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Methods for the Isolation and Identification of Salmonella from Foods and Environmental Samples
HPB Method MFHPB-20
March 2009
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(PDF
Version - 51 Kb)HEALTH PROTECTION BRANCH
OTTAWA
ISOLATION AND IDENTIFICATION OF SALMONELLA FROM FOOD
AND ENVIRONMENTAL SAMPLES
Anne Reid
Research Division
Health Products and Food Branch
Health Canada, Postal locator: 2204E
Ottawa, Ontario, K1A 0K9
e-mail: Anne_Reid@hc-sc.gc.ca
Microbiological Methods Committee
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204E
Ottawa, Ontario K1A 0K9
1. APPLICATION
This method is applicable to the detection of viable Salmonella in foods and environmental samples to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and specific Regulations (as summarized in the Interpretive Summary (7.15)). This revised method replaces MFHPB-20, dated April 1998. Due to health and safety issues surrounding the use of selenite cystine, this revised method should also be used in place of the Official Methods (Volume 1 of this Compendium) such as MFO-6, MFO-10, MFO-11 and MFO-12.
This revised method includes the following major changes in the analytical approach for the detection of foodborne Salmonella spp.:
1) The incorporation of Appendix J (November 2003) allowing the substitution of RVS enrichment broth for SC enrichment broth for most food commodities.
2) The incorporation of MFHPB-20A (April 2002), for the analysis of seed used to manufacture sprouts.
3) The inclusion of other selective media, including chromogenic media.
2. PRINCIPLE
The procedure consists of six distinct stages.
2.1 Non-Selective Enrichment (Pre-enrichment)
The initial handling of the food and the non-selective enrichment stage (pre-enrichment) vary according to the type of food examined (7.6). The test sample is inoculated into a non-inhibitory liquid medium to favour the repair and growth of stressed or sublethally-injured Salmonella arising from exposure to heat, freezing, desiccation, preservatives, high osmotic pressure or wide temperature fluctuations (7.1, 7.6).
2.2 Selective Enrichment
Replicate portions of each pre-enrichment culture are inoculated into two enrichment media to favour the proliferation of Salmonella through a selective inhibition of the growth of competing microorganisms (7.4). The enrichment broth RVS (7.25 - 7.29) may be substituted for SC broth for most food commodities (see the “Note” in Section 5, point 8 for exceptions).
2.3 Selective Plating
Enriched cultures are streaked onto selective and differential agars for the isolation of Salmonella (7.5, 7.30).
2.4 Purification
Presumptive Salmonella isolates are purified on MacConkey agar plates. This removes the possibility of viable but inhibited organisms from the selective agars contaminating the culture in further tests.
2.5 Biochemical Screening
Isolates are screened using determinant biochemical reactions.
2.6 Serological Identification
Polyvalent and/or grouping somatic antisera are used to support the identification of isolates as Salmonella. Cultures should be sent to a reference typing centre for complete serotyping.
3. DEFINITION OF TERMS
See Appendix A of Volume 2.
4. COLLECTION OF SAMPLES
4.1 Sampling
See Appendix B of Volume 2.
For routine sampling and analysis, 5 sample units of at least 100 g each should be collected from each lot unless stipulated in Table I. Refer to the Interpretive Summary (7.15) for Guidelines and Regulations specific to the foods being analysed for Salmonella.
For additional information concerning sampling plans in relation to degrees of risk and conditions of use refer to ICSMF (7.16).
5. MATERIALS AND SPECIAL EQUIPMENT
Note: The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with the International Standard referred to as “ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories".
The media and reagents listed below are commercially available and are to be used, prepared and/or sterilized according to the manufacturer's instructions. See Appendix G of Volume 2 for the media formula.
