Chapter 17: Salmonella

• Potential Food Safety Hazard
• Control Measures
• Recommended Microbiological Limits
• ICMSF Recommended Microbial Limits
• Caniadian Bacteriological Guidelines
• FDA Guidelines
• State Guidelines
• Growth
• Heat Resistance
• Analytical Procedures
• Food Sampling and Preparation of Sample Homogenate
• Salmonella
• Other analytical procedures
• Commercial Test Products
• References

Contents

Potential Food Safety Hazard

Salmonella is a rod-shaped, motile bacterium -nonmotile exceptions S. gallinarum and S. pullorum-, nonsporeforming and Gram-negative. There is a widespread occurrence in animals, especially in poultry and swine. Environmental sources of the organism include water, soil, insects, factory surfaces, kitchen surfaces, animal feces, raw meats, raw poultry, and raw seafoods, to name only a few.

S.typhi and the paratyphoid bacteria normally cause septicemia and produce typhoid or typhoid-like fever in humans. Other forms of salmonellosis generally produce milder symptoms.

Acute symptoms include nausea, vomiting, abdominal cramps, diarrhea, fever, and headache. Chronic consequences include arthritic symptoms that may follow 3 - 4 weeks after onset of acute symptoms. Onset of the illness is usually 6 - 48 h. The infective dose is as few as 15–20 cells; depends upon age and health of host, and strain differences among the members of the genus. Acute symptoms may last for 1 to 2 d or may be prolonged, again depending on host factors, ingested dose, and strain characteristics.

The disease is caused by penetration and passage of Salmonella organisms from gut lumen into epithelium of small intestine where inflammation occurs; there is evidence that an enterotoxin may be produced, perhaps within the enterocyte. Foods commonly associated with the disease include raw meats, poultry, eggs, milk and dairy products, fish, shrimp, frog legs, yeast, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and toppings, dried gelatin, peanut butter, cocoa, and chocolate.

Various Salmonella species have long been isolated from the outside of egg shells. The present situation with S. enteritidis is complicated by the presence of the organism inside the egg, in the yolk. This and other information strongly suggest vertical transmission, i.e., deposition of the organism in the yolk by an infected layer hen prior to shell deposition. Foods other than eggs have also caused outbreaks of S. enteritidis disease (FDA, 1998a).

Contents

Control Measures

Hazards from Salmonella can be prevented by: heating seafood sufficiently to kill the bacteria (e.g., 24 s at 165ºF), holding chilled seafood below 4.4ºC (40ºF), preventing post-cooking cross-contamination and prohibiting people who are ill or are carriers of Salmonella from working in food operations. (Ward et al., 1997).

Contents

Recommended Microbiological Limits

Contents

ICMSF Recommended Microbial Limits

Plate counts below "m" are considered good quality. Plate counts between "m" and "M" are considered marginally acceptable quality, but can be accepted if the number of samples does not exceed "c." Plate counts at or above "M" are considered unacceptable quality (ICMSF, 1986).

Contents

Salmonella (Andrews et al., 1998)

Several changes were introduced in the 8^th edition (revision A) of the Bacteriological Analytical Manual (Merker, 1998). The first change involved the expanded use of Rappaport-Vassiliadis (RV) medium. In the previous edition, RV medium was recommended only for the analysis of shrimp. Based on the completion of AOAC precollaborative (June et al., 1995) and collaborative (in review) studies, RV medium is now recommended for the analysis of raw flesh foods, highly contaminated foods, and animal feeds. With supporting data, RV medium will eventually replace selenite cystine broth for the analysis of all foods. Tetrathionate (TT) broth will continue to be used as the second selective enrichment broth. However, TT broth is to be incubated at 43ºC for the analysis of raw flesh foods, highly contaminated foods, and animal feeds, and at 35ºC for the analysis of all other food types.

The second change involved the option of refrigerating incubated preenrichments and selective enrichments of low-moisture foods for up to 72 h. With this option, sample analyses can be initiated as late as Wednesday or Thursday without weekend work being involved.

The third change involved reducing the period of incubation of the lysine iron agar (LIA) slants. In the former edition (BAM-7), triple sugar iron agar (TSI) and LIA slants were incubated at 35ºC for 24 ± 2 h and 48 ± 2 h, respectively. Unpublished data have demonstrated that the 48 h reading of LIA slants is without diagnostic value. Of 193 LIA slants examined, all gave definitive results within 24 ± 2 h of incubation. No significant changes altered the final test result when the slants were incubated an additional 24 h. Thus, both the TSI and LIA slants are now incubated for 24 ± 2 h.

The fourth change involved the procedure for surface disinfection of shell eggs. In the previous edition (BAM-7), egg shells were surface-disinfected by soaking in 0.1% mercuric chloride solution for 1 h followed by soaking in 70% ethanol for 30 min. Mercuric chloride is classified as a hazardous waste, and is expensive to dispose of according to Environmental Protection Agency guidelines. In this edition, egg shells are now surface-disinfected by soaking for 30 min in a 200 ppm Cl solution containing 0.1% sodium dodecyl sulfate.

A method for the analysis of guar gum has been included. When guar gum is preenriched at a 1:9 sample/broth ratio, a highly viscous, and nonpipettable mixture results. Addition of the enzyme cellulase to the preenrichment medium, however, results in a readily pipettable mixture. The effectiveness of this procedure for the analysis of other types of thickening agents is currently under investigation.

The directions for picking colonies from the selective plating agars have been made more explicit to reflect the intent of the method. In the absence of typical or suspect colonies on the selective plating agars, it is recommended that atypical colonies be picked to TSI and LIA slants. This recommendation is based on the fact that up to 4% of all Salmonella cultures isolated by FDA analysts from certain foods, especially seafoods, during the past several years have been atypical.

Finally, since the publication of BAM-7, a 6-way comparison was conducted of the relative effectiveness of the three selective plating agars recommended in the BAM (bismuth sulfite, Hektoen enteric, and xylose lysine desoxycholate agars) and three relatively new agars (EF-18, xylose lysine Tergitol 4, and Rambach agars). FDA studies (in press) indicated no advantage in replacing any of the BAM-recommended agars with one or more of the newer agars. Thus, the combination of selective plating agars recommended in BAM-7 remains unchanged.

1. Equipment and materials
1. Blender and sterile blender jars (see Chapter 9)
2. Sterile, 16 oz (500 ml) wide-mouth, screw-cap jars, sterile 500 ml Erlenmeyer flasks, sterile 250 ml beakers, sterile glass or paper funnels of appropriate size, and, optionally, containers of appropriate capacity to accommodate composited samples
3. Sterile, bent glass or plastic spreader rods
4. Balance, with weights; 2000 g capacity, sensitivity of 0.1 g
5. Balance, with weights; 120 g capacity, sensitivity of 5 mg
6. Incubator, 35ºC
7. Refrigerated incubator or laboratory refrigerator, 4 ± 1ºC
8. Water bath, 48-50ºC
9. Water bath, 43 ± 0.2ºC
10. Water bath, 42 ± 0.2ºC
11. Sterile spoons or other appropriate instruments for transferring food samples
12. Sterile culture dishes, 15 x 100 mm, glass or plastic
13. Sterile pipets, 1 ml, with 0.01 ml graduations; 5 and 10 ml, with 0.1 ml graduations
14. Inoculating needle and inoculating loop (about 3 mm id or 10 µl), nichrome, platinum-iridium, chromel wire, or sterile plastic
15. Sterile test or culture tubes, 16 x 150 mm and 20 x 150 mm; serological tubes, 10 x 75 mm or 13 x 100 mm
16. Test or culture tube racks
17. Vortex mixer
18. Sterile shears, large scissors, scalpel, and forceps
19. Lamp (for observing serological reactions)
20. Fisher or Bunsen burner
21. pH test paper (pH range 6-8) with maximum graduations of 0.4 pH units per color change
22. pH meter
23. Plastic bags, 28 x 37 cm, sterile, with resealable tape. (Items w-y are needed in the analysis of frog legs and rabbit carcasses.)
24. Plastic beakers, 4 liter, autoclavable, for holding plastic bag during shaking and incubation
25. Mechanical shaker, any model that can be adjusted to give 100 excursions/min with a 4 cm (1-1/2 inches) stroke, such as the Eberbach shaker with additional 33 and 48 cm clamp bars
2. Media and reagents

For preparation of media and reagents, refer to secs 967.25-967.28 in Official Methods of Analysis (AOAC, 1995a).

