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17.9.27 AOAC Official Method 2000.06 Salmonella in Foods with a Low Microbial Load, Detection

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17.9.27 AOAC Official Method 2000.06 Salmonella in Foods with a Low Microbial Load, Detection 17.9.27 (f) Water bath.—48–50°C. AOAC Official Method 2000.06 (g) Water baths.—Circulating, thermostatically controlled, Salmonella 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) Incubator.—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), 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) 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) Lysine iron agar (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. Isolation 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.
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