1) Nutrient Broth (NB)
2) Trypticase (Tryptic, Tryptone) Soy Broth
3) Brilliant Green Water
4) Buffered Peptone Water (BPW)
5) Skim Milk Medium
6) Tetrathionate Brilliant Green Broth (TBG)
7) Selenite Cystine Broth (SC) (optional)
8) Rappaport-Vassiliadis Soya Peptone Broth (RVS)
NOTE:
a. USE OF SELENITE CYSTINE BROTH: Due to safety and environmental issues, SC broth should be replaced with RVS broth in all methods used for the detection of Salmonella in foods, food ingredients and environmental samples, under most circumstances. SC broth must be used (using the conditions described below) when analysing guar gum and when the presence of Salmonella Typhi is suspected, such as in outbreaks or for imports from tropical countries where S. Typhi is prevalent. In general, it is recommended that SC be used in the investigation of food borne outbreaks.
b. HANDLING OF SELENITE CYSTINE BROTH AND PRODUCTS CONTAINING SC: Selenium salts can be teratogenic and should be weighed in a safety cabinet. All transfer and streaking of products containing SC MUST be done using sterile disposable materials and in a fume hood or a biohazard hood with appropriate ventilation.
c. DISPOSAL OF SELENITE CYSTINE (SC) BROTH AND PRODUCTS CONTAINING SC: All products containing SC MUST be disposed of through a waste management facility. This includes ALL dry product and reconstituted product. Any product that has been contaminated must be autoclaved to sterilization (using a properly ventilated system) prior to disposal.
UNDER NO CIRCUMSTANCES IS ANY SC PRODUCT TO BE DISPOSED OF THROUGH THE DRAINAGE OR SEWER SYSTEM.
9) Selective and Differential Agars:
a. Group 1 Agars (this group of agars has been studied extensively) Must use at least two of these agars:
Bismuth Sulfite (BS) Agar (BD)
Brilliant Green Sulfa (BGS) Agar (BD)
Brilliance™ Salmonella Agar (formerly know as OSCM2 - Oxoid)
b. Group 2 Agars (must be used in conjunction with Group 1 Agars)
Hektoen Enteric Agar
Xylose Lysine Deoxycholate Agar
Rambach Agar
CHROMagar (BD)
RapidSalmonella Agar (Bio-Rad)
Any others not listed above.
10) MacConkey Agar
11) Nutrient Agar
12) Triple Sugar Iron Agar (TSI)
13) Lysine Iron Agar (LIA)
14) Urea Agar (Christensen's)
15) Commercial biochemical test kits and rapid identification kits (such as Vitek, API, or equivalent)
16) Polyvalent somatic antisera or latex agglutination kits
17) Monovalent somatic (O) and flagellar (H) antisera (optional)
18) Physiological saline
19) Blender, stomacher or other homogenizing device
20) Incubators capable of maintaining 35°C and 42.5°C. Where appropriate, waterbaths may be substituted for incubators.
Note: It is the responsibility of each laboratory to ensure that incubators and waterbaths are maintained at the recommended temperatures. Where 35°C is recommended in the text of the method, the incubators and waterbaths may be at 35 +/- 1.0° C. Similarly, lower temperatures of 30 or 25 may be +/- 1.0°C. However, where higher temperatures are recommended, such as 43 or 45.5°C, it is imperative that the incubators and waterbaths be maintained within 0.5°C due to potential lethality of higher temperatures on the microorganism being isolated.
21) Controls. Where available, use microorganisms from a reliable source such as ATCC.
1. Positive controls:
b) atypical Salmonella (optional): Lactose- and/or sucrose-fermenting Salmonella strains etc.
2. Negative controls: use a non-Salmonella culture such as Pseudomonas or Escherichia
22) O.B.I.S. (Oxoid; optional)
6. PROCEDURE
Each sample unit may be analysed individually or the analytical units taken from each sample unit may be combined as appropriate.
The two types of controls (positive and negative) are to be carried through each step of the analysis.
6.1 Handling of Sample Units
6.1.1 Prior to analysis, except for shelf-stable foods, keep sample units refrigerated (2-8° C) or frozen, depending on how the product was received. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.
