Isolation and Identification of Salmonella from Foods

Proposed Official Method MFO-21
July 2002

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1. APPLICATION

This method is applicable to the detection of viable Salmonella food in cocoa, chocolate, milk powders and other dairy product powders, froglegs, liquid eggs and other egg products to determine compliance with the requirements of Sections B.04.012, B.08.011, B.21.031, and B.22.033 of the Regulations of the Food and Drugs Act. This revised method replaces MFOs -6, -10, -11, and -12, all dated November 30, 1981.

2. DESCRIPTION

2.1 This revised method includes the following major changes in the analytical approach for the detection of foodborne Salmonella spp.:

1. Use of the soak method for the preenrichment of skim milk and other powdered dairy products (8.1, 8.2).
2. Optional use of refrigerated preenrichment and enrichment cultures of specific dry foods (cocoa, chocolate, milk powder and dried egg products) for increased laboratory productivity and flexibility (8.8, 8.9, 8.11, 8.12).
3. Inclusion of buffered peptone water (BPW) as an equivalent to nutrient broth for the non-selective enrichment of foods (8.10).
4. Revision of the section 7.5 "Purification".

2.2 Equivalent Methods

The methods MFHPB-20 and MFHPB-24 are considered to be equivalent to the method presented here, can be used to determine the presence of Salmonella and to determine compliance with the Regulations of the Food and Drugs Act listed above and in Table I of this method. These methods are found in Volume 2 of the Compendium of Analytical Methods.

3. PRINCIPLE

The procedure consists of five to six distinct stages. The initial handling of the food and the non-selective enrichment stage (preenrichment) vary according to the type of food examined.

3.1 Non-Selective Enrichment (Pre-enrichment).

The test sample is initially inoculated into a non-inhibitory liquid medium to favour the repair and growth of stressed or sublethally-injured salmonellae arising from exposure to heat, freezing, desiccation, preservatives, high osmotic pressure or wide temperature fluctuations (8.1; 8.7).

3.2 Selective Enrichment

Replicate portions of each preenrichment culture are inoculated into two enrichment media to favour the proliferation of salmonellae through a selective repression or inhibition of the growth of competing microorganisms (8.5).

3.3 Selective Plating

Each enrichment culture is streaked onto a minimum of 2 selective agars for the isolation of salmonellae (8.6).

3.4 Purification

If suspect Salmonella colonies are not well isolated, they are purified on MacConkey or selective agar plates.

3.5 Biochemical Screening

Isolates are screened using determinant biochemical reactions.

3.6 Serological Identification

Polyvalent and/or single grouping somatic antisera are used to support the tentative identification of isolates as members of Salmonella spp. For confirmation, and if serology is not possible, cultures should be sent to a reference typing centre for complete serotyping.

4. DEFINITION OF TERMS

See Appendix A of Volume 1.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 1.

6. MATERIALS AND SPECIAL EQUIPMENT

The following media (1 to 15) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 1 and reference 8.3 for the formula of individual media.

1. Nutrient broth (NB) or Buffered peptone water (BPW)
2. Trypticase (Tryptic, Tryptone) soy broth
3. Brilliant green water
4. Skim milk medium
5. Tetrathionate brilliant green broth (TBG)
6. Selenite cystine broth (SC)
7. Bismuth sulfite agar (BS)
8. Brilliant green sulfa agar (BGS)
9. MacConkey agar
10. Nutrient agar slants or plates (NA) or equivalent
11. Triple sugar iron agar (TSI)
12. Lysine iron agar (LIA)
13. Urea agar (Christensen's)
14. Sterile physiological saline
15. Sterile 1N NaOH, 1N HCl
16. Positive control cultures: Salmonella (use ATCC cultures or equivalent)
17. Commercial biochemical test kits (optional)
18. Polyvalent somatic (O) antisera; Optional: single grouping somatic (O) antisera; flagellar (H) antisera
19. Blender, stomacher or equivalent
20. pH meter or paper capable of distinguishing between 0.3 to 0.5 pH units within the range of pH 5.0 to 8.0
21. Incubators or waterbaths capable of maintaining 35°C and 42.5°C

Note: It is the responsibility of each laboratory to ensure that the temperature of the incubators or waterbaths are maintained at the recommended temperatures. Where 35°C is recommended in the text of the method, the incubator or waterbath may be 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 or waterbaths be maintained within 0.5°C due to potential lethality of the higher temperatures on the microorganism(s) being isolated.