1. Lactose broth (M74)
2. Nonfat dry milk (reconstituted) (M111)
3. Selenite cystine (SC) broth (M134)
4. Tetrathionate (TT) broth (M145)
5. Rappaport-Vassiliadis (RV) medium (M132)
6. Xylose lysine desoxycholate (XLD) agar (M179)
7. Hektoen enteric (HE) agar (M61)
8. Bismuth sulfite (BS) agar (M19)
9. Triple sugar iron agar (TSI) (M149)
10. Tryptone (tryptophane) broth (M164)
11. Trypticase (tryptic) soy broth (M154)
12. Lauryl tryptose (LST) broth (M76)
13. Trypticase soy-tryptose broth (TSB) (M160)
14. MR-VP broth (M104)
15. Simmons citrate agar (M138)
16. Urea broth (M171)
17. Urea broth (rapid) (M172)
18. Malonate broth (M92)
19. Lysine iron agar (LIA) (Edwards and Fife) (M89)
20. Lysine decarboxylase broth (M87)
21. Motility test medium (semisolid) (M103)
22. Potassium cyanide (KCN) broth (M126)
23. Phenol red carbohydrate broth (M121)
24. Purple carbohydrate broth (M130)
25. MacConkey agar (M91)
26. Nutrient broth (M114)
27. Brain heart infusion (BHI) broth (M24)
28. Papain solution, 5% (M56a)
29. Tryptose blood agar base (M166)
30. Potassium sulfite powder, anhydrous
31. Chlorine solution, 200 ppm, containing 0.1% sodium dodecyl sulfate (R12a)
32. Ethanol, 70% (R23)
33. Kovacs' reagent (R38)
34. Voges-Proskauer (VP) test reagents (R89)
35. Creatine phosphate crystals
36. Potassium hydroxide solution, 40% (R65)
37. 1 N Sodium hydroxide solution (R73)
38. 1 N Hydrochloric acid (R36)
39. Brilliant green dye solution, 1% (R8)
40. Bromcresol purple dye solution, 0.2% (R9)
41. Methyl red indicator (R44)
42. Sterile distilled water
43. Tergitol Anionic 7 (R78)
44. Triton X-100 (R86)
45. Physiological saline solution, 0.85% (sterile) (R63)
46. Formalinized physiological saline solution (R27)
47. Salmonella polyvalent somatic (O) antiserum
48. Salmonella polyvalent flagellar (H) antiserum
49. Salmonella somatic group (O) antisera: A, B, C₁, C₂, C₃, D₁, D₂, E₁, E₂, E₃, E₄, F, G, H, I, Vi, and other groups, as appropriate
50. Salmonella Spicer-Edwards flagellar (H) antisera

0. Preparation of foods for isolation of Salmonella

1. Dried egg yolk, dried egg whites, dried whole eggs, liquid milk (skim milk, 2% fat milk, whole, and buttermilk), and prepared powdered mixes (cake, cookie, doughnut, biscuit, and bread), infant formula, and oral or tube feedings containing egg. Preferably, do not thaw frozen samples before analysis. If frozen sample must be tempered to obtain analytical portion, thaw suitable portion as rapidly as possible to minimize increase in number of competing organisms or to reduce potential of injuring Salmonella organisms. Thaw below 45ºC for 15 min with continuous agitation in thermostatically controlled water bath or thaw within 18 h at 2-5ºC. Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. For nonpowdered samples, add 225 ml sterile lactose broth. If product is powdered, add about 15 ml sterile lactose broth and stir with sterile glass rod, spoon, or tongue depressor to smooth suspension. Add 3 additional portions of lactose broth, 10, 10, and 190 ml, for total of 225 ml. Stir thoroughly until sample is suspended without lumps. Cap jar securely and let stand 60 min at room temperature. Mix well by swirling and determine pH with test paper.

Adjust pH, if necessary, to 6.8 ± 0.2 with sterile 1 N NaOH or 1 N HCl. Cap jar securely and mix well before determining final pH. Loosen jar cap about 1/4 turn and incubate 24 ± 2 h at 35ºC.

Continue as in 4, a-k, below.

2. Eggs
1. Shell eggs. Wash eggs with stiff brush and drain. Soak eggs in 200 ppm Cl solution containing 0.1% sodium dodecyl sulfate (SDS) for 30 min. Prepare the 200 ppm Cl /0.1-SDS solution by adding 8 ml commercial bleach (5.25% sodium hypochlorite) to 992 ml distilled water containing 1 g SDS. This disinfectant should be prepared immediately before analysis. Crack eggs aseptically and, using sterile egg separator, discard whites. Aseptically weigh 25 g egg yolk into sterile 500 ml Erlenmeyer flask or other appropriate container. Add 225 ml trypticase (tryptic) soy broth (TSB) and mix well by swirling. Let stand 60 min at room temperature. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Incubate 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
2. Liquid whole eggs (homogenized). Weigh 25 g into sterile 500 ml Erlenmeyer flask or other appropriate container. Add 225 ml TSB and mix well by swirling. Continue as described above.
3. Hard-boiled eggs (chicken, duck, and others). At this time, it is not known if the inhibitory factors for Salmonella in egg albumen are heat stable. Thus, aseptically separate egg yolk from egg white. Weigh 25 g pulverized egg yolk solid into sterile 500 ml Erlenmeyer flask or other appropriate container. Add 225 ml TSB (without ferrous sulfate) and mix well by swirling. Continue as described above.

3. Nonfat dry milk
1. Instant. Aseptically weigh 25 g sample into sterile beaker (250 ml) or other appropriate container. Using sterile glass or paper funnel (made with tape to withstand autoclaving), pour 25 g analytical unit gently and slowly over surface of 225 ml brilliant green water contained in sterile 500 ml Erlenmeyer flask or other appropriate container. Alternatively, 25 g analytical units may be composited and poured over the surface of proportionately larger volumes of brilliant green water. Prepare brilliant green water by adding 2 ml 1% brilliant green dye solution per 1000 ml sterile distilled water. Let container stand undisturbed for 60 ± 5 min. Incubate loosely capped container, without mixing or pH adjustment, for 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
2. Non-Instant. Examine as described for instant nonfat dry milk, except that the 25 g analytical units may not be composited.

