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Detection of Salmonella Spp. in Foods by the Vidas SLM Method
HPB Method MFHPB-24
November 2001
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(PDF Version - 62 K)HEALTH PRODUCTS AND FOOD BRANCH
OTTAWA
DETECTION OF SALMONELLA SPP. IN FOODS BY THE VIDAS SLM TM METHOD
Jean-Yves D'Aoust
Microbiology Research Division
Health Products and Food Branch (HPFB)
Health Canada, Postal Locator : 2204A2
Ottawa, Ontario, K1A 0L2
Jean-Yves_D'Aoust@hc-sc.gc.ca
1. APPLICATION
This method is applicable to the detection of viable Salmonella spp. in foods to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. Where an Official Method for foods is specified, that method shall be followed.
2. DESCRIPTION
The VIDAS SLMTM (Salmonella) assay, hereafter referred to as VIDAS, is an automated enzyme immunoassay method for the detection of Salmonella in foods and in agricultural products. Presumptive- positive results with the VIDAS system need to be culturally confirmed by the isolation and identification of viable salmonellae from the selective enrichment and postenrichment cultures involved in the VIDAS presumptive identification of a contaminated sample.
In April 1994, the Association Française de Normalisation (AFNOR) certified the VIDAS method on the strength of its demonstrated equivalence to the ISO 6579 method of the International Organization of Standardization (9.1). The technique was also collaboratively evaluated against the U.S. Food and Drug Administration BAM/AOAC method (9.2, 9.3), and adopted first action as method 996.08 by the Official Methods Board of the Association of Analytical Chemists (AOAC) in May 1996 (9.4). A Health Canada collaborative study in 1996-1997 (D'Aoust et al., unpublished data) favorably compared the VIDAS to the standard MFHPB-20 method for Salmonella (9.5) using naturally contaminated foods.
3. PRINCIPLE
The VIDAS (Vitek Immunodiagnostic Assay System) provides for the automated enzyme-linked immunofluorescent detection of motile and non-motile salmonellae. The efficacy of the system hinges on the affinity and specificity of antibodies for somatic (O) and flagellar (H) Salmonella antigens. The internal surface of the Solid Phase Receptacle (SPR), a disposable device that resembles a pipette tip, is pre- coated with Salmonella-specific monoclonal antibodies for the capture of target antigens in test samples.
The VIDAS assay configuration prevents non-specific reactions with the SPR. All assay reagents including wash solutions, alkaline phosphatase conjugate and enzyme substrate are contained in sealed, ready-to-use multi-chambered strips.
The VIDAS instrument performs all assay steps sequentially and automatically. Following the addition of a portion of boiled M broth postenrichment culture into the sample chamber of the reagent strip, the instrument repeatedly draws the chamber contents in and out of the SPR for a programmed period of time. Salmonella antigens present in a test sample bind to antibodies adsorbed onto the inner wall of the SPR; unbound sample material is removed by washing. Signal polyclonal antibodies conjugated with alkaline phosphatase are then drawn in and out of the SPR to facilitate their reaction with the Salmonella antigen-antibody complexes immobilized on the inner wall of the SPR. Unbound conjugate is removed by washing.The substrate, 4-methyl-umbelliferyl phosphate, is then drawn into the SPR where it is converted into a fluorescent reaction product (4-methyl-umbelliferone) by the enzyme conjugate. The intensity of emitted fluorescence is measured by an optical scanner and the results, expressed as relative fluorescence (Rf) values, are provided in a printed report. Samples with Rf $ 0.23 are flagged as presumptively positive for Salmonella spp.
VIDAS SLMTM is a registered trade mark of bioMérieux.
4. DEFINITIONS OF TERMS
See Appendix A of Volume 2.
5. COLLECTION OF SAMPLES
5.1 Sampling
See Appendix B of Volume 2.
5.2 Stringency of Sampling
Food control efforts frequently target processes and products presenting significant human health risks. The International Commission on Microbiological Specifications for Food (ICMSF) has categorized foods according to the degree of hazard associated with product use. Each food category carries an appropriately stringent sampling plan to determine the acceptability of a food product (Table I).
5.3 "Routine" versus "Investigational" Sampling Plans
The presence of Salmonella in foods and food ingredients presents one of three degrees of hazard (Table I). Circumstances supporting the "Routine" and "Investigational" sampling modes are outlined in Table II. The "Routine" sampling plans fulfill the role of detecting levels of contamination that may require regulatory action and/or a more thorough examination of the product. Different circumstances can justify the need for "Investigational" sampling. The choice of sampling plan may require some subjective judgment based on the number and kinds of factors that contribute to the degree of hazard.