7. PROCEDURE

7.1 Handling of Sample Units

7.1.1 Analyze samples as soon as possible. If necessary, store samples under time and temperature conditions that will prevent the growth or death of native microflora. If sample units have been abused in transit, re-sampling of the lot should be carried out.

1. Frozen Foods: Sample units that show no signs of thawing upon receipt may be stored in the freezer at -10°C to -20°C.
2. Dried and shelf stable foods may be stored at room temperature.
3. 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 samples at room temperature within 60 min; if this is not possible, thaw the samples at refrigerator (4 to 10°C) temperature. Enrich thawed samples as soon as possible after thawing to avoid potential overgrowth by psychrotrophic organisms which could mask growth of Salmonella if present in low numbers.

7.1.3 If the sample unit received for analysis is less than the recommended analytical unit, analyze the entire amount and adjust the volume of non-selective enrichment broth required to maintain a 1:10 dilution. Record both amounts.

7.1.4 Blending or stomaching of samples should be limited to the minimum time required to produce a homogeneous suspension. Excessive mixing could result in physical damage to endogenous microflora and adversely affect their viability.

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. Containment during the handling 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 test sample (25 g) is dispersed into a suitable non-selective enrichment broth (Table II). Nutrient broth (NB) and buffered peptone water (BPW) are equally reliable and can be used interchangeably as general purpose preenrichment media (8.10).

7.2.2.2 If the pH of the preenrichment mixture lies outside the range of 6.0 - 7.0, adjust with sterile 1N NaOH or 1N HCl.

7.2.2.3 A Salmonella culture positive control 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 after incubation would invalidate test results.

7.2.3 Optional:

Refrigeration of preenrichment cultures (dry foods: cocoa, chocolate, milk powder, dried egg products)

7.2.3.1 This novel approach involves the refrigeration of preenrichment cultures of low moisture foods for 72 h, thereby providing for increased laboratory productivity and analytical flexibility (8.8; 8.9; 8.11; 8.12). More specifically, weekend (72 h) refrigeration of preenrichment and enrichment (7.3.3) allows initiation of sample analyses on Mondays to Thursdays, inclusively, eliminating weekend work when practical.

7.2.3.2 Preenrichment cultures arising on Friday from sample analyses initiated the preceding day are refrigerated (4 to 10°C) over the weekend.

7.2.3.3 On the following Monday, the content of each refrigerated preenrichment culture is resuspended, and replicate portions (1 mL) inoculated into TBG42.5 and SC35.

7.2.3.4 Proceed as described in 7.3 to 7.7.

7.3 Selective Enrichment

7.3.1 With a sterile pipette, transfer 1.0 mL of the preenrichment culture into each of 9 mL of selenite cystine (SC) and tetrathionate brilliant green (TBG) broths.

7.3.2 Incubate SC and TBG broths for 24 ± 2 h at 35°C and 42.5°C, respectively.

Note: If screw-capped tubes are used, ensure tubes are loosely capped before incubation.

7.3.3 Optional:

Refrigeration of selective enrichment cultures (dry foods: cocoa, chocolate, milk powder, dried egg products)

7.3.3.1 This novel approach is complementary to that described in 7.2.3, and provides for greater laboratory productivity and analytical flexibility.

7.3.3.2 TBG42.5 and SC35 cultures arising on Friday from sample analysis initiated on the preceding Wednesday, are refrigerated (4 to 10°C) over the weekend.