4. Dry whole milk. Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml sterile distilled water and mix well by swirling. Cap jar securely and let stand 60 min at room temperature. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Cap jar securely and mix well before determining final pH. Add 0.45 ml 1% aqueous brilliant green dye solution and mix well. Loosen jar cap 1/4 turn and incubate jar 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
5. Casein. Aseptically weigh 25 g sample into sterile blender jar. Add 225 ml sterile lactose broth to sample and blend for 2 min. Aseptically transfer blended homogenate into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Cap jar securely and let stand 60 min at room temperature. Mix well by shaking and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Cap jar securely and mix well before determining final pH. Loosen jar cap 1/4 turn and incubate jar 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
6. Soy flour. Aseptically weigh 25 g sample into sterile beaker (250 ml) or other appropriate container. Using sterile glass or paper funnel (made with tape to withstand autoclaving), pour 25 g analytical unit gently and slowly over surface of 225 ml lactose broth contained in sterile 500 ml Erlenmeyer flask or other appropriate container. Analytical units (25 g) may not be composited. Let container stand undisturbed for 60 ± 5 min. Incubate loosely capped container, without mixing or pH adjustment, for 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
7. Egg-containing products (noodles, egg rolls, macaroni, spaghetti), cheese, dough, prepared salads (ham, egg, chicken, tuna, turkey), fresh, frozen, or dried fruits and vegetables, nut meats, crustaceans (shrimp, crab, crayfish, langostinos, lobster), and fish. Preferably, do not thaw frozen samples before analysis. If frozen sample must be tempered to obtain analytical portion, thaw below 45ºC for <15 min with continuous agitation in thermostatically controlled water bath or thaw within 18 h at 2-5ºC.

Aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile lactose broth and blend 2 min. Aseptically transfer homogenized mixture to sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container and let stand 60 min at room temperature with jar securely capped. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Mix well and loosen jar cap about 1/4 turn. Incubate 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.

8. Dried yeast. Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml sterile trypticase soy broth. Mix well to form smooth suspension. Let stand 60 min at room temperature with jar securely capped. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2, mixing well before determining final pH. Loosen jar cap 1/4 turn and incubate 24 ± 2 h at 35ºC. For dried inactive yeast, continue as in 4, a-k, below. For dried active yeast, mix incubated sample and transfer 1 ml each to 10 ml lauryl tryptose broth and to 10 ml tetrathionate broth. Incubate selective enrichment broths 24 ± 2 h at 35ºC. Vortex sample mixtures thoroughly and streak 3 mm loopful of each broth to each of 3 selective agars as described in 4, c and d, below. Continue as in 4, f-k, below.
9. Frosting and topping mixes. Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml nutrient broth and mix well. Cap jar securely and let stand 60 min at room temperature. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Loosen jar cap about 1/4 turn and incubate 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
10. Spices
1. Black pepper, white pepper, celery seed or flakes, chili powder, cumin, paprika, parsley flakes, rosemary, sesame seed, thyme, and vegetable flakes. Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml sterile trypticase soy broth (TSB) and mix well. Cap jar securely and let stand 60 min at room temperature. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Loosen jar cap about l/4 turn and incubate 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
2. Onion flakes, onion powder, garlic flakes. Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Preenrich sample in TSB with added K₂SO₃ (5 g K₂SO₃ per L TSB, resulting in final 0.5% K₂SO₃ concentration). Add K₂SO₃ to broth before autoclaving 225 ml volumes in 500 ml Erlenmeyer flasks at 121ºC for 15 min. After autoclaving, aseptically determine and, if necessary, adjust final volume to 225 ml. Add 225 ml sterile TSB with added K₂SO₃ to sample and mix well. Continue as in 3-ji.
3. Allspice, cinnamon, cloves, and oregano. At this time there are no known methods for neutralizing the toxicity of these 4 spices. Dilute them beyond their toxic levels to examine them. Examine allspice, cinnamon, and oregano at 1:100 sample/broth ratio, and cloves at 1:1000 sample/broth ratio. Examine leafy condiments at sample/broth ratio greater than 1:10 because of physical difficulties encountered by absorption of broth by dehydrated product. Examine these spices as described in 3-ji, above, maintaining recommended sample/broth ratios.

11. Candy and candy coating (including chocolate). Aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile, reconstituted nonfat dry milk and blend 2 min. Aseptically transfer homogenized mixture to sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container and let stand 60 min at room temperature with jar securely capped. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Add 0.45 ml 1% aqueous brilliant green dye solution and mix well. Loosen jar caps 1/4 turn and incubate. Continue as in 4, a-k, below.
12. Coconut. Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml sterile lactose broth, shake well, and let stand 60 min at room temperature with jar securely capped. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Add up to 2.25 ml steamed (15 min) Tergitol Anionic 7 and mix well. Alternatively, use steamed (15 min) Triton X-100. Limit use of these surfactants to minimum quantity needed to initiate foaming. For Triton X-100 this quantity may be as little as 2 or 3 drops. Loosen jar cap about l/4 turn and incubate 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
13. Food dyes and food coloring substances. For dyes with pH 6.0 or above (10% aqueous suspension), use method described for dried whole eggs (3-a, above). For laked dyes or dyes with pH below 6.0, aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml tetrathionate broth without brilliant green dye. Mix well and let stand 60 min at room temperature with jar securely capped. Using pH meter, adjust pH to 6.8 ± 0.2. Add 2.25 ml 0.1% brilliant green dye solution and mix thoroughly by swirling. Loosen jar cap about 1/4 turn and incubate 24 ± 2 h at 35ºC. Continue as in 4, c-k, below.
14. Gelatin. Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml sterile lactose broth and 5 ml 5% aqueous papain solution and mix well. Cap jar securely and incubate at 35°C for 60 min. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Loosen jar cap about 1/4 turn and incubate 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.
15. Meats, meat substitutes, meat by-products, animal substances, glandular products, and meals (fish, meat, bone). Aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile lactose broth and blend 2 min. Aseptically transfer homogenized mixture to sterile wide-mouth, screw-cap jar (500 ml) or other appropriate container and let stand 60 min at room temperature with jar securely capped. If mixture is powder or is ground or comminuted, blending may be omitted. For samples that do not require blending, add lactose broth and mix thoroughly; let stand for 60 min at room temperature with jar securely capped.

Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Add up to 2.25 ml steamed (15 min) Tergitol Anionic 7 and mix well. Alternatively, use steamed (15 min) Triton X-100. Limit use of these surfactants to minimum quantity needed to initiate foaming. Actual quantity will depend on composition of test material. Surfactants will not be needed in analysis of powdered glandular products. Loosen jar caps 1/4 turn and incubate sample mixtures 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.

