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Food and Nutrition

Detection of Clostridium botulinum in honey and syrups

Laboratory Procedure
MFLP-50
January 2012

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John W. Austin
Microbiology Research Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204E
Ottawa, Ontario K1A 0K9

E-mail: micro_methods_committee@hc-sc.gc.ca

1. Principle

The procedure involves the removal of botulinal spores from the liquid portion of honey or syrups by membrane filtration, cultivation of the membrane in a liquid medium, analysis of the culture for toxin, and identification of toxins with specific botulinal antisera. Of the common human types of C. botulinum, only types A and B are commonly involved in infant botulism. The procedure is therefore geared towards the detection of these 2 types. A rare human type (F) may be considered as the possible source of toxin if (a) injected mice show the typical signs of botulism, and (b) the toxin cannot be neutralized by Type A or B antisera. This revised method replaces MFLP-50, dated April 1998.

2. Definition of Terms

See Appendix A of Volume 3.

3. Collection of Samples

See Appendix B of Volume 3.

4. 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" (7.1).

Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.

  1. Millipore sterilfil holders XXII04710.
  2. Millipore membrane filters (MF) HAWP04700.
  3. 1 cc tuberculin syringes.
  4. ½" 27G needles.
  5. Botulinal antitoxins.
  6. Sterile beakers.
  7. Sterile dH2O.
  8. Sterile 1% Tween 80.
  9. 150 mL screw capped dilution bottles.
  10. 300 mL centrifuge bottles.
  11. Water bath set to 65°C.
  12. Centrifuge.
  13. Laminar flow cabinet.
  14. TPGYB medium.
  15. Anaerobic jars or anaerobic chamber.
  16. Paraffin oil.
  17. 0.45 μm filter with Luer lock.
  18. Gelatin phosphate buffer.
  19. White mice (approx 20 g).

4.1 Filtration Equipment

4.1.1 Millipore Sterifil holders XXII 04710. 

These are placed on 1-litre suction flasks. Two or more units may be linked, in parallel, to a manifold which is connected to a vacuum pump.

4.1.2 Millipore membrane filters (MF) HAWP 04700. 

These are retailed in boxes containing 4 packages of 25 filters each.

Note: Flow rate and volume of filterable material depend on the direction in which the MF are placed in the filter units, but the direction of optimum flow bears no relation to their orientation in the packages.

When a new box is opened, take 2 filters from a package and place them (in succession or in parallel) in filter units (a) in the same orientation as in the package (keeping the top side up) and (b) with top and bottom sides reversed. Filter 100 mL of diluted honey (20% w:v), heated to 65°C, through both and record the flow rates. Maintain the orientation with the higher flow rates for the remaining 25 MF in the first package. Examine at least one filter each of the remaining 3 packages in the same way to ascertain proper orientation.

4.2. Syringes and Needles

Recommended syringe: 1 cc tuberculin, reorder number 5602, Becton-Dickinson.
Recommended needle: ½" 27G; also B-D.

4.3. Botulinal antitoxins

Trivalent (A,B,E) antiserum
National Center for Infectious Diseases

Monovalent (A and B) antisera
Centers for Disease Control and Prevention
Biological Reference Reagents
1600 Clifton Road, N.E., Mailstop C21
Atlanta, GA  30333
(404) 639-3354

5. Procedure

5.1 Preparation of diluted samples

Weigh 25 g of honey (or syrup) into a sterile foil-covered beaker.  Add 100 mL of sterile distilled water with 1% Tween 80 and stir until the solution is homogeneous.

5.2 Spore activation, filtration and incubation

For syrups, transfer the 125 mL suspensions to 150 mL screw-capped dilution bottles, hold in a water bath at 65°C for 30 min. and filter through a membrane filter (MF).

For honeys, transfer the 125 mL suspensions to 300 mL centrifuge bottles. Hold in a water bath at 65°C for 30 min. and centrifuge at 15,000 xg for 20 min. Filter the supernate through a membrane filter.  Keep the sediment temporarily at 4°C. After filtration rinse dilution bottle and funnel with about 5 mL of sterile, cold distilled water through each MF. Transfer the MF in a laminar flow cabinet into 110 mL of TPGYB medium. In the analysis of honey, carefully add the sediment from the centrifugation to the dilution bottle containing TPGYB medium and the filter. Incubate at 35°C for 7 days under anaerobic conditions. Check the bottles daily. Cap loosely to prevent pressure build-up.

5.3. Modifications of 5.2 in case of clogged filters

In the rare event that the MF becomes clogged before the filtration of 125 mL is completed, transfer the unfiltered portion to a second filter unit. Rinse the funnel of the first unit with water, transfer the rinse water to the second unit and complete the filtration. Rinse, and transfer both filters to a single bottle of TPGYB medium.

