SOP: Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This method detects viable Listeria species in food and environmental samples. A portion of the product (or an environmental sample such as a sponge or swab) is enriched first in a primary broth, then in a secondary enrichment broth, plated onto a specified agar medium plus one additional plating medium, and incubated for set times and temperatures. Viable Listeria cells multiply under these conditions and form visible colonies that can be identified. A mandatory step requires the 24 h enriched LEB (UVM1 formulation) broth to be streaked directly to the selective agars, in addition to the transfer step into modified Fraser broth. The method serves to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act.

2. Apparatus required

• Stomacher, blender or equivalent
• Vortex mixer
• Microscope
• Incubators able to hold 30°C and 35°C
• Phase-contrast microscope with oil immersion objective (for wet-mount motility examination)

3. Media and reagents

Listeria broths and agars (base media and supplements are commercially available):

• Listeria enrichment broth (LEB) — UVM1 formulation
• Modified Fraser broth (MFB)
• Basal medium: tryptose — 10.0 g; beef extract — 5.0 g; yeast extract — 5.0 g; sodium chloride (NaCl) — 20.0 g; mono-potassium phosphate (KH2PO4) — 1.35 g; Na2HPO4 — 9.6 g; esculin — 1.0 g; lithium chloride — 3.0 g; nalidixic acid — 0.02 g; acriflavine HCl — 0.024 g; distilled water — 1.0 L. pH 7.2 ± 0.2. Supplement: ferric ammonium citrate (FAC) — 0.5 g in 10.0 mL of distilled water (filter-sterilized).
• To prepare: suspend the basal medium ingredients in 1.0 L of distilled water ↓ mix thoroughly ↓ heat to boiling to dissolve completely ↓ autoclave at 121 °C for 15 min ↓ cool to room temperature ↓ aseptically add 10.0 mL of the FAC solution to the cooled medium ↓ mix well and dispense ↓ store under refrigeration in the dark (acriflavine can photo-oxidize to form compounds inhibitory to Listeria sp.).
• Oxford agar (OXA) — mandatory plating medium
• Basal medium: Columbia blood agar base — 39.0 g; esculin — 1.0 g; ferric ammonium citrate — 0.5 g; lithium chloride (LiCl) — 15.0 g; agar — 2.0 g; distilled water — 1.0 L. pH 7.2 ± 0.2. Supplement per 10 mL (commercially available): cycloheximide — 400 mg; colistin sulphate — 20.0 mg; acriflavine — 5.0 mg; cefotetan — 2.0 mg; fosfomycin — 10.0 mg (dissolve in 10 mL of a 1:1 mixture of ethanol and distilled water; filter-sterilize).
• To prepare: suspend the basal medium ingredients in 1.0 L of distilled water ↓ heat to boiling with constant stirring to dissolve all ingredients ↓ autoclave at 121 °C for 15 min and cool to 45–50 °C ↓ aseptically add 10 mL of supplement and dispense ↓ store under refrigeration in the dark (acriflavine can photo-oxidize to form compounds inhibitory to Listeria spp.).
• Second selective agar (one of the following is mandatory):
• Agar Listeria according to Ottaviana and Agosti (ALOA)
• A.L. Agar (Bio-Rad)
• BBL CHROMagar Listeria (BD)
• Chromogeneic Listeria Agar Plate (OCLA; Oxoid)
• Lithium chloride-phenylethanol-moxalactam medium (LPM)
• Modified Oxford agar (MOX)
• PALCAM agar (PAL)
• RAPID’L.Mono (Bio-Rad)

Control cultures (use ATCC strains or equivalent):

• Positive controls: Listeria monocytogenes, Listeria ivanovii, Listeria innocua, Listeria grayi (Staphylococcus aureus and Rhodococcus equi — optional)

Confirmation media and reagents:

• Tryptose broth and agar (TA)
• Trypticase soy broth and agar, with 0.6% yeast extract (TSB-YE and TSA-YE)
• TSB-YE — Basal medium: tryptone — 17.0 g; soya peptone — 3.0 g; dextrose — 2.5 g; sodium chloride (NaCl) — 5.0 g; dipotassium phosphate (K2HPO4) — 2.5 g; yeast extract — 6.0 g (0.6%); distilled water — 1.0 L. pH 7.3 ± 0.2.
• To prepare (TSB-YE): suspend the basal medium ingredients in 1.0 L of distilled water ↓ heat to boiling with constant stirring to dissolve all ingredients ↓ dispense ↓ autoclave at 121 °C for 15 min.
• TSA-YE (agar form) — Basal medium: tryptone — 17.0 g; soya peptone — 3.0 g; dextrose — 2.5 g; sodium chloride (NaCl) — 5.0 g; dipotassium phosphate (K2HPO4) — 2.5 g; yeast extract — 6.0 g (0.6%); agar — 15.0 g; distilled water — 1.0 L. pH 7.3 ± 0.2.
• To prepare (TSA-YE): suspend the basal medium ingredients in 1.0 L of distilled water ↓ heat to boiling with constant stirring to dissolve all ingredients ↓ autoclave at 121 °C for 15 min.
• Horse or sheep blood agar (recommended for the haemolysis test)
• Basal medium: trypticase — 10.0 g; beef extract — 3.0 g; sodium chloride (NaCl) — 5.0 g; agar — 15.0 g; distilled water — 1.0 L. Final pH 7.2 ± 0.1. Supplement: sterile defibrinated horse or sheep blood — 50.0 mL.
• To prepare: suspend the basal ingredients in 1.0 L of distilled water ↓ heat to boiling to dissolve ↓ sterilize at 121 °C for 15 min ↓ cool to 45–50 °C and aseptically add the sterile defibrinated blood ↓ mix thoroughly without incorporating air bubbles ↓ dispense. The solidified, complete medium cannot be re-liquefied.
• Motility test medium
• Basal medium: beef extract — 3.0 g; pancreatic digest of gelatin — 10.0 g; NaCl — 5.0 g; agar — 4.0 g; distilled water — 1.0 L. pH 7.2 ± 0.2.
• To prepare: suspend the ingredients in water and mix thoroughly ↓ heat with frequent agitation and boil for 1 min to dissolve completely ↓ dispense and sterilize at 121 °C for 15 min.
• Carbohydrate fermentation agars or broths (mannitol, rhamnose and xylose) — these biochemicals may also be done with rapid identification kits
• 0.85% saline (wet-mount suspending medium)
• To prepare: dissolve 8.5 g of sodium chloride in distilled water and make up to 1.0 L.
• Gram stain solutions
• 3% hydrogen peroxide (catalase test)
• Use as supplied.
• Biochemicals — dextrose, esculin, maltose, α-methyl-D-mannoside
• Beta-lysine discs (e.g., Remel)

Optional:

• Rapid identification kits, such as the Vitek or API Listeria (bioMérieux Vitek, Inc.), Micro-ID Listeria (Organon Teknika Corp.), the Listeria Accuprobe Test (Gen-Probe; MFLP-88) or Oxoid Biochemical Identification System (O.B.I.S.) Mono kit (Oxoid) or equivalent
• Other chromogenic or novel agar — may be used, but only together with the mandatory plating media
• Sheep blood agar — for the CAMP test
• Basal medium: trypticase — 10.0 g; beef extract — 3.0 g; sodium chloride (NaCl) — 5.0 g; agar — 15.0 g; distilled water — 1.0 L. Final pH 7.2 ± 0.1. Supplement: sterile defibrinated sheep blood — 50.0 mL.
• To prepare: suspend the basal ingredients in 1.0 L of distilled water ↓ heat to boiling to dissolve ↓ sterilize at 121 °C for 15 min ↓ cool to 45–50 °C and aseptically add the sterile defibrinated blood ↓ mix thoroughly without incorporating air bubbles ↓ dispense. The solidified, complete medium cannot be re-liquefied.
• Latex agglutination kit (e.g., Oxoid Listeria Test Kit)
• Listeria monocytogenes antisera (e.g., Denka Seiken)