16. Frog legs. (This method is used for all domestic and imported frog legs.) Place 15 pairs of frog legs into sterile plastic bag and cover with sterile lactose broth (see 1, w-y, above). If single legs are estimated to average 25 g or more, examine only one leg of each of 15 pairs. Place bag in large plastic beaker or other suitable container and shake 15 min on mechanical shaker set for 100 excursions/min with stroke of 4 cm (1-1/2 inches). Pour off lactose broth from bag into another sterile plastic bag and add more lactose broth to total volume of 3500 ml. Mix well and let stand 60 min at room temperature. Adjust pH, if necessary, to 6.8 ± 0.2, using pH paper. Place plastic bag containing the lactose broth into plastic beaker or other suitable container. Incubate 24 ± 2 h at 35ºC. Continue examination as in 4, a-k, below.
17. Rabbit carcasses. (This method is used for all domestic and imported rabbit carcasses.) Place each of 3 rabbit carcasses into sterile plastic bag and cover with sterile lactose broth (see 1, w-y, above). Place bag in large plastic beaker or other suitable container and shake 15 min on mechanical shaker set for 100 excursions/min with stroke of 4 cm (1-1/2 inches). Composite lactose broth rinsings by pouring into another sterile container and add more lactose broth to total volume of 3500 ml. Mix well and let stand 60 min at room temperature. Adjust pH, if necessary, to 6.8 ± 0.2, using pH paper. Incubate 24 ± 2 h at 35ºC. Continue examination as in 4, a-k, below.
18. Guar gum. Aseptically weigh 25 g sample into sterile beaker (250 ml) or other appropriate container. Prepare a 1.0% cellulase solution (add 1 g cellulase to 99 ml sterile distilled water). Dispense into 150 ml bottles. (Cellulase solution may be stored at 2-5ºC for up to 2 weeks). Add 225 ml sterile lactose broth and 2.25 ml sterile 1% cellulase solution to sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. While vigorously stirring the cellulase/lactose broth with magnetic stirrer, pour 25 g analytical unit quickly through sterile glass funnel into the cellulase/lactose broth. Cap jar securely and let stand 60 min at room temperature. Incubate loosely capped container without pH adjustment, for 24 ± 2 h at 35ºC. Continue as in 4, a-k, below.

4. Isolation of Salmonella

1. Tighten lid and gently shake incubated sample.

Raw flesh foods, highly contaminated foods, and animal feeds. Transfer 0.1 ml mixture to 10 ml Rappaport-Vassiliadis (RV) medium and another 1 ml mixture to 10 ml tetrathionate (TT) broth.

Other foods. Transfer 1 ml mixture to 10 ml selenite cystine (SC) broth and another 1 ml mixture to 10 ml TT broth.

2. Incubate selective enrichment media as follows:

Raw flesh foods, highly contaminated foods, and animal feeds. Incubate RV medium 24 ± 2 h at 42 ± 0.2ºC (water bath). Incubate TT broth 24 ± 2 h at 43 ± 0.2ºC (water bath).

Other foods. Incubate SC and TT broths 24 ± 2 h at 35ºC.

3. Mix (vortex, if tube) and streak 3 mm loopful (10 µl) incubated TT broth on bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar. Prepare BS plates the day before streaking and store in dark at room temperature until streaked.
4. Repeat with 3 mm loopful (10 µl) of RV medium (for samples of raw flesh foods, highly contaminated foods, and animal feeds) and of SC broth (for samples other than raw flesh foods, highly contaminated foods, and animal feeds).
5. Refer to 994.04 in Official Methods of Analysis for option of refrigerating incubated sample preenrichments and incubated sample selective enrichments (SC and TT broths only) of low moisture foods. This option allows sample analyses to be initiated as late as Thursday while still avoiding weekend work.
6. Incubate plates 24 ± 2 h at 35ºC.
7. Examine plates for presence of colonies that may be Salmonella.

Typical Salmonella Colony Morphology

Pick 2 or more colonies of Salmonella from each selective agar after 24 ± 2 h incubation. Typical Salmonella colonies are as follows:

1. Hektoen enteric (HE) agar. Blue-green to blue colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies.
2. Xylose lysine desoxycholate (XLD) agar. Pink colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies.
3. Bismuth sulfite (BS) agar. Brown, gray, or black colonies; sometimes they have a metallic sheen. Surrounding medium is usually brown at first, but may turn black in time with increased incubation, producing the so-called halo effect.

If typical colonies are present on the BS agar after 24 ± 2 h incubation, then pick 2 or more colonies. Irrespective of whether or not BS agar plates are picked at 24 ± 2 h, reincubate BS agar plates an additional 24 ± 2 h. After 48 ± 2 h incubation, pick 2 or more typical colonies, if present, from the BS agar plates, only if colonies picked from the BS agar plates incubated for 24 ± 2 h give atypical reactions in triple sugar iron agar (TSI) and lysine iron agar (LIA) that result in culture being discarded as not being Salmonella. See sections 4-i and 4-j, below, for details in interpreting TSI and LIA reactions.

Atypical Salmonella Colony Morphology

In the absence of typical or suspicious Salmonella colonies, search for atypical Salmonella colonies as follows:

HE and XLD agars. Atypically a few Salmonella cultures produce yellow colonies with or without black centers on HE and XLD agars. In the absence of typical Salmonella colonies on HE or XLD agars after 24 ± 2 h incubation, then pick 2 or more atypical Salmonella colonies.

BS agar. Atypically some strains produce green colonies with little or no darkening of the surrounding medium. If typical or suspicious colonies are not present on BS agar after 24 ± 2 h, then do not pick any colonies but reincubate an additional 24 ± 2 h. If typical or suspicious colonies are not present after 48 ± 2 h incubation, then pick 2 or more atypical colonies.

Suggested Control Cultures

In addition to the positive control cultures (typical Salmonella), 3 additional Salmonella cultures are recommended to assist in the selection of atypical Salmonella colony morphology on selective agars. These cultures are a lactose-positive, H₂S-positive S. arizonae (ATCC 12325) and a lactose-negative, H₂S-negative S. abortus equi (ATCC 9842); OR a lactose-positive, H₂S-negative S. diarizonae (ATCC 29934). These cultures may be obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852-1776.

8. Lightly touch the very center of the colony to be picked with sterile inoculating needle and inoculate TSI slant by streaking slant and stabbing butt. Without flaming, inoculate LIA slant by stabbing butt twice and then streaking slant. Since lysine decarboxylation reaction is strictly anaerobic, the LIA slants must have deep butt (4 cm). Store picked selective agar plates at 5-8ºC.
9. Incubate TSI and LIA slants at 35ºC for 24 ± 2 h. Cap tubes loosely to maintain aerobic conditions while incubating slants to prevent excessive H₂S production. Salmonella in culture typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H₂S (blackening of agar) in TSI. In LIA, Salmonella typically produces alkaline (purple) reaction in butt of tube. Consider only distinct yellow in butt of tube as acidic (negative) reaction. Do not eliminate cultures that produce discoloration in butt of tube solely on this basis. Most Salmonella cultures produce H₂S in LIA. Some non-Salmonella cultures produce a brick-red reaction in LIA slants.
10. All cultures that give an alkaline butt in LIA, regardless of TSI reaction, should be retained as potential Salmonella isolates and submitted for biochemical and serological tests. Cultures that give an acid butt in LIA and an alkaline slant and acid butt in TSI should also be considered potential Salmonella isolates and should be submitted for biochemical and serological tests. Cultures that give an acid butt in LIA and an acid slant and acid butt in TSI may be discarded as not being Salmonella. Test retained, presumed-positive TSI cultures as directed in 4-k, below, to determine if they are Salmonella species, including S. arizonae. If TSI cultures fail to give typical reactions for Salmonella (alkaline slant and acid butt) pick additional suspicious colonies from selective medium plate not giving presumed-positive culture and inoculate TSI and LIA slants as described in 4-h, above.
11. Apply biochemical and serological identification tests to:
1. Three presumptive TSI cultures recovered from set of plates streaked from SC broth (or RV medium for appropriate foods), if present, and 3 presumptive TSI agar cultures recovered from plates streaked from TT broth, if present.
2. If 3 presumptive-positive TSI cultures are not isolated from one set of agar plates, test other presumptive-positive TSI agar cultures, if isolated, by biochemical and serological tests. Examine a minimum of 6 TSI cultures for each 25 g analytical unit.

