates at 5–8 C or at room temperature (20–25°C). found within the AOAC Official Methods of Analysis, 16th Edition (b) Presumptive reactions.—Incubate TSI, C(n), and LIA, C(o), [or later] may be used instead of the biochemical tests presented in this method) and serological identification tests to 3 presumptive slants at 35 ± 2°C for 24 ± 2 h. Cap tubes loosely to maintain aerobic positive TSI cultures picked from selective agar plates streaked from conditions while incubating slants to prevent excessive H S produc- 2 RV medium and to 3 presumptive positive TSI cultures picked from tion. Salmonella cultures typically have alkaline (red) slant and acid selective agar plates streaked from TT broth as directed in 967.27 (yellow) butt, with or without H2S (blackening of agar) in TSI. In LIA, Salmonella cultures typically have alkaline (purple) reaction in (17.9.03) and 967.28 (17.9.07). butt. Consider only a distinct yellow coloration in butt of tube as an If 3 presumptive positive TSI cultures are not isolated from one set acidic (negative) reaction. Do not eliminate cultures that produce of selective agar plates, test other presumptive positive TSI cultures, discoloration in butt solely on this basis. Most Salmonella cultures if isolated, by biochemical and serological tests. A minimum of 6 TSI cultures is examined for each 25 g test portion tested. produce H2S in LIA. Retain all presumptive positive Salmonella cul- tures on TSI (alkaline slant and acid butt) agar for biochemical and References: J. AOAC Int. 84, 65(2001). serological tests whether or not corresponding LIA reaction is posi- Official Methods of Analysis (1999) 16th Ed., AOAC tive (alkaline butt) or negative (acid butt). Do not exclude a TSI cul- INTERNATIONAL, Gaithersburg, MD, 967.25 ture that appears to be non-Salmonella if the reaction in LIA is (17.9.01); 967.26 (17.9.02); 967.27 (17.9.03); 967.28 typical (alkaline butt) for Salmonella. Treat these cultures as pre- (17.9.07).

© 2002 AOAC INTERNATIONAL