6. MATERIALS AND SPECIAL EQUIPMENT
1) A large capacity VIDAS 30 or a reduced capacity mini VIDAS
2) VIDAS Salmonella Assay kit (ref. 30 702)
The VIDAS Salmonella Assay kit contains the necessary reagents including standards and controls for the analysis of 60 test samples. The kit is available from bioMérieux Canada, Inc., 4535 Dobrin, St. Laurent, Québec H4R 2L8. Tel: (514) 336 7321; Fax: (514) 336 6450.
Kit components:
- 60 ready-to-use SLM Reagent Strips
- 60 ready-to-use SLM Solid Phase Receptacles (SPR)
- S1 , SLM Standard (1 x 6 mL)
- C1 , SLM Positive Control (1 x 6 mL)
- C2 , SLM Negative Control (1 x 6 mL)
- MLE card (Specifications sheet to calibrate the test)
3) Calibrated pipette with disposable tip to dispense $500 µL
4) Incubators or water baths capable of maintaining 350C and 42.50C
5) Water-bath (100°C) or equivalent system
Note: It is the responsibility of each laboratory to ensure that the temperature of incubators or water baths are maintained at the required temperatures. When 35°C is stipulated, the incubator may be operated at 35 ± 1.0°C. However, it is imperative that temperature variations for incubators or water baths operated at 42.5°C not exceed ± 0.5°C because of the potential lethality of higher temperatures on the target microorganism.
6) Preenrichment broth media
Buffered peptone water (BPW)
Lactose broth (LAC)
Brilliant green water
Skim milk medium
Trypticase (tryptic) soy broth with 0.5% K2SO3
7) Enrichment broth media
Selenite Cystine (SC)
8) Postenrichment broth medium
9) Plating media
Brilliant Green Sulfa (BGS)
10) Purification agar media
Nutrient
11) Biochemical screening
Lysine Iron agar (LIA)
Urea agar (Christensen's)
Commercial biochemical test kits
12) Serological confirmation
13) Blender, stomacher and stomacher bags with filter
14) pH meter or paper with a minimum sensitivity of 0.3 to 0.5 units within a pH range of 5.0 to 8.0
15) 1N NaOH, 1 N HCl
16) Physiological saline
7. PROCEDURE
7.1 Handling of Sample Units
7.1.1 Analyze sample units as soon as possible. If necessary, store sample units under time and temperature conditions that will prevent the growth or death of native microflora. If sample units have been abused in transit, resampling of the lot should be carried out.
a. Frozen Foods: Sample units that show no signs of thawing upon receipt may be stored in the freezer at - 10 to - 20EC.
b. Dry and shelf stable foods may be stored at room temperature.
c. Refrigerate all other foods, including those that are received in a partially thawed condition: analyze these samples as soon as possible preferably within 24 h of receipt.
7.1.2 Thaw frozen sample units at room temperature within 60 min; if this is not possible, thaw the sample units at refrigerator temperature (4 -10EC).
Note: a) Large sample units (e.g. whole turkey) may not readily thaw at refrigerator temperatures. For greater expediency, enclose the frozen sample unit in a heavy walled paper bag and thaw overnight at room temperature. This technique maintains the product surface cold during the thawing process.
b) Appropriate containers should ensure that the drippings from the product do not contaminate the laboratory environment.
7.1.3 If the sample unit received for analysis is less than the required analytical unit, analyze the entire amount and record the weight used.
7.1.4 Blending or stomaching of analytical units should be limited to the minimum time required to produce a homogeneous suspension. Excessive blending could adversely affect the viability and recovery of salmonellae.
For products that do not require blending, disperse the analytical unit into the appropriate preenrichment broth.
7.1.5 Use aseptic techniques and sterile equipment at all stages of analysis. The physical containment of powdered products is critical if cross-contamination of the work environment is to be avoided.
7.2 Non-selective Enrichment (Preenrichment)
7.2.1 Compositing of Analytical Units
To reduce the workload, up to 15 x 25 g (mL) analytical units may be composited into a single test sample (e.g. 375 g or mL).
If a sample unit consists of more than one container, aseptically mix the contents of the containers prior to withdrawal of the analytical unit. If not possible or practical, the analytical unit shall then consist of equal portions from each of the containers.
7.2.2 Sample Analysis
7.2.2.1 The analytical unit (25 g) is dispersed into a suitable non-selective enrichment broth (Table III). Nutrient (NB), buffered peptone water (BPW) and lactose (LAC) broths are equally reliable and can be used interchangeably as general purpose preenrichment media (9.6, 9.7).