5. Identification of Salmonella

1. Mixed cultures. Streak TSI agar cultures that appear to be mixed on MacConkey agar, HE agar, or XLD agar. Incubate plates 24 ± 2 h at 35ºC. Examine plates for presence of colonies suspected to be Salmonella.
1. MacConkey agar. Typical colonies appear transparent and colorless, sometimes with dark center. Colonies of Salmonella will clear areas of precipitated bile caused by other organisms sometimes present.
2. Hektoen enteric (HE) agar. See 4-gi, above.
3. Xylose lysine desoxycholate (XLD) agar. See 4-gii, above. Transfer at least 2 colonies suspected to be Salmonella to TSI and LIA slants as described in 4-g, above, and continue as in 4-i, above.

2. Pure cultures
1. Urease test (conventional). With sterile needle, inoculate growth from each presumed-positive TSI slant culture into tubes of urea broth. Since occasional, uninoculated tubes of urea broth turn purple-red (positive test) on standing, include uninoculated tube of this broth as control. Incubate 24 ± 2 h at 35ºC.
2. Optional urease test (rapid). Transfer two 3 mm loopfuls of growth from each presumed-positive TSI slant culture into tubes of rapid urea broth. Incubate 2 h in 37 ± 0.5ºC water bath. Discard all cultures giving positive test. Retain for further study all cultures that give negative test (no change in color of medium).

3. Serological polyvalent flagellar (H) test
1. Perform the polyvalent flagellar (H) test at this point, or later, as described in 5-d, below. Inoculate growth from each urease-negative TSI agar slant into either 1) BHI broth and incubate 4-6 h at 35ºC until visible growth occurs (to test on same day); or 2) trypticase soy-tryptose broth and incubate 24 ± 2 h at 35ºC (to test on following day). Add 2.5 ml formalinized physiological saline solution to 5 ml of either broth culture.
2. Select 2 formalinized broth cultures and test with Salmonella polyvalent flagellar (H) antisera. Place 0.5 ml of appropriately diluted Salmonella polyvalent flagellar (H) antiserum in 10 x 75 mm or 13 x 100 mm serological test tube. Add 0.5 ml antigen to be tested. Prepare saline control by mixing 0.5 ml formalinized physiological saline solution with 0.5 ml formalinized antigen. Incubate mixtures in 48-50ºC water bath. Observe at 15 min intervals and read final results in 1 h.

Positive--agglutination in test mixture and no agglutination in control.

Negative--no agglutination in test mixture and no agglutination in control.

Nonspecific--agglutination in both test mixture and control. Test the cultures giving such results with Spicer-Edwards antisera.

4. Spicer-Edwards serological test. Use this test as an alternative to the polyvalent flagellar (H) test. It may also be used with cultures giving nonspecific agglutination in polyvalent flagellar (H) test. Perform Spicer-Edwards flagellar (H) antisera test as described in 5, cii, above. Perform additional biochemical tests (5, ei-iii, below) on cultures giving positive flagellar test results. If both formalinized broth cultures are negative, perform serological tests on 4 additional broth cultures (5, ci, above). If possible, obtain 2 positive cultures for additional biochemical testing (5, ei-iii, below). If all urease-negative TSI cultures from sample give negative serological flagellar (H) test results, perform additional biochemical tests (5, ei-iii, below).
5. Testing of urease-negative cultures
1. Lysine decarboxylase broth. If LIA test was satisfactory, it need not be repeated. Use lysine decarboxylase broth for final determination of lysine decarboxylase if culture gives doubtful LIA reaction. Inoculate broth with small amount of growth from TSI slant suspicious for Salmonella. Replace cap tightly and incubate 48 ± 2 h at 35ºC but examine at 24 h intervals. Salmonella species cause alkaline reaction indicated by purple color throughout medium. Negative test is indicated by yellow color throughout medium. If medium appears discolored (neither purple nor yellow) add a few drops of 0.2% bromcresol purple dye and re-read tube reactions.
2. Phenol red dulcitol broth or purple broth base with 0.5% dulcitol. Inoculate broth with small amount of growth from TSI culture. Replace cap loosely and incubate 48 ± 2 h at 35ºC, but examine after 24 h. Most Salmonella species give positive test, indicated by gas formation in inner fermentation vial and acid pH (yellow) of medium. Production of acid should be interpreted as a positive reaction. Negative test is indicated by no gas formation in inner fermentation vial and red (with phenol red as indicator) or purple (with bromcresol purple as indicator) color throughout medium.
3. Tryptone (or tryptophane) broth. Inoculate broth with small growth from TSI agar culture. Incubate 24 ± 2 h at 35ºC and proceed as follows:
1. Potassium cyanide (KCN) broth. Transfer 3 mm loopful of 24 h tryptophane broth culture to KCN broth. Heat rim of tube so that good seal is formed when tube is stoppered with wax-coated cork. Incubate 48 ± 2 h at 35ºC but examine after 24 h. Interpret growth (indicated by turbidity) as positive. Most Salmonella species do not grow in this medium, as indicated by lack of turbidity.
2. Malonate broth. Transfer 3 mm loopful of 24 h tryptone broth culture to malonate broth. Since occasional uninoculated tubes of malonate broth turn blue (positive test) on standing, include uninoculated tube of this broth as control. Incubate 48 ± 2 h at 35ºC, but examine after 24 h. Most Salmonella species cultures give negative test (green or unchanged color) in this broth.
3. Indole test. Transfer 5 ml of 24 h tryptophane broth culture to empty test tube.

Add 0.2-0.3 ml Kovacs' reagent. Most Salmonella cultures give negative test (lack of deep red color at surface of broth). Record intermediate shades of orange and pink as ±.

4. Serological flagellar (H) tests for Salmonella. If either polyvalent flagellar (H) test (5-c, above) or the Spicer-Edwards flagellar (H) test tube test (5-d, above) has not already been performed, either test may be performed here.
5. Discard as not Salmonella any culture that shows either positive indole test and negative serological flagellar (H) test, or positive KCN test and negative lysine decarboxylase test.

6. Serological somatic (O) tests for Salmonella. (Pre-test all antisera to Salmonella with known cultures.)
1. Polyvalent somatic (O) test. Using wax pencil, mark off 2 sections about 1 x 2 cm each on inside of glass or plastic petri dish (15 x 100 mm). Commercially available sectioned slides may be used. Emulsify 3 mm loopful of culture from 24-48 h TSI slant or, preferably, tryptose blood agar base (without blood) with 2 ml 0.85% saline. Add 1 drop of culture suspension to upper portion of each rectangular crayon-marked section. Add 1 drop of saline solution to lower part of one section only. Add 1 drop of Salmonella polyvalent somatic (O) antiserum to other section only. With clean sterile transfer loop or needle, mix culture suspension with saline solution for one section and repeat for other section containing antiserum. Tilt mixtures in back-and-forth motion for 1 min and observe against dark background in good illumination. Consider any degree of agglutination a positive reaction. Classify polyvalent somatic (O) test results as follows:

Positive--agglutination in test mixture; no agglutination in saline control.

Negative--no agglutination in test mixture; no agglutination in saline control.