6.1.2 Analyse sample units as soon as possible after their receipt in the laboratory.
Note: Large samples (e.g., whole turkey) may not readily thaw at refrigerator temperatures. For greater expediency, enclose the frozen, packaged sample in a heavy-walled paper bag and thaw overnight at room temperature. This technique maintains a cold product surface during the thawing process.
6.1.3 During consumer complaint and other investigations, the required sample size may not be available. If individual sample units received for analysis are less than the recommended analytical unit weight, analyse the entire amount and record the weight used.
6.2 Non-selective Enrichment (Pre-enrichment)
6.2.1 Compositing of Analytical Units
From each sample unit, an analytical unit (usually 25 g or mL) is taken. To reduce the workload, up to 15 analytical units may be composited into a single test sample (e.g. 375 g or mL).
If an individual sample unit consists of more than one container, aseptically combine and mix the contents of the containers prior to withdrawal of the analytical unit. If this is not possible or practical, the analytical unit shall then consist of equal portions from each of the containers.
Note: For analysis of sprouts, follow directions given in Appendix A of this method before proceeding with sample analysis.
6.2.2 Sample Analysis
6.2.2.1 The required analytical unit (tested individually or composited as appropriate) is dispersed into a suitable non-selective enrichment broth (Table I). Nutrient broth (NB) and buffered peptone water (BPW) are equally reliable and for those commodities not specifying another broth, can be used interchangeably as general purpose pre-enrichment media (7.9). Stomach, or blend if applicable (see Table I).
Note: When analysing composites, pre-warm the enrichment broth to approximately 35°C.
6.2.2.2 If the pH of the pre-enrichment mixture lies outside the range of 6.0 to 7.0, adjust with 1N NaOH or 1N HCl.
Note: If a sample unit consists of a container with little food material, thoroughly rinse the interior of the container with a suitable amount of pre-enrichment broth medium. Incubate the rinse in a suitable sterile container, such as a flask or stomacher bag.
6.2.2.3 Incubate the pre-enrichment mixture and controls at 35oC for 18 to 24 h.
6.2.3 Refrigeration of incubated pre-enrichment broths (dry foods) - OPTIONAL
Note: The refrigeration of the pre-enrichment and enrichment broths has been validated for low moisture dry foods (e.g., whole egg powder, milk chocolate, animal feed and instant skim milk powder) only. This step must be validated for other foods. Once validation is completed, please forward the data to Chair, Microbiological Methods Committee so this step can be extended to other foods.
6.2.3.1 This approach allows for the refrigeration of incubated pre-enrichment broth of low moisture dry foods (e.g., whole egg powder, milk chocolate, animal feed and instant skim milk powder) for 72 h, thereby providing increased flexibility (7.7, 7.8, 7.10, 7.11).
6.2.3.2 Incubated pre-enrichment broths from sample analyses initiated the preceding day may be refrigerated (2 to 8°) for up to 72 hours until analysis can proceed (e.g., after a weekend).
6.3 Selective Enrichment
6.3.1 Vortex or resuspend the pre-enrichment broths. Transfer 1.0 mL of the incubated pre-enrichment broth into 9 mL of Tetrathionate Brilliant Green (TBG) broth and 0.1 mL into 9 mL of Rappaport-Vassiliadis Soya Peptone (RVS) broth. If substituting SC for RVS broth, transfer 1.0 mL of the pre-enrichment broth into 9 mL of SC broth.
Note: If using SC broth, see required precautions in Section 5.
6.3.2 Incubate RVS and TBG broths at 42.5 °C for 24 ± 2 h. If using SC broth, incubate this broth for 24 ± 2 h at 35 °C.
6.3.3 Refrigeration of incubated enrichment broth (dry foods) - OPTIONAL
6.3.3.1 This approach is complementary to that described in Section 6.2.3, and provides greater flexibility.
6.3.3.2 Selective enrichment broths can be refrigerated (2 to 8 °C) for up to 72 hours (e.g., over a weekend).
6.4 Selective Plating
6.4.1 Vortex or resuspend the broths. Streak each selective enrichment broth onto selective agars to obtain well isolated colonies. Choose at least two media from the Group 1 Agars (Section 5). Other media from the Group 2 Agars may be used but only in conjunction with the Group 1 Agars.