7.2.2.2 If the pH of the preenrichment mixture lies outside the range of 6.0 - 7.0, adjust with 1N NaOH or 1N HCl.
Note: If the sample unit consists of a container with little food material, thoroughly rinse the interior of the container with a suitable preenrichment broth medium and incubate the rinse in a sterile flask. This eventuality is more frequently encountered in situations involving consumer complaints or food poisoning investigations.
7.2.2.3 A positive Salmonella and a negative medium control should be set up in parallel with the test samples.
7.2.2.4 Incubate the preenrichment mixture and the positive and negative controls at 35°C for 18 - 24 h.
Note: Any evidence of growth in the negative control and/or the absence of growth in the positive control would invalidate test results.
7.3 Selective Enrichment
7.3.1 Sample analysis
7.3.1.1 With a sterile pipette, transfer 1.0 mL of the preenrichment culture into each of 9.0 mL tetrathionate (TBG) and 9.0 mL selenite cystine (SC) broths.
7.3.1.2 Incubate TBG broth at 42.5° C and SC at 35° C for 6-8 h (dry foods and other products containing low levels of background microflora) or 18-24 h (raw meats and other products containing high levels of background microflora).
7.4 Post Enrichment
7.4.1 Sample analysis
7.4.1.1 Transfer 1.0 mL of SC and 1.0 mL of TBG broth cultures into separate tubes each containing 10 mL of M broth. Store the remaining SC and TBG cultures of raw meats and other products with high levels of background microflora at 4-10°C for use in confirmatory tests (8.1).However, reincubate the 6-8h enrichment cultures of dry foods and other products with low levels of background microflora for a total of 18 -24h and refrigerate the resulting cultures for use in confirmatory tests (8.1).
7.4.1.2 Incubate the inoculated M broth tubes for 6 - 8 h (raw meats and products with high levels of background microflora) or 18 h (dry foods and products with low levels of background microflora) at 42.5°C.
7.4.1.3 After incubation, resuspend the contents of each M broth culture and combine 1.0 mL from two homologous M broth cultures into a single tube; store the original M broth cultures at 4 -10°C for use in confirmatory tests (8.1).
7.4.1.4 Heat the tube containing the mixture of two M broth cultures for 15 minutes in a water-bath at 100°C.
7.4.1.5 Perform the VIDAS SLM test (7. 5) on a portion of the heat-treated culture (7.4.1.4).
7.5 Vidas SLM Procedure
7.5.1 Remove the VIDAS SLM kit from the refrigerator and allow the kit components to equilibrate at room temperature (minimum 30 min). Unused reagents should be stored at 2 - 8° C.
7.5.2 Label the SLM Reagent Strips with the appropriate sample identification numbers in the space provided.
7.5.3 Create a Work List by entering the "SLM" assay code and the number of tests to be run.
7.5.4 If a standard needs to be tested, enter "S1" for the sample identification. A standard must be entered at the beginning of the Work List and should be tested in duplicate if it is to be stored in memory. If a standard is to be tested on a mini VIDAS, enter "S" then "1" for the sample identification.
7.5.5 Mix /vortex the standard, controls and heat-treated samples.
7.5.6 Dispense 500 µL of standard, control or heat-treated sample into the sample well of SLM Reagent Strips.
7.5.7 Position the VIDAS SLM solid phase receptacles (SPR) and reagent strips as indicated on the screen and initiate the assay as described in the operator's manual. The assay will be completed within 45 minutes.
8. INTERPRETATION
VIDAS SLM immunoassays yielding relative fluorescence values (Rf) of > 0.23 indicate the presumptive presence of Salmonella in the test sample. All presumptive-positive results must be confirmed culturally.
8.1 Cultural confirmation of VIDAS presumptive - positive results
8.1.1 Streak replicate loopsful (4 mm) from previously refrigerated SC, TBG and M broth cultures of a test sample (7.4.1.1 and 7.4.1.3) onto BS and BGS agar plates as described in MFHPB-20 (9.5).
8.1.2 Streak up to three suspect colonies from each presumptively-positive plates onto MacConkey agar for purification. Isolates are screened biochemically (Table IV) and confirmed serologically as described in MFHPB-20 (9.5).
Caution: Non-agglutinating cultures that show biochemical traits suggestive of Salmonella spp. should not be discarded but sent to a Reference Typing Center for complete identification.
9. REFERENCES
9.1 International Organization for Standardization (ISO). 1993. Microbiology - General guidance on methods for the detection of Salmonella. International Standard ISO 6579. Geneva.
9.2 U.S. Foods and Drug Administration. 1995. Salmonella (Chapter 5). Bacteriological Analytical Manual (BAM), 8th edition. AOAC International, Gaithersburg, Md.