Nonspecific--agglutination in test and in control mixtures. Perform further biochemical and serological tests as described in Edwards and Ewing's Identification of Enterobacteriaceae (Ewing, 1986).

2. Somatic (O) group tests. Test as in 5-fi, above, using individual group somatic (O) antisera including Vi, if available, in place of Salmonella polyvalent somatic (O) antiserum. For special treatment of cultures giving positive Vi agglutination reaction, refer to sec. 967.28B in Official Methods of Analysis (1). Record cultures that give positive agglutination with individual somatic (O) antiserum as positive for that group. Record cultures that do not react with individual somatic (O) antiserum as negative for that group.

7. Additional biochemical tests. Classify as Salmonella those cultures which exhibit typical Salmonella reactions for tests 1-11, shown in Table 17-6. If one TSI culture from 25 g analytical unit is classified as Salmonella, further testing of other TSI cultures from the same 25 g analytical unit is unnecessary. Cultures that contain demonstrable Salmonella antigens as shown by positive Salmonella flagellar (H) test but do not have biochemical characteristics of Salmonella should be purified (5-a, above) and retested, beginning with 5-b, above.
Table 17-6. Biochemical and serological reactions of Salmonella

Test or substrate	Result		Salmonella species reactiona
	Positive	Negative
1. Glucose (TSI)	Yellow butt	Red butt	+
2. Lysine decarboxylase (LIA)	Purple butt	Yellow butt	+
3. H2S (TSI and LIA)	blackening	no blackening	+
4. Urease	purple-red color	no color change	-
5. Lysine decarboxylase broth	purple color	yellow color	+
6. Phenol red dulcitol broth	yellow color and/or gas	no gas; no color change	+b
7. KCN broth	growth	no growth	-
8. Malonate broth	blue color	no color change	-c
9. Indole test	violet color at surface	yellow color at surface	-
10. Polyvalent flagellar test	agglutination	no agglutination	+
11. Polyvalent somatic test	agglutination	no agglutination	+
12. Phenol red lactose broth	yellow color and/or gas	no gas; no color change	-c
13. Phenol red sucrose broth	yellow color and/or gas	no gas; no color change	-
14. Voges-Proskauer test	pink-to-red color	no color change	-
15. Methyl red test	diffuse red color	diffuse yellow color	+
16. Simmons citrate	growth; blue color	no growth; no color change	v

^a+, 90% or more positive in 1 or 2 d; -, 90% or more negative in 1 or 2 d; v, variable.
^bMajority of S. arizonae cultures are positive.
^cMajority of S. arizonae cultures are negative.

Perform the following additional tests on cultures that do not give typical Salmonella reactions for tests 1-11 in Table 17-6 and that consequently do not classify as Salmonella.

1. Phenol red lactose broth or purple lactose broth.
1. Inoculate broth with small amount of growth from unclassified 24-48 h TSI slant. Incubate 48 ± 2 h at 35ºC, but examine after 24 h.

Positive--acid production (yellow) and gas production in inner fermentation vial. Consider production of acid only as positive reaction. Most cultures of Salmonella give negative test result, indicated by no gas formation in inner fermentation vial and red (with phenol red as indicator) or purple (with bromcresol purple as indicator) throughout medium.

2. Discard as not Salmonella, cultures that give positive lactose tests, except cultures that give acid slants in TSI and positive reactions in LIA, or cultures that give positive malonate broth reactions. Perform further tests on these cultures to determine if they are S. arizonae.

2. Phenol red sucrose broth or purple sucrose broth. Follow procedure described in 5, gi), above. Discard as not Salmonella, cultures that give positive sucrose tests, except those that give acid slants in TSI and positive reactions in LIA.
3. MR-VP broth. Inoculate medium with small amount of growth from each unclassified TSI slant suspected to contain Salmonella. Incubate 48 ± 2 h at 35ºC.
1. Perform Voges-Proskauer (VP) test at room temperature as follows: Transfer 1 ml 48 h culture to test tube and incubate remainder of MR-VP broth an additional 48 h at 35ºC. Add 0.6 ml a-naphthol and shake well. Add 0.2 ml 40% KOH solution and shake. To intensify and speed reaction, add a few crystals of creatine. Read results after 4 h; development of pink-to-ruby red color throughout medium is positive test. Most cultures of Salmonella are VP-negative, indicated by absence of development of pink-to-red color throughout broth.
2. Perform methyl red test as follows: To 5 ml of 96 h MR-VP broth, add 5-6 drops of methyl red indicator. Read results immediately. Most Salmonella cultures give positive test, indicated by diffuse red color in medium. A distinct yellow color is negative test. Discard, as not Salmonella, cultures that give positive KCN and VP tests and negative methyl red test.

4. Simmons citrate agar. Inoculate this agar, using needle containing growth from unclassified TSI agar slant. Inoculate by streaking slant and stabbing butt. Incubate 96 ± 2 h at 35ºC. Read results as follows:

Positive--presence of growth, usually accompanied by color change from green to blue. Most cultures of Salmonella are citrate-positive.

Negative--no growth or very little growth and no color change.

8. Classification of cultures. Classify, as Salmonella, cultures that have reaction patterns of Table 17-6.

Discard, as not Salmonella, cultures that give results listed in any subdivision of Table 17-7. Perform additional tests described in Edwards and Ewing's Identification of Enterobacteriaceae (Ewing, 1986) to classify any culture that is not clearly identified as Salmonella by classification scheme in Table 17-6 or not eliminated as not being Salmonella by test reactions in Table 17-7. If neither of 2 TSI cultures carried through biochemical tests confirms the isolate as Salmonella, perform biochemical tests, beginning with 5-e, on remaining urease-negative TSI cultures from same 25 g analytical unit.

Table 17-7. Criteria for discarding non-Salmonella cultures.

Test or substrate	Results
1. Urease	positive (purple-red color)
2. Indole test and     Polyvalent flagellar (H) test; or     Indole test and     Spicer-Edwards flagellar test	positive (violet color at surface) negative (no agglutination); positive (violet color at surface) negative (no agglutination)
3. Lysine decarboxylase and     KCN broth	negative (yellow color) positive (growth)
4. Phenol red lactose broth	positive (yellow color and/or gas) a,b
5. Phenol red sucrose broth	positive (yellow color and/or gas)b
6. KCN broth,     Voges-Proskauer test, and     Methyl red test	positive (growth) positive (pink-to-red color) negative (diffuse yellow color)

^aTest malonate broth positive cultures further to determine if they are S. arizonae.
^bDo not discard positive broth cultures if corresponding LIA cultures give typical Salmonella reactions; test further to determine if they are Salmonella species.

9. Presumptive generic identification of Salmonella. As alternative to conventional biochemical tube system, use any of 5 commercial biochemical systems (API 20E, Enterotube II, Enterobacteriaceae II, MICRO-ID, or Vitek GNI) for presumptive generic identification of food-borne Salmonella. Choose a commercial system based on a demonstration in analyst's own laboratory of adequate correlation between commercial system and biochemical tube system delineated in this identification section. Commercial biochemical kits should not be used as a substitute for serological tests. Assemble supplies and prepare reagents required for the kit.

Inoculate each unit according to sec. 978.24 (API 20E, Enterotube II, and Enterobacteriaceae II), sec. 989.12 (MICRO-ID), and sec. 991.13 (Vitek GNI in Official Methods of Analysis (AOAC, 1995a), incubating for time and temperature specified. Add reagents, observe, and record results. For presumptive identification, classify cultures, according to (AOAC, 1995a), above, as Salmonella or not Salmonella.