4. Sample handling and preparation

• In the laboratory, keep sample units refrigerated or frozen (except shelf-stable foods), depending on the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions that prevent microbial growth or death.
• Analyse sample units as soon as possible after receipt in the laboratory.
• Have sterile LEB ready, pre-warmed to 30 ± 1°C before use.
• Clean the surface of the working area with disinfectant.
• To take a representative analytical unit: agitate liquids or free-flowing materials until the contents are homogeneous; for solid sample units, take portions from several locations within the sample unit.
• Each sample unit may be analysed individually, or the analytical units may be composited as described in the Listeria Policy. Maintain a ratio of 1 part sample material to 9 parts sterile enrichment broth. Analysing each analytical unit separately gives information on Listeria distribution.
• For environmental samples, follow the sampling procedures given in MFLP-41.

5. Procedure

Incubation-time flexibility: times of 24 h are ± 2 h; times of 48 h are ± 4 h.

Environmental samples: add the environmental sponge or large swabs to 100 mL of LEB, or composite up to 10 sponges with 100 mL of LEB per sponge; place smaller environmental swabs (e.g., cotton-tip) in 10 mL portions of LEB, or composite up to 10 swabs with 10 mL of LEB per swab

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Food samples: add 25 g or mL of the food (the analytical unit) to 225 mL of LEB in a blender jar or stomacher bag; for composite samples, combine analytical units up to 125 g or mL (e.g., 125 g or mL of food into 1,125 mL of LEB); keep the 1:9 ratio of sample material to LEB for any other analytical-unit size

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Stomach or vortex the environmental samples and the food blend as needed for thorough mixing; the LEB culture may be incubated in the stomacher bag or another sterile container; incubate the LEB culture for 48 h at 30°C

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Optional: the incubated LEB enrichment broth may be refrigerated for up to 4 days; mix thoroughly after refrigeration before continuing

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At 24 h and at 48 h, mix the LEB culture by swirling or vortexing, and inoculate 10 mL of modified Fraser broth with 0.1 mL of the LEB culture; incubate 24–26 h at 35°C (the method’s flow diagram prints “up to 48 h” for this incubation); at the same time as the 24 h transfer, directly streak the 24 h LEB to the selective agars as below (streaking the 48 h LEB enrichment to selective plates is optional)

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Helpful hint: vortex the MFB at 20 to 24 h, then reincubate for a further 2 to 6 h before reading; reading the MFB at 26 h can substantially reduce the plating done at 48 h

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Streak MFB that has turned positive onto plates: a positive broth has darkened and may be black, dark brown or dark green; a negative MFB keeps the straw colour of a freshly made broth; reincubate negative broths another 24 h and streak all broths that turn positive

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After vortexing, streak the positive MFB onto two different plating media: Oxford agar (mandatory) and one of ALOA formulation agar, A.L. Agar, BBL CHROMagar Listeria, Chromogeneic Listeria Agar Plate, lithium chloride-phenylethanol-moxalactam medium, modified Oxford agar, PALCAM agar, or RAPID’L.Mono; incubate LPM plates at 30°C and all other selective agars at 35°C for 48 h, unless the manufacturer directs a different time and temperature; examine plates at 24 h for typical colonies, and again at 48 h as suited to the medium

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Note: there are up to 2 plates per selective agar — one from the streaking of the 24 h LEB enriched broth (always present) and potentially one from a positive MFB tube

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Read the colonies by agar:

• OXA agars: after 24 h, 1 mm diameter black colonies surrounded by black haloes; after 48 h, colonies are 2–3 mm in diameter, black with a black halo and a sunken centre; they can also appear brown-black or green-black; before 24 h, growth is sometimes apparent but without the characteristic blackening; some strains of the genus, other than L. monocytogenes, are inhibited on this medium when incubated at 35°C
• Agar Listeria according to Ottaviani & Agosti: colonies appear blue-green, with L. monocytogenes and L. ivanovii colonies showing opaque halos around them after 24 h; all other Listeria species are blue-green but have no halo (consult the manufacturer insert for a more detailed description)
• A.L. agar: all Listeria spp. form blue to blue-green colonies, with L. monocytogenes and L. ivanovii colonies having opaque halos around them after 24 and 48 h, respectively
• BBL CHROMagar: L. monocytogenes and L. ivanovii are blue-green colonies surrounded by an opaque white halo; other Listeria spp. are blue-green colonies without a halo
• LPM: examine for suspect colonies using beamed white light strong enough to illuminate the plate well, striking the plate bottom at a 45° angle; under optimum transillumination the more isolated, larger (48 h old) Listeria colonies look like whitish piles of crushed glass, often showing mosaic-like internal structures, occasionally with blue-grey iridescence that tends to sparkle; alternatively the colonies can look smooth with a blue tinge around the perimeter; when growth becomes nearly confluent, an even blue-grey iridescent sheen can be seen
• MOX agars: as for OXA — after 24 h, 1 mm diameter black colonies surrounded by black haloes; after 48 h, 2–3 mm black colonies with a black halo and sunken centre; brown-black or green-black also possible; growth before 24 h may lack the blackening; some non-monocytogenes strains are inhibited at 35°C
• Brilliance Listeria Agar (formerly OCLA agar): L. monocytogenes and L. ivanovii appear as blue colonies surrounded by an opaque halo; other Listeria species produce blue colonies without a halo after 24 h
• PAL agar: Listeria species form 2 mm grey-green colonies with a black sunken centre and a black halo on a cherry-red background; some Enterococcus and Staphylococcus strains form grey colonies with a brown-green halo, or yellow colonies with a yellow halo
• RAPID’L.Mono: L. monocytogenes forms blue colonies without a yellow halo; L. ivanovii are greenish-blue colonies with a yellow halo; other Listeria species are yellow to white in colour

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Confirmation — pick colonies as follows:

• If the colonies are well isolated on the selective agars: pick a minimum of 5 typical colonies from each selective plate onto blood agar, at each step of the method that selective plating is done (e.g., after the 24 h LEB, after darkening of the MFB broth)
• If the colonies are not well isolated: pick a minimum of 5 typical colonies from each selective plate onto Tryptose agar or Trypticase soy agar with 0.6% yeast extract, streaking for separation; as above, pick a minimum of 5 typical colonies from each step that required purification from selective agars; incubate the plates at 30°C for 24–48 h or until growth is satisfactory; examine the plates for typical colonies using the beamed-white-light arrangement described for LPM

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Confirm Listeria spp. with motility, haemolysis and the 3 carbohydrate agars (mannitol, rhamnose and xylose), or other valid confirmation steps published in the Compendium of Analytical Methods as equivalent; other biochemical tests are optional; rapid identification kits may help reinforce the confirmation and differentiate the Listeria species

Haemolysis

On blood agar plates (sheep or horse), draw a grid of 20–25 spaces on the plate bottom; pick typical colonies from the selective agars (if well isolated) or from the TA or TSA-YE plates (if streaked for purity) and inoculate the blood agars by stabbing one colony per grid; stab positive and negative controls (L. monocytogenes, L. ivanovii and L. innocua or L. grayi) on each plate; incubate for 24 h at 35°C

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Note: stab the blood agar plates and the carbohydrate plates concurrently from the same colony (the motility agar may also be stabbed at this time); keep each colony in the same position on all grid plates

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Examine the culture stabs by transillumination with a bright light (hold the plate so the light shines through from the back): L. monocytogenes produces a slight cleared zone around the stab; L. innocua shows no zone of haemolysis; L. ivanovii produces a well-defined zone of clearing around the stab