5.4 Preparation of culture filtrate

After 7 days of incubation, select the bottles with signs of growth and withdraw about 20 mL of culture. Centrifuge at 20,000 x g for 20 min and decant the supernate. Take about 10 mL of supernate up in a disposable syringe and sterilize by filtration through a Millex HA 0.45 μm membrane filter (Millipore) fitted on the syringe.

5.5 Detection of toxin

Dilute 4 mL of sterile filtrate with 4 mL of gelatin phosphate buffer. Inject intraperitoneally two mice (about 24 g) each with 0.5 mL of diluted filtrate and observe for 4 days. Store the unused portions of diluted and undiluted filtrate at 4°C.

Notes:

  1. Dilution of filtrate is required to prevent anaphylactic shock from the high protein content of the medium.
  2. 95% of the mice killed by botulinal toxin in TPGYB medium will be dead or near death after 24 h.

5.6 Confirmation of botulinal toxin

Select all samples causing death in 1/2 or 2/2 mice. Place 1.5 mL each of diluted filtrate in four 10 x 75 mm test tubes. Add 0.15 mL of botulinal antiserum (Appendix B, 4): trivalent A, B, E to the first, monovalent A to the second, monovalent B to the third, none to the fourth. Mix, and keep the mixtures at ambient temperature for 45 min. to 1 h. Inject two mice each with 0.55 mL of each filtrate/antiserum mixtures and 0.5 mL of filtrate without antiserum. Observe for 4 days. If a sample kills only 1/2 mice, inject 2 more mice, if possible within 24 h after the first injection. Samples are considered positive for toxin if 2/2 or at least 2/4 mice are killed.  Clostridium botulinum type A is confirmed if mice are protected with trivalent A, B, E and monovalent A antisera; C. botulinum type B is confirmed if mice are protected with trivalent A, B, E and monovalent B antisera.

Notes:

  1. If there are signs of botulism prior to death (ruffled fur, laboured abdominal breathing, weak or paralysed limbs) and none of the antisera has a protective effect, C. botulinum type F may be the source of toxin. In that case, ship the remaining filtrate to the Botulism Reference Service for identification and store the original culture at 4°C for future reference.
  2. Trivalent (A, B, E) antiserum is used in lieu of divalent (A,B) antiserum.

6. Preparation of media

6.1 Trypticase-peptone-glucose-yeast extract-beef extract (TPGYB) medium

  • Trypticase (BBL)*
    50 g
  • Peptone (Difco)
    5 g
  • Dextrose (Difco)
    4 g
  • Yeast extract (Difco)
    20 g
  • Beef extract (Difco)
    10 g
  • Sodium thioglycollate
    1 g
  • Distilled water
    1 L

* May be substituted with special peptone L72 (Oxoid)

Note:    If the medium is not used on the day of autoclaving, deaerate prior to use by steaming at 100°C in the autoclave for 10 min, or by placing the dilution bottles in boiling water, about 6 cm deep, for 10 min.

6.2. Tween 80 diluent

  • Tween 80 (polyethylene sorbitan monooleate)
    10 g
  • Distilled water
    1 L

Filter-sterilize.

6.3. Gelatin phosphate buffer

  • Gelatin
    2 g
  • Disodium hydrogen phosphate (Na2HPO4)
    10 g
  • Distilled water
    1 L

Adjust pH to 6.6 with 1 N HCl. Autoclave at 121°C and 15 lb pressure for 15 min.

Potential hazards to the investigator

Liquid cultures of C. botulinum contain high levels of toxin and should be handled only by experienced personnel after immunization with botulinal toxoid.

Caution: the toxoid supplied by CDC protects only against C. botulinum of types A to E, not against type F which may be, though rarely, involved in food-borne or infant botulism. Contaminated sealed products (canned or vacuum-packaged) may be under pressure and must be opened in a fume hood or safety cabinet for protection from aerosols.  Goggles must be worn whenever an accidental splash may be expected.

Caution: immunization does not assure protection of the eye from botulinal toxin, and splashes may result in blindness. Disposable gloves should be worn and pipetting by mouth is to be avoided. Used glassware and other supplies in contact with toxin are placed in a sturdy, heat-resistant container which should be placed in the autoclave by the investigator. Disposable material such as gloves, cotton or tissue paper is collected in autoclave bags for hazardous waste and are autoclaved. If accidental spills occur, the toxin may be inactivated with saturated or dry sodium bicarbonate.

Incriminated foods (excepting sealed products) and clinical specimens may be analyzed by experienced personnel without the need for immunization; if toxic, they usually contain relatively low levels of toxin.

Clostridium botulinum is considered a Risk Group 2 organism according to the Laboratory Biosafety Guidelines published by Health and Welfare Canada and Medical Research Council of Canada. As such, the laboratory should meet the requirements of a Containment Level 2 laboratory, as outlined in the Laboratory Biosafety Guidelines.

End of Document

Date Modified: 2012-06-20

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