For confirmation of cultures presumptively identified as Salmonella, perform the Salmonella serological somatic (O) test (5-f, above) and the Salmonella serological flagellar (H) test (5-c, above) or the Spicer-Edwards flagellar (H) test (5-d, above), and classify cultures according to the following guidelines:

1. Report as Salmonella those cultures classified as presumptive Salmonella with commercial biochemical kits when the culture demonstrates positive Salmonella somatic (O) test and positive Salmonella (H) test.
2. Discard cultures presumptively classified as not Salmonella with commercial biochemical kits when cultures conform to AOAC criteria (AOAC, 1995a) for classifying cultures as not Salmonella.
3. For cultures that do not conform to a or b, classify according to additional tests specified in 5, b-g, above, or additional tests as specified by Ewing (2), or send to reference typing laboratory for definitive serotyping and identification.

10. Treatment of cultures giving negative flagellar (H) test. If biochemical reactions of certain flagellar (H)-negative culture strongly suggest that it is Salmonella, the negative flagellar agglutination may be the result of nonmotile organisms or insufficient development of flagellar antigen. Proceed as follows: Inoculate motility test medium in petri dish, using small amount of growth from TSI slant. Inoculate by stabbing medium once about 10 mm from edge of plate to depth of 2-3 mm. Do not stab to bottom of plate or inoculate any other portion. Incubate 24 h at 35ºC. If organisms have migrated 40 mm or more, retest as follows: Transfer 3 mm loopful of growth that migrated farthest to trypticase soy-tryptose broth. Repeat either polyvalent flagellar (H) (5-c, above) or Spicer-Edwards (5-d, above) serological tests. If cultures are not motile after the first 24 h, incubate an additional 24 h at 35ºC; if still not motile, incubate up to 5 d at 25ºC. Classify culture as nonmotile if above tests are still negative. If flagellar (H)-negative culture is suspected of being a species of Salmonella on the basis of its biochemical reactions, FDA laboratories should submit the culture to the Midwest Laboratory for Microbiological Investigations (HFR-MW460), Minneapolis, MN 55401, for further identification and/or serotyping. Laboratories other than FDA should make arrangements with a reference laboratory for the serotyping of Salmonella cultures.
11. Submission of cultures for serotyping. Submit 2 isolates of each somatic group recovered from each analytical unit, unless otherwise instructed. Submit cultures on BHI agar slants in screw-cap tubes (13 x 100 mm or 16 x 125 mm) with caps secured tightly. Label each tube with sample number, subsample (analytical unit) number, and code, if applicable. Submit copies of the following records for each sample: 1) Collection Report, FD-464, or Import Sample Report, FD-784; 2) Analyst's Worksheet, FD-431; and 3) Salmonella Record Sheet, FD-431g. Place cultures in culture container with official FDA seal. Place accompanying records (5-k, above) inside shipping carton but not within officially sealed culture container. Submit memo or cover letter for each sample number to expedite reporting of results. Prepare cultures for shipment according to requirements for shipment of etiological agents (Federal Register, 1971). Label secondary shipping container according to Federal Register (1972). Send container by most rapid mail service available. Maintain duplicate cultures of those submitted for serotyping only on those samples under consideration for legal action.

6. Rapid screening methods for Salmonella

Table 17-8. Rapid methods approved by AOAC for detection of Salmonella

Rapid screening method		Manufacturer	AOAC Official Methods of Analysis section numbera
Generic name	Trade name
Fluorescent antibody	Not applicable	Not applicable	975.54
Hydrophobic grid membrane filter	ISO-GRID	QA Laboratories Ltd. 6645 Nancy Ridge Drive San Diego, CA 92121	99l.12
DNA hybridization enzyme immunoassay	GENE-TRAK	GENE-TRAK Systems, Inc. Salmonella Assay 94 South Street Hopkington, MA 01748 Framingham, MA 01701	990.13
Colorimetric monoclonal	Salmonella-Tek	Organon Teknika Corp. 100 Akzo Avenue Durham, NC 27704	986.35, 987.11, and 993.08
Colorimetric polyclonal enzyme immunoassay	TECRA Salmonella Visual Immunoassayb	Bioenterprises Pty Ltd, 28 Barcoo Street Roseville, NSW 2069 Australia	989.14
	Assurance Salmonella Enzyme Immunoassay	BioControl Systems, Inc. 12822 Southeast 32nd Street Bellevue, WA 98005	992.11
Immunodiffusion	1-2 TEST	BioControl Systems, Inc. 12822 Southeast 32nd Street Bellevue, WA 98005	989.13
Automated conductance	Malthus Microbial Detection System	Malthus Instruments, LTD The Manor, Manor Royal Crawley, West Sussex RH10 2PY, UK	991.38

^a See (AOAC, 1995a)
^bAlso distributed as BioPro Salmonella Visual Immunoassay.

Contents

Other analytical procedures

• Motile and non-motile Salmonella in foods: Polyclonal enzyme immunoassay screening method (AOAC, 1995b).
• Motile Salmonella in all foods: Immunodiffusion (1-2 Test) method (AOAC, 1995c).
• Salmonella, Escherichia coli, and other Enterobacteriaceae in foods: Biochemical system identification (Vitek GNI) screening method (AOAC, 1995d).
• Salmonella in cocoa and chocolate: Motility enrichment on modified semi-solid Rappaport-Vassiliadis (MSRV) medium (AOAC, 1995e).
• Salmonella in dried milk products: Motility enrichment on modified semi-solid Rappaport-Vassiliadis (MSRV) medium (AOAC, 1995f).
• Salmonella in dry foods: Refrigerated preenrichment and selective enrichment broth culture methods (AOAC, 1995g).
• Salmonella in foods: Automated conductance method (AOAC, 1995h).
• Salmonella in foods: Colorimetric deoxyribonucleic acid hybridization method (GENE-TRAK) (AOAC, 1995i).
• Salmonella in foods: Colorimetric monoclonal EIA (Salmonella-Tek) screening method (AOAC, 1995j).
• Salmonella in foods: Colorimetric monoclonal enzyme immunoassay (Salmonella Tek) (AOAC, 1995k).
• Salmonella in foods: Colorimetric polyclonal enzyme immunoassay screening method (AOAC, 1995l).
• Salmonella in foods: Detection (AOAC, 1995m).
• Salmonella in foods: DNA hybridization screening method (AOAC, 1995n).
• Salmonella in foods: Fluorescent antibody (FA) screening method (AOAC, 1995o).
• Salmonella in foods: Fluorogenic and colorimetric monoclonal enzyme immunoassay (Q-Trol) screening methods (AOAC, 1995p).
• Salmonella in foods: Hydrophobic grid membrane filter (iso-grid) screening method (AOAC, 1995q).
• Salmonella in foods: Identification (AOAC, 1995r).
• Salmonella in foods: Preparation of culture media and reagents (AOAC, 1995s).
• Salmonella in foods: Serological tests (AOAC, 1995t).
• Salmonella in low-moisture foods (AOAC, 1995u).
• Salmonella in raw, highly contaminated foods and animal feeds, detection (AOAC, 1995v).
• Salmonella spp., Escherichia coli, and other enterobacteriaceae in foods: Biochemical identification kit method (AOAC, 1995w).
• Salmonella spp. in foods: Biochemical identification kit method (AOAC, 1995x).