9.3 Curiale, M.S., V. Gangar, and C. Cravens. 1997. VIDAS enzyme-linked immunofluorescent assay for detection of Salmonella in food: collaborative study. J. AOAC Intern. 80:491-504.
9.4 Association of Official Analytical Chemists. 1996. For your information. Methods adopted First Action. J. AOAC Intern. 79:76a.
9.5 D'Aoust, J.-Y. and U. Purvis. 1998. Isolation and Identification of Salmonella from Foods. MFHPB-20. ( Vol. 2 ). Compendium of Analytical Methods. http://www.hc-sc.gc.ca/food -aliment.
9.6 D'Aoust, J.-Y. and C. Maishment. 1979. Preenrichment conditions for effective recovery of Salmonella in foods and feed ingredients. J. Food Prot. 42: 153-157.
9.7 D'Aoust, J.-Y. 2000. Salmonella (Chapter 45; Vol. II).The Microbiological Safety and Quality of Food. B.M. Lund, A.C. Baird-Parker and G.W. Gould (eds). Aspen Publishers, Inc., Gaithersburg, Md.
9.8 D'Aoust, J.-Y. 1977. Effect of storage conditions on the performance of bismuth sulfite agar. J. Clin. Microbiol. 5:122-124.
9.9 International Commission on Microbiological Specifications for Foods (ICMSF). 1986. Microorganisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications. Second edition. University of Toronto Press, Toronto, ON.
10. MEDIA AND REAGENTS
10.1 Antisera, Salmonella
a. Somatic Polyvalent Antisera
b. Somatic Single Grouping Antisera
The antisera should be prepared according to manufacturer's instructions. Antisera should be checked periodically with control Salmonella cultures.
10.2 Bismuth Sulfite Agar (BS)
| Peptone | 10.0 g |
|---|---|
| Beef extract | 5.0 g |
| Dextrose | 5.0 g |
| Disodium phosphate | 4.0 g |
| Ferrous sulfate | 0.3 g |
| Bismuth sulfite indicator | 8.0 g |
| Brilliant green | 0.025 g |
| Agar | 20.0 g |
Suspend ingredients in one litre of distilled water and mix thoroughly. Uniformly heat the medium to incipient boiling using a hotplate rather than an open flame. Frequently swirl medium during heating. Do not autoclave. Cool to 45 - 50 0C and swirl medium to evenly disperse the heavy precipitate just prior to pouring the medium into plates.
Note: Bismuth sulfite agar can delay the growth of Salmonella spp. unless the prepared plates are refrigerated at 4 -100C for 24 h before inoculation. If no growth appears after 24 h of incubation, reincubate plates for an additional 24 h. If freshly poured medium is used, incubate inoculated plates for 48 h (9.8).
10.3 Brilliant Green Sulfa Agar (BGS)
| Yeast extract | 3.0 g |
|---|---|
| Peptone | 10.0 g |
| Sodium chloride | 5.0 g |
| Lactose | 10.0 g |
| Sucrose | 10.0 g |
| Phenol red | 0.08 g |
| Brilliant green | 12.5 mg |
| Sulfapyridine | 1.0 g |
| Agar | 20.0 g |
| Final pH | 6.9 ± 0.2 |
Suspend the ingredients in one litre of distilled water. Heat to boiling with frequent swirling. Autoclave at 1210C for 15 minutes and cool to 45 - 50 0C. Dispense medium into plates and allow to dry with lids partially removed.
10.4 Brilliant Green Water
| Brilliant green dye | 0.02 g |
|---|---|
| Sterile water | 1 L |
Dissolve 1.0 g brilliant green dye in 100 mL sterile water (1% w/v). Add 2.0 mL 1% brilliant green solution to one litre of sterile water (final concentration of 0.002% w/v).
10.5 Buffered Peptone Water (BPW)
| Peptone | 10.0 g |
|---|---|
| Sodium chloride | 5.0 g |
| Disodium phosphate | 3.5 g |
| Potassium dihydrogen phosphate | 1.5 g |
| Final pH | 7.2 ± 0.2 |
Dissolve 20.0 g in one litre of distilled water. Dispense into appropriate glassware and autoclave at 1210C for 15 minutes.
10.6 Lactose Broth (LAC)
| Beef extract | 3.0 g |
|---|---|
| Peptone | 5.0 g |
| Lactose | 5.0 g |
| Final pH | 6.9± 0.2 |
Dissolve 13.0g in one litre of distilled water. Dispense into appropriate glassware and autoclave at 1210C for 15 minutes.