Motility

Stab motility test medium from the selective agars, TA or TSA-YE; incubate the motility media for a minimum of 48 h at room temperature and observe daily; if results are ambiguous after 48 h, continue incubation for up to 7 days; only Listeria spp. give the typical umbrella growth pattern

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and/or — wet mount: pick at least one typical colony from selective agar, TA or TSA-YE incubated at 30°C or less, and examine as a wet mount in 0.85% saline with the oil immersion objective of a phase-contrast microscope

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Alternatively: inoculate TSB-YE or TB broths and incubate overnight at 30°C; transfer a loopful of the overnight cultures to a fresh TSB-YE or TB and incubate at 25°C for 6 h; put a drop of each 6 h culture on a glass slide and examine for typical Listeria motility with the oil immersion objective of a phase-contrast microscope: Listeria appear as slim, short rods with tumbling motility; always compare to a known Listeria culture; cocci, large rods, or rods with rapid swimming motility are not Listeria species

Carbohydrate utilisation

Plates: on carbohydrate (mannitol, rhamnose and xylose) agar plates, draw a grid of 20–25 spaces on the plate bottom; pick typical colonies from the selective agars, TA or TSA-YE plates and inoculate the agars by stabbing one culture per grid; keep each colony in the same position on all grid plates; always stab positive and negative controls (L. ivanovii, L. monocytogenes and L. grayi); incubate for 24 h at 35°C

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and/or — broths: from the TSB-YE culture, inoculate the following carbohydrates set up as 0.5% solutions in purple carbohydrate broth: dextrose, esculin, maltose, mannitol, rhamnose, α-methyl-D-mannoside and xylose; incubate 7 days at 35°C and examine daily; Listeria spp. produce acid with no gas, or no reaction; consult the carbohydrate-reactions table for the Listeria spp.

Note: the carbohydrate plates and broths may also be replaced by rapid identification kits.

Optional tests

• PCR: from a single colony on selective agar, follow a validated PCR confirmation method for detection of Listeria spp.; it is suggested that colonies identified by PCR be streaked onto TSA or TSA-YE from the blood agar to obtain isolates of the positives; biochemical assays may be required if mentioned in the PCR method — check the specific PCR method for guidance and for interpretation of PCR results
• Rapid identification kits: such as the Vitek or API Listeria, Micro-ID Listeria or the Listeria Accuprobe Test, or equivalent; follow the manufacturer’s instructions for use
• Catalase: transfer a typical colony onto a clean glass slide and add one drop of 3% hydrogen peroxide; development of bubbles indicates a positive reaction; Listeria cells are catalase-positive; avoid picking test colonies from agars containing blood, which can produce a false positive
• Gram stain: Listeria is a small Gram-positive rod
• CAMP test: streak fresh isolates of beta-haemolytic Staphylococcus aureus and Rhodococcus equi vertically on a sheep blood agar plate; keep the vertical streaks apart so test strains can be streaked horizontally between them without touching the vertical streaks; after 24–48 h incubation at 35°C, examine the plates for haemolysis in the zone of the vertical streaks:
• The haemolysis of L. monocytogenes and L. seeligeri is enhanced near the Staphylococcus streak, while L. ivanovii haemolysis is enhanced near the Rhodococcus streak; the other Listeria species are CAMP-test negative; the test can differentiate L. ivanovii from L. seeligeri, and a weakly-haemolytic L. seeligeri from L. welshimeri
• Alternative: a convenient CAMP test may be done using the S. aureus factor in commercially prepared sterile beta-lysine discs — place a beta-lysine disc in the centre of the sheep blood plate and streak 4–5 Listeria cultures as radiating lines from the disc, taking care not to touch the disc with the inoculum; after 18–24 h incubation at 35°C, a very sharp CAMP reaction between L. monocytogenes or L. seeligeri cultures and the disc can be observed; L. ivanovii are strongly haemolytic and form a clear beta haemolytic line along the entire streak
• Serology: follow the manufacturer’s instructions provided with the antisera