7.3.3.3 On the following Monday, the contents of the refrigerated TBG42.5 and SC35 cultures are resuspended, and replicate loopsful from each culture are plated onto bismuth sulfite (BS) and brilliant green sulfa (BGS) agar media.

7.3.3.4 Proceed as described in 7.4 to 7.7.

7.4 Selective Plating

7.4.1 Streak replicate 10 L loopsful of each selective enrichment culture onto BS and BGS agars to obtain well isolated colonies. The enrichment cultures may also be streaked onto additional plating media for the isolation of Salmonella.

7.4.2 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.

7.4.3 Examine incubated plates for suspect Salmonella colonies. 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.

Note:

1. Lactose-and/or sucrose-fermenting Salmonella strains develop a coliform-like (greenish) appearance on BGS agar. A heavy growth of non-salmonellae may also mask the presence of Salmonella on this medium.
2. BS agar can retard the growth of Salmonella serovars, other than S. typhi , unless poured plates are refrigerated (4 to 10°C) for 24 h prior to streaking (8.4).

7.4.4 If suspect colonies are absent on the plating media, the analytical test unit is considered to be negative for Salmonella spp.

7.5 Purification

If suspect colonies are not well isolated, proceed with steps 7.5.1 to 7.5.4. If colonies on the selective plates appear well isolated proceed to Sec. 7.6.

7.5.1 Streak a minimum of 2 colonies from each presumptively-positive plate onto MacConkey agar or onto a selective agar for purification.

7.5.2 Incubate plates at 35°C for 24 ± 2 h.

7.5.3 Typical Salmonella colonies are lactose-negative and will appear as colourless colonies on MacConkey agar. However, lactose-positive biotypes will occur as pink colonies.

7.5.4 Proceed to 7.6.

7.6 Biochemical Screening

7.6.1 Using a sterile needle, pick isolated colonies from the MacConkey plates (7.5) or a minimum of 2 well isolated typical colonies from the selective agars. Using the same inoculum, inoculate suspect colonies into triple sugar iron agar (TSI) and lysine iron agar (LIA) by stabbing the butt and streaking the slant and Christensen's urea agar by inoculating the entire surface of the slant. These biochemicals and biochemical reactions are listed in Table III. Commercial diagnostic kits that yield equivalent results may also be used.

7.6.2 To ensure the purity of colonies which were transferred to biochemistry directly from the selective agars, inoculate a MacConkey plate for isolated colonies with a portion of each colony, preferably going from the biochemicals directly onto the plate.

7.6.3 If commercial kits are used, inoculate a portion of each colony onto a nutrient agar (NA) slant or equivalent for use in serological determination (7.7).

7.6.4 Incubate biochemicals and plates at 35°C for 18-24 h.

Note: Erroneous biochemical results may be obtained if tubes are not loosely capped during incubation.

7.6.5 If none of the isolates from a particular analytical unit are typical of Salmonella, the analytical unit is considered to be free of salmonellae.

Note: When interpreting the TSI and LIA reactions, attention must be paid to the possibility of atypical reactions. These include H2S and lysine negative, sucrose and lactose positive. If an atypical Salmonella is suspected, perform polyvalent and somatic (if possible) serology, as well as more detailed biochemistry.

7.6.6 If the presence of Salmonella is suspected, proceed with serological testing using growth from the TSI or LIA or NA slants.

7.6.7 If serological testing is not to be performed within 72 h, inoculate suspect isolates onto nutrient agar slants and incubate at 35°C for 24 ± 2 h.

7.6.8 Store the agar slants at refrigerator (4 to 10°C) temperature.

7.6.9 Slants that have been stored for more than 72 h should not be used for serological testing. Prepare fresh agar slants for this purpose.

7.7 Serological Identification

7.7.1 Testing with somatic polyvalent antisera

7.7.1.1 Mark the following areas on an agglutination plate: C+ (positive control), C- (negative control) and T (test culture).