10.7 Lysine Iron Agar (LIA)
| Peptone | 5.0 g |
|---|---|
| Yeast extract | 3.0 g |
| Dextrose | 1.0 g |
| L-Lysine | 10.0 g |
| Ferric ammonium citrate | 0.5 g |
| Sodium thiosulfate | 0.04 g |
| Brom cresol purple | 0.02 g |
| Agar | 15.0 g |
| Final pH | 6.7 ± 0.2 |
Suspend ingredients in one litre of distilled water and heat to boiling with frequent swirling.
Dispense appropriate volume of medium into tubes, and autoclave at 1210 C for 15 minutes. Cool in slanted position to form deep butts.
10.8 M Broth
| Yeast extract | 5.0 g |
|---|---|
| Tryptone | 12.5 g |
| D-Mannose | 2.0 g |
| Sodium citrate | 5.0 g |
| Sodium chloride | 5.0 g |
| Dipotassium phosphate | 5.0 g |
| Manganese chloride | 0.14 g |
| Magnesium sulfate | 0.8 g |
| Ferrous sulfate | 0.04 g |
| TweenR 80 | 0.75 g |
| Final pH | 7.0 ± 0.2 |
Suspend ingredients in one litre of distilled water and heat to boiling for 1-2 minutes. Autoclave at 121°C for 15 minutes ; cool and dispense as appropriate.
10.9 MacConkey Agar
| Peptone | 17.0 g |
|---|---|
| Proteose peptone | 3.0 g |
| Lactose | 10.0 g |
| Bile salts mixture | 1.5 g |
| Sodium chloride | 5.0 g |
| Neutral red | 0.03 g |
| Crystal violet | 0.001 g |
| Agar | 13.5 g |
| Final pH | 7.1 ± 0.2 |
Suspend ingredients in one litre of distilled water and heat to boiling with frequent swirling. Autoclave at 121°C for 15 minutes and cool to 45 - 50 °C. Dispense medium into plates and allow to dry with lids partially removed.
10.10 Nutrient Agar
| Peptone | 5.0 g |
|---|---|
| Beef extract | 3.0 g |
| Agar | 15.0 g |
| Final pH | 6.8 ± 0.2 |
Suspend ingredients in one litre of distilled water and heat to boiling with frequent swirling.
Dispense into tubes and autoclave at 121°C for 15 minutes. Cool tubes in a slanted position.
10.11 Nutrient Broth (NB)
| Peptone | 5.0 g |
|---|---|
| Beef extract | 3.0 g |
| Final pH | 6.8 ± 0.2 |
Dissolve ingredients in one litre of distilled water. Dispense into appropriate glassware and autoclave at 121°C for 15 minutes.
10.12 Physiological Saline
| Sodium chloride | 8.5 g |
|---|
Dissolve ingredient in one litre of distilled water. Dispense into appropriate glassware and autoclave at 121°C for 15 minutes.
10.13 Selenite Cystine Broth (SC)
| Tryptone or peptone | 5.0 g |
|---|---|
| Lactose | 4.0 g |
| Disodium phosphate | 10.0 g |
| Sodium acid selenite | 4.0 g |
| L-Cystine | 0.01 g |
| Final pH | 7.0 ± 0.2 |
Suspend ingredients in one litre of distilled water and heat for 10 minutes in flowing steam or boil on a hotplate with frequent swirling. Do not autoclave. Cool to 45 - 50 °C and dispense medium into appropriate glassware. Use medium on the day of preparation.
Note: Selenium salts can be teratogenic and should be weighed in a safety cabinet.
10.14 Skim Milk Medium
| Instant nonfat dry milk | 100.0 g |
|---|---|
| Brilliant green dye | 0.02 g |
Dissolve instant nonfat dry milk in one litre of distilled water and add 20 mL of 0.1% (w/v) aqueous brilliant green solution (final concentration of 0.002% w/v). Autoclave at 121°C for 15 minutes.
10.15 Tetrathionate Brilliant Green Broth (TBG)
a. Basal Medium
| Peptone or proteose peptone | 5.0 g |
|---|---|
| Bile Salts | 1.0 g |
| Calcium carbonate | 10.0 g |
| Sodium thiosulphate | 30.0 g |
| Final pH | 8.4 ± 0.2 |
Suspend ingredients of basal medium in one litre of distilled water and heat to boiling. Cool to 45 - 50°C.
b. Brilliant green solution (1% w/v)
Dissolve 1.0 g of brilliant green dye in 100 mL of distilled water.
c. Iodine solution
| Potassium iodide | 25.0 g |
|---|---|
| Iodine | 30.0 g |
| Distilled water | 100.0 mL |
Suspend ingredients in 100 mL of distilled water.Do not heat.The iodine solution should be prepared well in advance because dissolution of iodine is slow.