6. Interpretation of results for speciation

• Listeria spp. are small, Gram-positive motile rods that are catalase-positive, urea-negative, and produce an acid slant and butt in TSI without producing H₂S. They utilise dextrose, esculin and maltose, with some species also using mannitol, rhamnose and xylose, producing acid. All species give +/+ reactions in MR-VP broth. L. grayi and L. murrayi are the only two species that utilise mannitol. L. murrayi is the only species that can reduce NO₃⁻ to NO₂⁻. L. grayi and L. murrayi are proposed to be members of a single species.
• L. monocytogenes, L. ivanovii and L. seeligeri (weak) produce haemolysis in horse or sheep blood agar and are also positive in the CAMP test. Of the three, only L. monocytogenes cannot utilise xylose, but is rhamnose-positive. L. ivanovii is differentiated from L. seeligeri by the CAMP test: L. seeligeri shows enhanced haemolysis only at the Staphylococcus streak, and L. ivanovii shows enhanced haemolysis in the area of the R. equi streak.
• L. innocua is differentiated from L. monocytogenes by its lack of haemolysis on blood agar plates and its negative reaction in the CAMP test. L. welshimeri that is rhamnose-negative may be confused with a weakly-haemolytic L. seeligeri unless the CAMP test is run.
• All biochemical, serological and pathogenicity data are collected in the two tables below: the characteristics differentiating the species of the genus Listeria, and the serology, haemolytic activity and mouse virulence data.

Characteristics differentiating the species of the genus Listeria:

• = ≥90% of strains positive; − = ≥90% negative; ND = not determined; V = variable.
  ++ = usually a wider zone of haemolysis observed.

Serology, haemolytic activity and mouse virulence for Listeria species:

* un = undefined.

7. Reporting of results

• A final report states the presence or absence of Listeria monocytogenes in the test portion analysed. If other Listeria species are isolated, these may be noted in the test report, if agreed between the parties concerned.
• The final report also includes all information necessary for the complete identification of the sample, the method used (including any deviations), and information specifying the mass in grams or the volume in millilitres. Any information that may have influenced the outcome of the test is included in the final report.

8. Precautions

• The Laboratory Supervisor must ensure the analysis is carried out in accordance with ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories.
• If the analyst uses any variations of the media listed (commercial product or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency; equivalency data is forwarded to the Editor of the Compendium of Analytical Methods for consideration of modification of the method.
• Incubator and water-bath temperatures are the laboratory’s responsibility: where 35°C is recommended, the incubator may be at 35 ± 1.0°C; lower temperatures of 30 or 25°C may be ± 1.0°C; where higher temperatures are recommended, such as 43 or 45.5°C, the incubators or water baths must be held within 0.5°C, because of the potential lethality of higher temperatures on the microorganism being isolated.

9. References

Health Canada, Compendium of Analytical Methods, Volume 2 — HPB Method MFHPB-30, February 2011: “Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples.” (Replaces MFHPB-30, dated September 2010, the Supplement to MFHPB-30, dated March 2002, and Appendix L (Supplement to All Listeria Methods), dated August 2005.)

Bibliography (the method’s own references, 8.1–8.15; the source’s running headers print “Febuary 2011” — the title page prints February 2011):

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• Health Canada, Health Protection and Food Branch, Food Directorate. 2010. Policy on Listeria monocytogenes in Ready-to-Eat Foods.
• Hitchins, A.D. 1995. Listeria monocytogenes. Chapter 10. Revised 1998. Bacteriological Analytical Manual (BAM). AOAC International. Gaithersburg, MD.
• Lovett, J. 1987. Listeria isolation. Chapter 29. Bacteriological Analytical Manual (BAM). AOAC. Arlington, Virginia.
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