7.7.1.2 Prepare somatic polyvalent antisera as directed by the manufacturer; add one drop to each of the areas marked T and C+; add one drop of physiological saline to the area marked C-.

7.7.1.3 Remove sufficient culture material from a triple sugar iron, lysine iron or nutrient agar slant to prepare a moderately dense suspension in the test area (T) and in the negative control (C-) area. The inoculum should be withdrawn from the slope portion of agar slants.

7.7.1.4 For the positive control, prepare a similar suspension of a known Salmonella culture in the area marked C+.

7.7.1.5 Mix the culture-antiserum suspensions in T and C+ and the saline-culture mixture in C- with a sterile needle or loop. To avoid cross-contamination, use a separate (or re-sterilized) sterile loop or needle for each suspension. Tilt the slide preparation back and forth for 1 min.

7.7.1.6 Hold the slide under or against a well illuminated dark background and observe for agglutination. Salmonella cultures usually agglutinate within 1 min.

7.7.1.7 False positive reactions from microorganisms that are closely related to Salmonella may occur. Such misleading reactions can usually be resolved through further testing with somatic grouping and flagellar antisera. Additional biochemistry may also be required.

7.7.1.8 The serological test for a given culture is invalidated if the negative control shows agglutination (autoagglutination).

7.7.2 Testing with Somatic Grouping Antisera

It is advantageous to test presumptive Salmonella cultures with somatic grouping antisera whenever possible. 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.

Note: It should be stressed that any non-agglutinating culture possessing the biochemical reactions suggestive of Salmonella should be sent to a reference typing centre for identification.

7.7.2.1 Mark the following areas on an agglutination plate: C- (negative control) and T (test culture).

7.7.2.2 If a Salmonella control culture is available for each somatic group tested, prepare C+ (positive control) as described in 7.7.1.

7.7.2.3 Prepare somatic group antiserum as directed by the manufacturer; add one drop to each of the areas marked T and C+; add one drop of physiological saline to the area marked C-.

7.7.2.4 Remove sufficient culture material from a triple sugar iron, lysine iron or nutrient agar slant to prepare a moderately dense suspension in the test area and in the negative control area. The inoculum should be withdrawn from the slope portion of the agar slants.

7.7.2.5 Mix the culture-antiserum suspensions in T and C+ and the saline-culture mixture in C- with a sterile needle or loop. To avoid cross-contamination, use a separate (or re-sterilized) sterile loop or needle for each suspension. Tilt the slide preparation back and forth for 1 min.

7.7.2.6 Hold the slide under or against a well illuminated dark background and observe for agglutination. Salmonella cultures usually agglutinate within 1 min.

7.7.2.7 If the culture-antiserum mixture in the area marked T does not agglutinate, repeat the procedure with another somatic group antiserum.

7.7.2.8 If the serological test is positive, the culture should be sent to a Salmonella typing centre for complete serotyping.

7.7.2.9 The serological test for a given culture is invalidated if the negative control shows agglutination (autoagglutination).

7.7.2.10 A biochemically suspect Salmonella isolate (Table III) that fails to yield any positive serological reaction should be sent to a reference typing centre for identification.

7.7.3 Testing with Flagellar (H) Antisera

In instances where the services of a reference typing centre are not available, Salmonella isolates agglutinable with somatic antisera should be further identified by testing with polyvalent H antiserum.. Follow manufacturer's instructions on the preparation and use of antisera. See also, Microorganisms in Food, Vol. 1 (8.13).

8. REFERENCES

8.1 Andrews, W.H. 1989. Methods for recovering injured "classical" enteric pathogenic bacteria (Salmonella, Shigella, and enteropathogenic Escherichia coli) from foods. (Chapter 3). Injured Index and Pathogenic Bacteria. B. Ray (ed.). CRC Press, Boca Raton, FL. pp. 55-113.

8.2 Association of Official Analytical Chemists (AOAC) International. 1998. FDA Bacteriological Analytical Manual. Eighth edition, Revision A. AOAC International, Arlington, VA.