To prepare the complete medium on the day of use, aseptically add 1.0 mL of 1% brilliant green solution and 20.0 mL of iodine solution to one litre of basal medium. Do not heat the medium after the addition of iodine.
10.16 Triple Sugar Iron Agar (TSI)
| Beef extract | 3.0 g |
|---|---|
| Yeast extract | 3.0 g |
| Peptone | 15.0 g |
| Proteose peptone | 5.0 g |
| Lactose | 10.0 g |
| Sucrose | 10.0 g |
| Dextrose | 1.0 g |
| Ferrous sulfate | 0.2 g |
| Sodium chloride | 5.0 g |
| Sodium thiosulfate | 0.3 g |
| Agar | 12.0 g |
| Phenol red | 0.024 g |
| Final pH | 7.4 ± 0.2 |
Suspend ingredients in one litre of distilled water and heat to boiling with occasional swirling. Dispense appropriate volume of medium into tubes and autoclave at 121°C for 15 minutes. Cool tubes in a slanted position to form deep butts.
10.17 Trypticase (Tryptic, Tryptone) Soy Broth
| Tryptone | 17.0 g |
|---|---|
| Soytone | 3.0 g |
| Dextrose | 2.5 g |
| Sodium chloride | 5.0 g |
| Dipotassium phosphate | 2.5 g |
| Final pH | 7.3 ± 0.2 |
Dissolve ingredients in one litre of distilled water. Dispense into appropriate glassware and autoclave at 121°C for 15 minutes.
10.18 Urea Agar (Christensen's)
a. Agar Base
| Peptone | 1.0 g |
|---|---|
| Dextrose | 1.0 g |
| Sodium chloride | 5.0 g |
| Potassium dihydrogen phosphate | 2.0 g |
| Phenol Red | 0.012 g |
| Agar | 15.0 g |
| Final pH | 6.8 ± 0.2 |
Dissolve ingredients of basal medium in 900 mL of distilled water. Autoclave at 121°C for 15 minutes and cool to 45 - 50° C.
b. Urea solution
Dissolve 20 g of urea in 100 mL distilled water and sterilize by filtration.
To prepare the complete medium, add 100 mL of urea solution to 900 mL of tempered agar base. Dispense appropriate volumes of the complete medium in tubes to obtain deep butts upon cooling in a slanted position.
TABLE I. Stringency of ICMSF sampling plans for Salmonella in foodsa
| ICMSF | BAMe | ||||
|---|---|---|---|---|---|
| Hazard in usea | Case numberb | Number of sample units (n) | Hazard Category | Sample units (n) | |
| Routinec | Investigationalc,d | ||||
| Reduced | 10 | 5 | 15 | III | 15 |
| Unchanged | 11 | 10 | 30 | II | 30 |
| Increased | 12 | 20 | 60 | I | 60 |
a Relates to the degree of hazard associated with the conditions under which the food is expected to be handled and consumed after sampling. See Tables 6 (p.43) and 7 (p.48) in reference 9.9.
b The case numbers are ICMSF terminologies relating to the degrees of concern for foods and food ingredients that may contain Salmonella spp. and that may be implicated in outbreaks of human salmonellosis.The stringency of food sampling increases with increasing case number under the routine and investigational modes of sampling.
c See Tables 10 (p.74) and 11 (p.77) in reference 9.9. All 2-class sampling plans (C=0).
d Applies to foods distributed to susceptible consumers (Table 8, p. 55 in reference 9.9) or for investigational purposes (Table 11, p. 77). All 2-class sampling plans (C=0).
e FDA Bacteriological Analytical Manual (9.2), Chapter 1.
TABLE II. Conditions for "Routine" or "Investigational" sampling of foodsa
| Routine sampling | Investigational samplingb |
|---|---|
| A. THE FOOD | |
| Previous test satisfactory. | Prior tests frequently unsatisfactory. |
| Indicator tests give no evidence of severe contamination. | Indicator tests have revealed severe contamination. |
| Not commonly involved in disease. | Of a type frequently involved in disease outbreak. |
| Not specifically suspect in an outbreak. | Food from same manufacturer currently involved in
a disease outbreak. Circumstances point to possible involvement in an outbreak. |
| Not primarily destined to sensitive populations. | Suspect and destined to sensitive populations. New type of food or new formulation with some rationale for hazard. Conflicting analytical results from different laboratories. |
| B. THE MANUFACTURER | |
| Records satisfactory. | No records. |
| Sanitation control known to be normally adequate. | Known or suspected to exercise unsatisfactory plant
control. Knowledge of temporary hazardous conditions at the plant. |
| C. COUNTRY OF ORIGIN | |
| Known to exercise competent control of plant operations. | Endemic or epidemic situations potentially hazardous. |
| Not in areas endemic or currently epidemic for pertinent foodborne disease. | High carrier rate. Sewage pollution usually severe. Food control systems primitive. |
a Adapted from "Microorganisms in Foods" Vol. 2. Table 11, p. 77 (9.9). b To be appraised on the basis of all available information. Any single factor would not necessarily warrant investigational sampling.