Note: If using SC broth, see required precautions in Section 5. Transfers must be done in an appropriate safety cabinet.
6.4.2 Incubate plates at 35 °C for 24± 2 h. If colonies suggestive of Salmonella have not developed on BS plates, incubate for an additional 24 ± 2 h.
6.4.3 Examine incubated plates for colonies suggestive of Salmonella. Typical Salmonella usually occur as pink to fuchsia colonies surrounded by red medium on BGS agar, and as black colonies on BS agar with or without a metallic sheen, and showing a gradual H2S-dependent blackening of the surrounding medium with increasing incubation time. Atypical Salmonella may show poor growth on one or both of the media used, and may appear differently than described. On Brilliance Salmonella Agar typical Salmonella are bright purple while most other enterics may be blue or colourless. Follow manufacturer’s instructions on the use of Group 2 agars as well as identifying presumptive Salmonella.
Note:
a. Lactose-and/or sucrose-fermenting Salmonella strains develop a coliform-like (greenish) appearance on BGS agar. Other atypical Salmonella strains may appear green or brown on BS agar (e.g., S. Enteritidis), while others not typically found in food may grow poorly or not at all on one or both media used. All strains of Salmonella, including lactose- and/or sucrose-fermenting strains, are bright purple on Brilliance Salmonella Agar.
b. A heavy growth of non-Salmonella may mask the presence of Salmonella.
c. BS agar can retard the growth of Salmonella serovars other than S. Typhi unless poured plates are refrigerated ( 2 to 8°C) for 24 h prior to streaking (7.3). Freshly poured plates may be used if they are incubated for at least 48 h.
6.4.4 Where the suspect colonies are well isolated and large enough, 1 to 3 colonies per agar type (or more if needed to identify different Salmonella serotypes etc.) should be carried forward through biochemical testing. Simultaneously, purify isolates as in Section 6.5.
Where colonies are not well isolated or are small (possibly a slow growing serotype), streak suspect colonies onto MacConkey agar for purity as in Section 6.5, and then continue.
6.4.5 The absence of suspect colonies (either typical or atypical in appearance) on the plates indicates that the analytical unit or composite test sample did not contain Salmonella spp. capable of growth on the media used.
6.5 Purification
6.5.1 Streak suspect colonies onto MacConkey agar for purification.
6.5.2 Incubate plates at 35oC for 24 ± 2 h.
6.5.3 Typical Salmonella colonies are lactose-negative and will appear as colourless colonies on this medium. However, lactose-positive biotypes will occur as pink colonies.
6.6 Biochemical Screening
6.6.1 With a sterile needle, inoculate suspect colonies into the biochemical media listed in Table II. Incubate the biochemical media for 18 to 24 h at 35oC. Cap tubes loosely.
Note: Erroneous biochemical results may result if tubes are not loosely capped during incubation.
6.6.2 Alternatively, commercially available diagnostic kits may be used to obtain more detailed biochemical profiles of bacterial isolates.
6.6.3 If none of the isolates are suggestive of Salmonella, the analytical unit or sample (if compositing) is considered to be free of Salmonella. If the presence of Salmonella is suspected, proceed with serological testing.
6.6.4 If serological testing is not to be performed within 72 h, inoculate suspect isolates onto nutrient agar slants and incubate at 35oC for 24 ± 2 h.
6.6.5 Other Biochemical Tests (Optional)
Other biochemical tests and kits (e.g., O.B.I.S.). May be used in conjunction with those listed above.
6.7 Serological Identification
6.7.1 Testing with Somatic (O) Polyvalent Antiserum or Latex Agglutination Kits
Follow manufacturer’s instructions using inoculum that is less than 72 hours old.
Note: Non-agglutinating cultures possessing the biochemical reactions suggestive of Salmonella should be sent to a reference typing centre for identification.