8.3 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.

8.4 D'Aoust, J.-Y. 1977. Effect of storage conditions on the performance of bismuth sulfite agar. J. Clin. Microbiol. 5:122-124.

8.5 D'Aoust, J.-Y. 1981. Update on preenrichment and selective enrichment conditions for detection of Salmonella in foods. J. Food Prot. 44:369-374.

8.6 D'Aoust, J.-Y. 1984. Effective enrichment-plating conditions for detection of Salmonella in foods. J. Food Prot. 47:588-590.

8.7 D'Aoust, J.-Y. 1989. Salmonella. (Chapter 9). Foodborne Bacterial Pathogens. M.P. Doyle (ed.). Marcel Dekker Inc., New York, NY. pp. 327-445.

8.8 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.

8.9 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.

8.10 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.

8.11 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. AOAC Int. 76:814-821.

8.12 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.

8.13 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. pp. 169-170.

8.14 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.

Table I.Criteria and sampling plans for Salmonella in specific foods

Determination Food Regulations of the Food and Drug Act Criteria: No. of sample units (n) Criteria: Acceptance Number (c) Criteria: Concentration of Microorganisms (m) Criteria: Maximum Concentration of Microorganisms (M) Salmonella cocoa and chocolate B.04.012 10 0 0 - milk powders, and other dairy product powders B.08.011 20 0 0 - froglegs B.21.031 5 0 0 - egg products and liquid eggs B.22.033 10 0 0 -

Lot:

A batch or production unit which may be identified by the same code. When there is no code identification, a lot may be considered as (a) that quantity of product produced under essentially the same conditions, at the same establishment and representing no more than one day's production; or (b) the quantity of the same variety of product from one and the same manufacturer available for sampling at a fixed location.

n:

The number of sample units usually but not always selected at random from a lot and examined in order to satisfy the requirements of a particular acceptance plan used. This is the sample.

m:

The numerical value of "m" represents acceptable concentrations of microorganisms, usually per g or mL. In a 2-class plan (as for Salmonella), "m" separates sample units of acceptable and defective quality; in a 3-class plan, "m" separates sample units of acceptable quality from those of marginally acceptable quality. The "m" values listed in the table are based on levels achievable under GMP.

M:

(Only in a 3-class plan), the numerical value of "M" represents unacceptable concentrations of microorganisms, usually per g or mL, that indicate a (potential) health or injury hazard, imminent spoilage or gross insanitation; "M" separates sample units of marginally acceptable quality from those of defective quality. A value determined for any one sample unit of a sample that is greater than that of "M" renders the pertaining lot unacceptable.

c:

The maximum allowable number of marginally acceptable sample units. "c" is the acceptance number of a plan. When this number is exceeded, the lot becomes unacceptable.

Table II.Procedures for non-selective enrichment (preenrichment)

Type of Product	Number of Sample Units	Sample Unit	Analysis of the individual analytical unit
Chocolate and cocoa	10	100 g	Suspend 25 g in 225 mL of skim milk medium and blend
Milk powders, and other dairy product powders	20	100 g	Gently add 25 g to 225 mL of sterile brilliant green water and allow to soak undisturbed; do not mix. Ensure that the test material rehydrates completely during soaking. Use of the soak method for milk powder composites of low solubility is contradicted because of the propensity for incomplete wetting of the test material.
Egg products and liquid egg products	10	100 g	Suspend 25 g in 225 mL NB or BPW and blend.
Froglegs	5	> or = 25 g	Place 25 g in 225 mL NB or BPW.

Table III. 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.	Negative (some strains 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 strains
	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

The method described above, being comprised of 13 pages and identified as MFO-21 and dated July 2002, is hereby designated the "Official Method" referred to in Sections B.04.012, B.08.011, B.21.031 and B.22.033 of the Regulations of the Food and Drugs Act for the microbiological examination of cocoa, chocolate, milk powders and other dairy product powders, froglegs, liquid eggs and other egg products.