TABLE III. Procedures for non-selective enrichment (preenrichment)
| Type of Product | Degree of Hazarda (number of sample units) |
Sample Unitc | Preparation of the analytical unit |
|---|---|---|---|
Alimentary Pastes |
Unchanged (10) | 100 g | Suspend 25 g in 225 mL of NB, BPW or LAC and blend. Dry blending at reduced speed can also be used to obtain a fine particulate test material. |
| Alimentary Pastes e.g. spaghetti, noodles. b. Requires cooking |
Reduced (5) | 100 g | Suspend 25 g in 225 mL of NB, BPW or LAC and blend. Dry blending at reduced speed can also be used to obtain a fine particulate test material. |
| Coconut | Unchanged (10) | 100 g | Suspend 25 g in 225 mL of NB, BPW or LAC and blend. |
| Confectionery a. Chocolate and cocoab |
Unchanged (10) | 100 g | Suspend 25 g in 225 mL of skim milk medium and blend. |
| Confectionery b. Candy |
Unchanged (10) | 100 g | Suspend 25 g in 225 mL of skim milk medium and blend. |
| Dairy Products a. Cheese |
Unchanged (10) | 100 g | Suspend 25 g in 225 mL of NB, BPW or LAC and blend. |
| Dairy Products b. Fluid milk |
Increased (20) | 100 mL | Add 25 mL to 225 mL of brilliant green water. |
| Dairy Products c. Ice cream |
Unchanged (10) | 100 g | Suspend 25 g in 225 mL of brilliant green water. |
| Dairy Products d. Powdered products e.g. whey, buttermilk |
Increased (20) | 100 g | Gently add 25 g to 225 mL of sterile brilliant green water and allow to soak undisturbedd. |
| Dairy Products e. Skim milk powderb |
Increased (20) | 100 g | Gently add 25 g to 225 mL of sterile brilliant green water and allow to soak undisturbedd . |
| Liquid Egg Productsb | Unchanged (10) | 100 g | Suspend 25 g in 225 mL NB, BPW or LAC and blend. |
| Froglegsb | Reduced (5) | ≥25 g | Place 25 g in 225 mL NB, BPW or LAC. |
| Meats a. Raw |
Reduced (5) | ≥ 100 g package | Suspend 25 g in 225 mL NB, BPW or LAC and blend. |
| Meats b. Cooked |
Unchanged (10) | ≥ 100 g package | Suspend 25 g in 225 mL NB, BPW or LAC and blend. |
| Poultry a. Raw |
Reduced (5) | i) Whole bird | Place thawed or fresh bird, drippings and giblets (if present) into a heavy sterile plastic bag. Add 1.0 litre NB, BPW or LAC and shake vigorously so that all sample surfaces come into contact with the broth medium. |
| ii) Cut-up: ≥ 100 g package | As described above for "whole bird". | ||
| iii) Giblets: ≥ 100 g package | Suspend 25 g in 225 mL NB, BPW or LAC and blend. | ||
| Poultry b. Cooked |
Unchanged (10) | 100 g | Suspend 25 g in 225 mL NB, BPW or LAC and blend. |
| Prepared Foods e.g. meat pies, TV dinners |
Unchanged (10) | ≥ 100 g package | Combine 25 g of each food component (if applicable) into a single analytical unit; add nine volumes of NB, BPW or LAC and blend. |
| Spices and Seasonings a. To be cooked |
Reduced (5) | ≥ 100 g package | Suspend 25 g in 225 mL NB, BPW or LAC and mix thoroughlyf. With onions and garlic, use trypticase (tryptic) soy broth containing 0.5% (w/v) K2SO3. |
| Spices and Seasonings b. Ready to eate |
Unchanged (10) | ≥ 100 g package | Suspend 25 g in 225 mL NB, BPW or LAC and mix thoroughlyf. With onions and garlic, use trypticase (tryptic) soy broth containing 0.5% (w/v) K2SO3. |
| Yeast | Unchanged (10) | ≥ 100 g package | Suspend 25 g in 225 mL, NB, BPW or LAC and mix thoroughly. |
| Foods not listed above a. Raw |
Reduced (5) | ≥ 100 g package | Suspend 25 g in 225 mL NB, BPW or LAC; blend or mix as appropriate. |
|
Foods not listed above |
Unchanged (10) |
≥ 100 g package |
Suspend 25 g in 225 mL NB, BPW or LAC; blend or mix as appropriate. |
a Refer to Table I for stringency of sampling and degree of hazard associated with use.