6.7.2 Testing with Somatic (O) Grouping Antisera (Optional)
It is sometimes advantageous to test presumptive Salmonella cultures with somatic grouping antisera. Many foodborne Salmonella belong to somatic groups B, C, D, or E. Nevertheless, it is important to recognize that unless a complete set of grouping antisera is available, Salmonella belonging to uncommon serogroups may be missed.
Follow manufacturer’s instructions.
6.7.3 Testing with Flagellar (H) Antisera (Optional)
In instances where the services of a reference typing centre are not available, Salmonella isolates should be further identified by testing with polyvalent H antiserum according to the method described in “Microorganisms in Food” (7.12).
7. REFERENCES
7.1 Andrews, W.H. 1989. Methods for recovering injured "classical" enteric pathogenic bacteria (Salmonella, Shigella, and enteropathogenic Escherichia coli) from foods. Chapter 3. In: B. Ray (ed.) Injured, Index and Pathogenic Bacteria, CRC Press, Boca Raton, FL. pp. 55-113.
7.2 Association of Official Analytical Chemists (AOAC) International. 1995. FDA Bacteriological Analytical Manual, Eighth Edition. AOAC International, Arlington, VA.
7.3 D'Aoust, J.-Y. 1977. Effect of storage conditions on the performance of bismuth sulfite agar. J. Clin. Microbiol. 5:122-124.
7.4 D'Aoust, J.-Y., 1981. Update on preenrichment and selective enrichment conditions for detection of Salmonella in foods. J. Food Prot. 44:369-374.
7.5 D'Aoust, J.-Y. 1984. Effective enrichment-plating conditions for detection of Salmonella in foods. J. Food Prot. 47:588-590.
7.6 D'Aoust, J.-Y. 1989. Salmonella. Chapter 9. In: M.P. Doyle (ed.). Foodborne Bacterial Pathogens, Marcel Dekker Inc., New York, NY. pp. 327-445.
7.7 D'Aoust, J.-Y., C. Maishment, D.M. Burgener, D.R. Conley, A. Loit, M. Milling and U. Purvis. 1980. Detection of Salmonella in refrigerated preenrichment and enrichment broth cultures. J. Food Prot. 43:343-345.
7.8 D'Aoust, J.-Y., H.J. Beckers, M. Boothroyd, A. Mates, C.R. McKee, A.B. Moran, P. Sado, G.E. Spain, W.H. Sperber, P. Vassiliadis, D.E. Wagner, and C. Wiberg. 1983. ICMSF Methods Studies. XIV. Comparative study on recovery of Salmonella from refrigerated preenrichment and enrichment broth cultures. J. Food Prot. 46:391-399.
7.9 D'Aoust, J.-Y., A.M. Sewell and D.W. Warburton. 1992. A comparison of standard cultural methods for the detection of foodborne Salmonella. Int. J. Food Microbiol. 16: 41-50.
7.10 D'Aoust, J.-Y., A.M. Sewell and P. Greco. 1993. Detection of Salmonella in dry foods using refrigerated preenrichment and enrichment broth cultures: interlaboratory study. J. of AOAC Int. 76:814-821.
7.11 D'Aoust. J.-Y., A.M. Sewell and C. McDonald. 1995. Recovery of Salmonella spp. from refrigerated preenrichment cultures of dry food composites. J. AOAC Int. 78: 1322-1324.
7.12 International Commission on Microbiological Specifications for Foods (ICMSF). 1978. Microorganisms in Foods 1. Their significance and methods of enumeration. Second edition. University of Toronto Press, Toronto, ON.
7.13 ICMSF. 1986. Microorganisms in Foods 2. Sampling for microbiological analysis: Principles and specific applications. Second edition. University of Toronto Press, Toronto, ON.
7.14 ICMSF. 2005. Microorganisms in Foods. Volume 6. Microbial Ecology of Food Commodities. Second edition. Kluwer Academic/Plenum Publishers. New York.
