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BAM: Salmonella
NOTICE:
If you are looking for BAM Chapter 5: Salmonella (December Edition) that is incorporated by reference in 21 CFR Parts 16 and 118: Federal Register Final Rule1 (July 9, , 74 FR 33030): Prevention of Salmonella Enteritidis in Shell Eggs During Production, Storage, and Transportation, please use these versions of the BAM Salmonella Chapter2 (PDF, 189 Kb) and Appendix 1: Rapid Methods for Detecting Foodborne Pathogens3 (PDF, 195 Kb).
The most recent Edition of BAM Chapter 5: Salmonella is available below this notice.
November Version
Bacteriological Analytical Manual
Chapter 5
Salmonella
Authors: Wallace H. Andrews and Thomas Hammack
Revision History:
· November - Addition to Section C: Preparation of foods for isolation of Salmonella: Leafy green vegetables and herbs.
· February – Removed link to Appendix 1: Rapid Methods for Detecting Foodborne Pathogens (now archived).
· December – Mamey pulp method added, and Section D revised.
· June – Eggs method revised for shell eggs and liquid whole eggs.
· April – Frog legs method, Lactic casein, Rennet casein, Sodium caseinate and Rabbit carcass methods revised, top ears and other dog chew toys added. Removed section A.25, Mechanical shaker.
· October 25, – Extension of the applicability of the orange juice method in section C.19 to apple juice and apple cider.
· 1999-DEC, -MAR, and -AUG Final revision on -NOV-14 (see the Introduction for a summary of changes).
To obtain a copy of a prior version not currently posted, please contact Thomas Hammack
Chapter Contents
· Introduction
· Equipment and Materials
· Media and Reagents
· Preparation of foods for isolation of Salmonella
· Isolation of Salmonella
· Identification of Salmonella
· References
Introduction
Several changes are being introduced in this edition of BAM (8th Edition). The first change involves the expanded use of Rappaport-Vassiliadis (RV) medium4 for foods with both high and low levels of competitive microflora. In the previous edition, RV medium was recommended only for the analysis of shrimp. Based on the completion of AOAC precollaborative (5, 6) and collaborative (7, 8) studies, RV medium is now being recommended for the analysis of high microbial and low microbial load foods. RV medium replaces selenite cystine (SC) broth for the analysis of all foods, except guar gum. In addition, RV medium replaces lauryl tryptose broth for use with dry active yeast. Tetrathionate (TT)5 broth continues to be used as the second selective enrichment broth. However, TT broth is to be incubated at 43°C for the analysis of high microbial load foods and at 35°C for the analysis of low microbial load foods, including guar gum.
The second change involves 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 involves reducing the period of incubation of the lysine iron agar (LIA)6 slants. In the former edition (BAM-7), triple sugar iron agar (TSI)7 and LIA slants were incubated at 35°C 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 involves 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 (BAM-8) egg shells are now surface-disinfected by soaking for at least 10 sec in a 3:1 solution consisting of 3 parts of 70% alcohol (ethyl or isopropyl) to 1 part of iodine/potassium iodide solution.
The fifth change involves the sample preparation of eggs. Egg contents (yolk and albumen) are thoroughly mixed before analysis. After mixing the egg contents, 25 g (ml) are added to 225 ml trypticase (tryptic) soy broth supplemented with ferrous 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, nonpipettable mixture results. Addition of the enzyme cellulase to the preenrichment medium, however, results in a readily pipettable mixture.
A method for orange juice (pasteurized and unpasteurized) has been included due to recent orange juice-related outbreaks.
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 sulfite8, Hektoen enteric9, and xylose lysine desoxycholate agars10) and three relatively new agars (EF-18, xylose lysine Tergitol 4, and Rambach agars). Our results (9) 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.
A. Equipment and Materials
1. Blender and sterile blender jars (see Chapter 111)
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; g capacity, sensitivity of 0.1 g
5. Balance, with weights; 120 g capacity, sensitivity of 5 mg
6. Incubator, 35 ± 2 °C
7. Refrigerated incubator or laboratory refrigerator, 4 ± 2°C
8. Water bath, 49 ± 1°C
9. Water bath, circulating, thermostatically-controlled, 43 ± 0.2°C
10. Water bath, circulating, thermostatically-controlled,42 ± 0.2°C
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 5l), 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 23-24 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. Sponges, non-bactericidal (Nasco cat # B01299WA), or equivalent.
26. Swabs, non-bactericidal, cotton-tipped.
B. Media12 and Reagents13
For preparation of media and reagents, refer to Methods 967.25-967.28 in Official Methods of Analysis (1).
1. Lactose broth (M7414)
2. Nonfat dry milk (reconstituted) (M11115)
3. Selenite cystine (SC) broth (M13416)
4. Tetrathionate (TT) broth (M14517)
5. Rappaport-Vassiliadis (RV) medium (M13218). NOTE: RV medium must be made from its individual ingredients. Commercial formulations are not acceptable.