Contents

Commercial Test Products

Table 17-9. Commercial test products for Salmonella.

Test Kit	Analytical Technique	Approx. Total Test Time1	Supplier
1-2 Test2	Immunodiffusion	36 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
Assurance Gold Salmonella EIA	Enzyme immunoassay	29.5 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
Assurance Salmonella EIA2	Enzyme immunoassay	48 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
BAX® for Screening/Salmonella2	Polymerase chain reaction	26-30 h	Qualicon, Inc.  P.O. Box 80357  Wilmington, DE  19880-0357  Phone: 800/863-6842; 302/695-9400  E-mail: info@qualicon.com  Web: www.qualicon.com
Bind® Salmonella2	Bacteriophage with gene for ice crystal proteins and indicator dye	22 h	IDEXX Laboratories, Inc.  Contact: Greg Getchell  One Idexx Dr.  Westbrook, ME  04092  Phone: 800/321-0207; 207/856-0580  E-mail: greg-getchell@idexx.com  Web: www.idexx.com/fed/home/start.asp
CHECK 3 Salmonella	Chemical, visual detection	4-18 h	Contamination Sciences LLC  Contact: Robert Steinhauser  4230 East Towne Blvd., Suite 191  Madison, WI  53704  Phone: 608/825-6125  E-mail: bsteinha@contam-sci.com Web: www.contam-sci.com
CheckPoint Colony Lift Immunoassay Kit for Presumptive Detection of Group D Salmonella	Colony lift immunoassay	15-18 h	Kirkegaard & Perry Laboratories, Inc.  Contact: Diagnostic Technical Services  2 Cessna Court  Gaithersburg, MD  20879-4174  Phone: 800/638-3167; 301/948-7755  E-mail: diagnostics@kpl.com  Web: www.kpl.com
Dynabeads® anti-Salmonella2	Immunomagnetic separation	48 or 72 h	Dynal Inc.  Contact: Technical Service  5 Delaware Dr.  Lake Success, NY  1042  Phone: 516/326-3270  E-mail: techserv@dynalusa.attmail.com  Web: www.dynal.no
EIAFoss Salmonella2	Combination ELISA and immunomagnetic separation	22-24 h	Foss North America, Inc.  7682 Executive Dr.  Eden Prairie, MN  55344  Phone: 612/974-9892  E-mail: sales@fossnorthamerica.com  Web: www.fossnorthamerica.com
GENE-TRAK Salmonella Assay2	Nucleic acid hybridization	48 h	GENE-TRAK Systems  Contact: Linda Dragone  94 South St.  Hopkinton, MA  01748  Phone: 508/435-7400 E-mail: MCyr@vysis.com
ISO-GRID Method for Salmonella Detection using EF-18 Agar2	Enrichment followed by membrane filtration with selective and differential culture medium based on lysine decarboxylase and sucrose fermentation	42-72 h (42-48 h for negative screen and 24 h additional to confirm presumptive positive result)	QA Life Sciences, Inc.  Contact: Phyllis Entis  6645 Nancy Ridge Dr.  San Diego, CA  92121  Phone: 800/788-4446; 619/622-0560  E-mail: bugsy@qalife.com
Oxoid Salmonella Latex Test2	Polyvalent latex agglutination	42 h	Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca
Oxoid Salmonella Rapid Test2	Rapid differential culture with serological confirmation	42 h	Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca
PATH-STICK One Step Rapid Salmonella Test	Immunochromatography	38 to 56 h	Celsis, Inc.  Contact: Susan Moffa  165 Fieldcrest Ave.  Edison, NJ 08837  Phone: 800/222-8260; 732-346-5100  E-mail: smoffa@celsis.com  Web: www.celsis.com
Probelia PCR System	Polymerase chain reaction	22 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
Reveal® Microbial Screening Test for Salmonella2	Sandwich ELISA	21 h	Neogen Corporation  620 Lesher Pl.  Lansing, MI 48912  Phone: 517/372-9200  E-mail: NeogenCorp@aol.com  Web: www.neogen.com
Salmonella2	Culture	48 h	Contamination Sciences LLC  Contact: Robert Steinhauser  4230 East Towne Blvd., Suite 191  Madison, WI  53704  Phone: 608/825-6125  E-mail: bsteinha@contam-sci.com Web: www.contam-sci.com
Salmonella Antigen Detection Test	Antibody-dye conjugate complex	20-48 h	Morningstar Diagnostics, Inc.  1832 Centre Point Circle, Suite 103  Naperville, IL  60563  Phone: 630/577-0700  E-mail: information@mstarusa.com  Web: www.mstarus.com
GENE-TRAK Salmonella DLP2	Nucleic acid hybridization	48 h	GENE-TRAK Systems  Contact: Linda Dragone  94 South St.  Hopkinton, MA  01748  Phone: 508/435-7400 E-mail: MCyr@vysis.com
Salmonella Screen/Salmonella Verify2	Immunomagnetic separation   latex agglutination (for positive samples only)	24 h	Vicam, L.P. Contact: Brian Kraus  313 Pleasant St.  Watertown, MA  02472  Phone: 800/338-4381 E-mail: vicam@vicam.com  Web: www.vicam.com
Salmonella Screen/SE Verify2	Immunomagnetic separation   latex agglutination (for positive samples only)	24 h	Vicam, L.P.  Contact: Brian Kraus  313 Pleasant St.  Watertown, MA  02472  Phone: 800/338-4381 E-mail: vicam@vicam.com  Web: www.vicam.com
Salmonella-TekTM 24-Hour Method2	Bead immunocapture	24 h	Organon Teknika Corp.  100 Akzo Ave.  Durham, NC 27712  Phone: 800/654-0331; 919/620-2000 E-mail: casey@orgtek.com
Salmonella-TekTM 48-Hour Method2	ELISA	48 h	Organon Teknika Corp.  100 Akzo Ave.  Durham, NC 27712  Phone: 800/654-0331; 919/620-2000 E-mail: casey@orgtek.com
TECRA Salmonella Immunocapture	Immunocapture prior to using TECRA SALM VIA	24 h	International BioProducts  Contact: Mike Yeager 14780 NE 95th St.  Redmond, WA  98052  Phone: 800/729-7611; 425/883-1349 E-mail: myeager@intlbioproducts.com Web: intlbioproducts.com
TECRA Salmonella Unique 2	Immunoenrichment/ ELISA	22 h	International BioProducts  Contact: Bob Ward  14780 NE 95th St.  Redmond, WA  98052  Phone: 800/729-7611; 425/883-1349  E-mail: myeager@intlbioproducts.com Web: intlbioproducts.com
TECRA Salmonella Visual Immunoassay2	ELISA	48 h	International BioProducts  Contact: Bob Ward  14780 NE 95th St.  Redmond, WA  98052  Phone: 800/729-7611; 425/883-1349 E-mail: myeager@intlbioproducts.com Web: intlbioproducts.com
Vidas ICS	Immunoconcentration	24 h	bioMérieux Inc. Contact:  bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO 63042  Phone: 800/638-4835; 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com
Vidas SLM2	Enzyme linked fluorescent assay	48 h	bioMérieux Inc. Contact:  bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO 63042  Phone: 800/638-4835; 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com
VIP for Salmonella [Detects Salmonella spp.]	Lateral flow immunoassay	28 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com

¹Includes enrichment
²AOAC Approved

Contents

References

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