7.15 Health Canada. 2008. Health Products and Food Branch Standards (HPFB) and Guidelines for Microbiological Safety of Food - Interpretive Summary. In: Volume 1. The Compendium of Analytical Methods. Ottawa. http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index_e.html
7.16 ICMSF. 2002. Microorganisms in Foods. Volume 7. Microbiological Testing in Food Safety Management. Kluwer Academic/Plenum Publishers. New York.
7.17 Ponka, A., Y. Andersson, A. Siitonen, B. de Jong, B, M. Jahkola, O. Haikala, A. Kuhmonen, and P. Pakkala. 1995. Salmonella in alfalfa sprouts. Lancet-British-Edition. 345(8947): 462-463.
7.18 Gill, C. J., W. E. Keene, J. C. Mohle Boetani, J. A. Farrar, P. L. Waller, C. G. Hahn, and P. R. Cieslak. 2003. Alfalfa seed decontamination in a Salmonella outbreak. Emerging Infectious-Diseases. 9(4):474-479.
7.19 Liao, C.-H., and W. F. Fett. 2003. Isolation of Salmonella from Alfalfa Seed and demonstration of Impaired Growth of Heat-Injured Cells in Seed Homogenates. Int. J. Food Microbiol. 82:245-253.
7.20 Inami, G. B., S. M. C. Lee, R. W. Hogue, and R. A. Brenden. 2001. Two Processing Methods for the Isolation of Salmonella from Naturally Contaminated Alfalfa seeds. J. Food Prot. 64(8):1240-1243.
7:21 Dong, Y., A. L. Iniguez, B. M. M. Ahmer, and E. W. Triplett. 2003. Kinetics and Strain Specificity of Rhizophere and Endophytic Colonization by Enteric Bacteria on Seedlings of Medicago sativa and Medicago truncatula. Appl. Environ. Microbiol. 69(3):1783-1790.
7.22 Jaquette, C. B., L. R. Beuchat, and B. E. Mahon. 1996. Efficacy of Chlorine and Heat Treatment in Killing Salmonella stanley Inoculated onto Alfalfa Seeds and Growth and Survival of the Pathogen During Sprouting and Storage. 62(6):2212-2215.
7.23 Monteville, R., and D. Schaffner. 2005. Monte Carlo Stimulation of Pathogen Behavior During the Sprout Production Process. Appl. Environ. Microbiol. 71(2):746-753.
7.24 Gandhi, M., S. Golding, S. Yaron, and K. R. Matthews. 2001. Use of Green Fluorescent Protein Expressing Salmonella Stanley to Investigate Survival, Spatial Location, and Control on Alfalfa Sprouts. J. Food Prot. 64(12):1891-1898.
7.25 International Organization for Standardization (ISO). 2002. ISO 6579:2002. Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for the Detection of Salmonella, International Organization for Standardization, Geneva, ISO Central secretariat. CP 56 CH-1211. Geneva 20. Switzerland.
7.26 ISO. 2002. ISO 6579:2002/Amd 1:2007. Annex D: Microbiology of food and animal feeding stuffs - Detection of Salmonella spp. in animal faeces and in environmental samples from the primary production stage. International Organization for Standardization. Geneva . ISO Central secretariat. CP 56 CH-1211. Geneva 20. Switzerland
7.27 ISO. 1993. ISO 6579:1993. Microbiology - General Guidance on Methods for the Detection of Salmonella, International Organization for Standardization, Geneva, Geneva, ISO Central secretariat. CP 56 CH-1211. Geneva 20. Switzerland.
7.28 ISO. 2001. ISO 6785:2001. Microbiology of Food and Animal Feeding Stuffs, Milk and milk products -- Detection of Salmonella sp. International Organization for Standardization, Geneva, Geneva, ISO Central secretariat. CP 56 CH-1211. Geneva 20. Switzerland.
7.29 ISO. 1995. ISO 6340:1995. Water quality - Detection and enumeration of Salmonella. International Organization for Standardization, Geneva, Geneva, ISO Central secretariat. CP 56 CH-1211. Geneva 20. Switzerland.