6. Xylose lysine desoxycholate (XLD) agar (M17919)
7. Hektoen enteric (HE) agar (M6120)
8. Bismuth sulfite (BS) agar (M1921)
9. Triple sugar iron agar (TSI) (M14922)
10. Tryptone (tryptophane) broth (M16423)
11. Trypticase (tryptic) soy broth (M15424)
12. Trypticase soy broth with ferrous sulfate (M18625)
13. Trypticase soy-tryptose broth (M16026)
14. MR-VP broth (M10427)
15. Simmons citrate agar (M13828)
16. Urea broth (M17129)
17. Urea broth (rapid) (M17230)
18. Malonate broth (M9231)
19. Lysine iron agar (LIA) (Edwards and Fife) (M8932)
20. Lysine decarboxylase broth (M87)33
21. Motility test medium (semisolid) (M10334)
22. Potassium cyanide (KCN) broth (M12635)
23. Phenol red carbohydrate broth (M12136)
24. Purple carbohydrate broth (M13037)
25. MacConkey agar (M9138)
26. Nutrient broth (M11439)
27. Brain heart infusion (BHI) broth (M2440)
28. Papain solution, 5% (M56a41)
29. Cellulase solution, 1% (M18742)
30. Tryptose blood agar base (M16643)
31. Universal preenrichment broth (M18844)
32. Universal preenrichment broth (without ferric ammonium citrate) (M188a45)
33. Buffered peptone water (M19246)
34. Dey-Engley broth (M19347)
35. Potassium sulfite powder, anhydrous
36. Chlorine solution, 200 ppm, containing 0.1% sodium dodecyl sulfate (R12a48)
37. Ethanol, 70% (R2349)
38. Kovacs' reagent (R3850)
39. Voges-Proskauer (VP) test reagents (R8951)
40. Creatine phosphate crystals
41. Potassium hydroxide solution, 40% (R6552)
42. 1 N Sodium hydroxide solution (R7353)
43. 1 N Hydrochloric acid (R3654)
44. Brilliant green dye solution, 1% (R855)
45. Bromcresol purple dye solution, 0.2% (R956)
46. Methyl red indicator (R4457)
47. Sterile distilled water
48. Tergitol Anionic 7 (R7858)
49. Triton X-100 (R8659)
50. Physiological saline solution, 0.85% (sterile) (R6360)
51. Formalinized physiological saline solution (R2761)
52. Salmonella polyvalent somatic (O) antiserum
53. Salmonella polyvalent flagellar (H) antiserum
54. 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
55. Salmonella Spicer-Edwards flagellar (H) antisera
C. Preparation of foods for isolation of Salmonella
The following methods are based on the analysis of a 25 g analytical unit at a 1:9 sample/broth ratio. Depending on the extent of compositing, add enough broth to maintain this 1:9 ratio unless otherwise indicated. For samples not analyzed on an exact weight basis, e.g., frog legs, refer to the specific method for instructions.
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 45°C for 15 min with continuous agitation in thermostatically controlled water bath or thaw within 18 h at 2-5°C. 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 broth65. 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 ± 5 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 35°C. Continue as in D, 1-11 , below.
2. Eggs
a. Shell eggs. Remove any adherent material from the shell surface. Disinfect eggs with 3:1 solution consisting of 3 parts of 70% alcohol (ethyl or isopropyl) to 1 part iodine/potassium iodide solution. Prepare 70% alcohol solution either by diluting 700 ml 100% alcohol with sterile distilled water for a final volume of 1,000 ml or by diluting 700 ml 95% alcohol with sterile distilled water for a final volume of 950 ml. Prepare iodine/potassium iodide solution by dissolving 100 g potassium iodide in 200-300 ml sterile distilled water. Add 50 g iodine and heat gently with constant mixing until the iodine is dissolved. Dilute the iodine/potassium iodide solution to 1,000 ml with sterile distilled water. Store iodine/potassium iodide solution in amber glass-stoppered bottle in the dark. Prepare the disinfection solution by adding 250 ml iodine/potassium iodide solution to 750 ml 70% alcohol solution and mix well. Submerge eggs in disinfection solution for at least 10 seconds. Remove eggs and allow to air dry. Eggs with chipped, cracked, or broken shells are not included in the sample. Each sample shall consist of twenty (20) eggs cracked aseptically into a Whirl-Pak bag, for a total of fifty (50) samples per poultry house. Eggs are cracked aseptically by gloved hands, with a change of gloves between samples. Mix samples thoroughly by gloved hands, with a change of gloves between samples. Mix samples thoroughly by hand until yolks are completely mixed with the albumen. Samples are held at room temperature (20-24°C) for 96 ± 2 h. After 96 ± 2 h, remove 25 ml portion from each sample of pooled eggs, and preenrich 25 ml test portion in 225 ml sterile trypticase soy broth (TSB) supplemented with ferrous sulfate66 (35 mg ferrous sulfate added to 1000 ml TSB) and mix well by swirling. Let stand 60 ± 5 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 35°C. Continue as in D, 1-11, below.
b. Liquid whole eggs (homogenized). Combine fifteen (15) 25 ml test portions into a 375 ml composite contained in a 6-liter Erlenmeyer flask. Composites are held at room temperature (20-24°C) for 96 ± 2 h. After 96 ± 2 h, add 3,375 ml sterile TSB supplemented with ferrous sulfate67, as described above, and mix well by swirling. Let stand 60 ± 5 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 35°C. Continue as in D, 1-11, below.
c. Hard-boiled eggs (chicken, duck, and others). If the egg shells are still intact, disinfect the shells as described above and aseptica
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