b Official Method available.
c Where consumer size package is less than 100 g (mL), sample unit will consist of more than one package.
d Ensure that the test material rehydrates completely during soaking. Use of the soak method for milk powder composites of low solubility is contraindicated because of the propensity for incomplete wetting of the test material.
e Relates to condiments added to prepared foods prior to consumption i.e. pepper (black, white, cayenne, lemon), paprika, chilies, parsley flakes, cinnamon etc.
f The ratio of spice to medium should be 1:10 (w/v). However, higher ratios are required for spices that exhibit antibacterial activity, and for spices that absorb large amounts of broth medium (Table V).
TABLE IV. Determinant biochemical tests
| Medium | Reaction | Observation | Typical Salmonella reaction |
|---|---|---|---|
| Triple Sugar Iron Agar (TSI) | Lactose and/or sucrose utilization | Positive reaction: Slant turns yellow Negative reaction: Colour of slant unchanged. |
Negativea |
| Dextrose utilization | Positive reaction: Butt turns yellow with /without
gas pockets Negative reaction: Colour of butt unchanged |
Positive | |
| H2S production | Positive reaction: Blackening of butt and/or slant Negative reaction: No blackening |
Positiveb | |
| Gas formation | Positive reaction: Gas pockets in the medium Negative reaction: No gas pockets |
Positive | |
| Lysine Iron Agar (LIA) | H2S production | Positive reaction: Blackening of butt and/or slant Negative reaction: No blackening |
Positive |
| Lysine decarboxylase | Positive reaction: Butt remains purple. Negative reaction: Butt turns yellow |
Positive | |
| Lysine deaminase | Positive reaction: Slant turns wine-red Negative reaction: Colour of slant unchanged |
Negative | |
| Christensen's Urea agar | Urease | Positive reaction: Slant turns pink/red Negative reaction: Colour of slant unchanged |
Negative |
a. Some strains can utilize one or both saccharides.
b. Slow H2S producers may be encountered. The H2S reaction may be inhibited with lactose and/or sucrose-utilizing strains.
TABLE V. Ratio of spices and seasonings to preenrichment broth (w/v)
| Spices and Seasonings | Ratio |
|---|---|
| Allspice | 1:10 and 1:100 |
| Allspice powder | 1:20 and 1:100 |
| Anise seed | 1:10 |
| Arrowroot powder | 1:10 |
| Basil leaves | 1:20 |
| Bay leaves | 1:20 |
| Caraway seed | 1:10 |
| Cardamon seed | 1:10 |
| Cassia buds | 1:10 |
| Cassia powder | 1:50 |
| Cayenne pepper | 1:10 |
| Celery flakes | 1:20 |
| Celery seed | 1:10 |
| Chervil leaves | 1:20 |
| Chilies whole | 1:10 |
| Chives chopped | 1:20 |
| Cinnamon sticks | 1:10 and 1:100 |
| Cloves | 1:50 and 1:1000 |
| Coriander seed | 1:10 |
| Cumin seed | 1:10 |
| Curry powder | 1:10 |
| Dill seed | 1:10 |
| Fennel seed | 1:10 |
| Ginger root | 1:10 |
| Italian seasoning | 1:20 |
| Lemon peel | 1:10 |
| Lemon pepper | 1:50 |
| Mace powder | 1:10 |
| Marjoram | 1:10 |
| Mint flakes | 1:20 |
| Mint ground | 1:10 |
| Mushroom slices | 1:20 |
| Mustard seeds | 1:10 |
| Nutmeg whole | 1:10 |
| Onion powder | 1:10 |
| Orange bits | 1:10 |
| Oregano leaves | 1:50 |
| Paprika | 1:10 |
| Parsley flakes | 1:20 |
| Peppercorns | 1:10 |
| Pepper black | 1:10 |
| Pepper white | 1:10 |
| Poppy seed | 1:10 |
| Pumpkin pie seasoning | 1:50 |
| Rosemary leaves | 1:10 |
| Sesame seed | 1:10 |
| Salad herbs | 1:20 |
| Salad supreme | 1:10 |
| Sage leaves | 1:20 |
| Savory ground | 1:50 |
| Saffron | 1:20 |
| Tarragon | 1:20 |
| Tartar (cream of) | 1:10 |
| Thyme powder | 1:50 |
| Turmeric powder | 1:10 |
| Vegetable flakes | 1:20 |