7.30 Warburton, D.W., B. Bowen, A. Konkle, C. Crawford, S. Durzi, R. Foster, C. Fox, L. Gour, G. Krohn, et al. 1994. A comparison of six different plating media used in the isolation of Salmonella. Int. J. Food Microbiol. 22:277-289.
Appendix A
PROCEDURE FOR THE ANALYSIS OF SEED USED TO MANUFACTURE SPROUTS
Analysis of Alfalfa and other Seeds
Experience in the laboratory has shown that the probability of recovery of Salmonella from dry intact seeds, such as alfalfa, radish, clover, mustard, etc., is very low when the standard culture method is used (CFIA and HC unpublished data, 7.17, 7.20). Research also indicates that the pathogens are dispersed heterogeneously, at very low levels (0.3 to 0.9 cells per 25 g), have been severely injured during seed production or storage, and often reside within the seed itself (7.18, 7.19, 7.21).
The probability of recovery may be greatly improved by first sprouting the seeds under sterile conditions, as sprouting acts as a highly effective enrichment step (CFIA and HC, unpublished data, 7.20, 7.22, 7.23, 7.24).
Procedure for Germination of Seeds
Aseptically weigh 125 g of seeds into a sterile container and add sterile distilled water until the level reaches approximately 1 inch above the seeds. Aseptically cover and incubate at 30o C for 3 days.
The first addition of water will be absorbed after approximately 2 hours. Add more water to restore the level. Continue monitoring the water level for the remainder of the incubation period, adding more water as necessary.
After 3 days of incubation the seed coats should have split and some degree of germination should be evident. Some seeds take longer than others to fully sprout, but 3 days should be adequate for most. Aseptically weigh out 125 g (or recommended sample size which may vary with the type of sample, e.g., investigational or monitoring) of the germinated "slurry" (i.e., mixture of germinated seeds and water) and add to 1125 mL nutrient broth for overnight incubation at 35°C for 18 to 24 h. Proceed with MFHPB-20 at Section 6.3.
TABLE I. Procedures for non-selective enrichment (pre-enrichment)
| Type of Product | Number of Sample Units | Preparation of the analytical unit from each sample unit a |
|---|---|---|
|
Alimentary Paste e.g., pasta and noodles etc.
|
5 |
Suspend 25 g in 225 mL of NB or BPW and blend. Dry blending at reduced speed can also be used to obtain a fine particulate test material. |
| Coconut | 5 | Suspend 25 g in 225 mL of NB or BPW and blend. |
| Confectionery | ||
| a. Chocolate and cocoa | 10 | Suspend 25 g in 225 mL of skim milk medium and blend. |
| b. Candy | 5 | Suspend 25 g in 225 mL of skim milk medium and blend. |
| Dairy Products | ||
| a. Cheese | 5 | Suspend 25 g in 225 mL of NB or BPW and blend. |
| b. Fluid milk | 5 | Add 25 mL to 225 mL of brilliant green water. |
| c. Ice cream | 5 | Suspend 25 g in 225 mL of brilliant green water. |
| d. Powdered products e.g., whey and buttermilk etc. |
5 | Gently add 25 g to 225 mL of sterile brilliant green water and allow to soak undisturbed. Do not mix. |
| e. Skim milk powder | 20 | Gently add 25 g to 225 mL of sterile brilliant green water and allow to soak undisturbed. Do not mix. |
| Liquid Egg Products | 10 | Suspend 25 g in 225 mL NB or BPW and blend. |
| Frog Legs | 5 | Place 25 g in 225 mL NB or BPW. |
| Meats | 5 | Suspend 25 g in 225 mL NB or BPW and blend. |
| Poultry | ||
| a. Raw (Whole bird or cut up with packages > 100 g) | 5 | Place thawed or fresh bird, drippings and giblets (if present) into a heavy sterile plastic bag. Add 1 L NB or BPW and shake vigorously so that all sample surfaces come into contact with the broth medium and/or massage the poultry in the bag. Pour off the broth into a sterile container for incubation. |
| b. Giblets (any size package) and raw pieces (< 100 g packages) | 5 | Suspend 25 g in 225 mL NB or BPW and blend. |
| c. Cooked | 5 | Suspend 25 g in 225 mL NB or BPW and blend. |
| Prepared Foods e.g., meat pies and TV dinners |
5 | Combine 25 g of each food component (if applicable) into a single analytical unit; add nine volumes of NB or BPW and blend. |
| Spices and Seasonings | 10 | Suspend 25 g in 225 mL NB or BPW and mix thoroughly. 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 III). With onions and garlic, use trypticase (tryptic) soy broth containing 0.5% (w/v) K2SO3. |
| Seeds used to manufacture sprouts | 5 | See Appendix A. |
| Yeast | 5 | Suspend 25 g in 225 mL NB or BPW and mix thoroughly. |
| Foods not listed above | 5 | Suspend 25 g in 225 mL NB or BPW and blend or mix as appropriate. |
a Where consumer size package is less than 100 g (mL), sample unit will consist of more than one package.
| 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. |
Negative (some serovars can utilize one or both substrates). |
| Dextrose utilization | Positive reaction: Butt turns yellow with or without gas pockets Negative reaction: Colour of butt unchanged |
Positive | |
| H2S production | Positive reaction: Blackening of butt and/or slant Negative reaction: No blackening |
Positive (slow H2S producers may be encountered. The H2S reaction may be inhibited in lactose and/or sucrose-utilizing serovars. Some serovars do not produce H2S) | |
| Gas formation | Positive reaction: Gas pockets in the medium Negative reaction: No gas pockets |
Positive (some serovars are anaerogoenic) | |
| Lysine Iron Agar (LIA) | H2S production | Positive reaction: Blackening of butt and/or slant Negative reaction: No blackening |
Positive (some serovars do not produce H2S) |
| Lysine decarboxylase | Positive reaction: Butt remains purple Negative reaction: Butt turns yellow |
Positive (some serovars are negative for lysine decarboxylase) | |
| 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 |
TABLE III. Ratio of spices and seasonings to preenrichment broth (w:v) a
Allspice powder
Anise seed
Arrowroot powder
Basil leaves
Bay leaves
Caraway seed
Caradamon seed
Cassia buds
Cassia powder
Cayenne pepper
Celery flakes
Celery seed
Chervil leaves
Chilies whole
Chives chopped
Cinnamon sticks
Cloves
Corriander seed
Cumin seed
Curry powder
Dill seed
Fennel seed
Ginger root
Italian seasoning
Lemon peel
Lemon pepper seasoning
1:20 and 1:100
1:10
1:10
1:20
1:20
1:10
1:10
1:10
1:50
1:10
1:20
1:10
1:20
1:10
1:20
1:10 and 1:100
1:50 and 1:1000
1:10
1:10
1:10
1:10
1:10
1:10
1:20
1:10
1:50
Marjoram
Mint flakes
Mint ground
Mushroom slices
Mustard seeds
Nutmeg whole
Onion powder
Orange bits
Oregano leaves
Paprika
Parsley flakes
Peppercorns
Pepper black
Pepper white
Poppy seed
Pumpkin pie seasoning
Rosemary leaves
Sesame seed
Salad herbs
Salad supreme
Sage leaves
Savory ground
Saffron
Tarragon
Tartar (cream of)
Thyme powder
Turmeric powder
Vegetable flakes
1:10
1:20
1:10
1:20
1:10
1:10
1:10
1:10
1:50
1:10
1:20
1:10
1:10
1:10
1:10
1:50
1:10
1:10
1:20
1:10
1:20
1:50
1:20
1:20
1:10
1:50
1:10
1:20
a For spices and seasonings only: The ratio is given in weight to volume (w:v), so that 1:10 means for every 1 g of spice 10 mL of enrichment broth is added. Therefore, for a 25 g sample unit, 250 mL of enrichment broth is added.
