Supplement to MFHPB-21

October 2020

HEALTH PRODUCTS AND FOOD BRANCH

OTTAWA

SUPPLEMENT TO METHOD MFHPB-21

Microbiological Methods Committee

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

Contact the Microbiological Methods Committee: hc.mmc-cmm.sc@canada.ca

1. Application

The following information is offered as a supplement to the method MFHPB-21, dated September 2005,
and may be used with this method. The purpose of this supplement is to provide additional information
(such as source(s) of materials, optional steps, critical steps, confirmation steps, or helpful hints etc.).

2. Principle

This method determines the presence of Staphylococcus aureus by plating known quantities of (dilutions
of) a food sample onto a selective agar (Baird-Parker agar). The use of Baird-Parker agar has been
validated for all foods.

The method has also been validated with the use of Baird-Parker agar supplemented with acriflavine and
polymyxin-B (BP+ agar) for raw milk cheeses and the frozen food type of the fish and seafood products
category, in order to reduce the growth of background organisms. When raw milk cheeses or frozen fish
and frozen seafood products are tested, BP+ agar may be used instead of BP agar.

Compendium of Analytical Methods, Volume 2

 2 Supplement to MFHPB-21
October 2020

9. Media

The media listed below are commercially available and are to be prepared and sterilized according to the
manufacturer's instructions. See also Appendix G of Volume 1 for the formulae of individual media.

Baird-Parker Supplemented with Polymyxin-B-Sulphate and Acriflavine (BP+) Agar

Basal Medium
Baird Parker agar 63.0 g
Distilled water 900 mL
pH 7.0 ± 0.2
Supplements
50% Egg yolk/tellurite emulsion 50.0 mL
PBS 0.1M pH 7.2 + polymyxin-B-sulphate and acriflavine (PBS-PA) 50.0 mL
Basal Medium:
Add components to water. Bring to a boil to dissolve completely. Autoclave at 121°C for 15 min.
Cool to 45–50°C.
Complete Medium:
Egg yolk tellurite emulsion is commercially available and usually contains about 50% egg yolk. Use
as per manufacturer's instructions, for example, aseptically add 50.0 mL of pre-warmed (45–50°C)
commercial egg yolk/tellurite emulsion to the medium. Add 50.0 mL PBS-PA to the basal medium.
Mix thoroughly and pour plates.
Note:
The surface of the agar should be dried before inoculation. It has been observed that freshly
prepared BP medium may be toxic to injured cells. It is therefore advisable to store the plates
overnight before inoculation (at room temperature or refrigerated). The medium should be opaque;
do not use non-opaque plates. BP media can have a shelf-life of one to several weeks under
specified storage conditions.

Phosphate Buffered Saline 0.1M pH 7.2 + Polymyxin + Acriflavine (PBS-PA)

Phosphate Buffered Saline Buffer (PBS)
Disodium Phosphate anhydrous (Na2HPO4) 12.0 g
Monosodium Phosphate monohydrate (NaH2PO4.H2O) 2.2 g
Sodium chloride (NaCl) 85.0 g
Distilled water 1.0 L
pH 7.2 ± 0.2
Supplements (per 1 L PBS)
Polymyxin-B-sulphate 0.4 g
Acriflavine 0.14 g
Complete solution:
Dissolve the ingredients of the PBS buffer in 800 mL of distilled water and adjust the pH to 7.2 with
1N NaOH. Add supplements and complete to 1 L with distilled water. Filter-sterilize the solution
(0.22 µm filter membrane). PBS-PA can be stored under refrigeration in the dark for 2 months
(acriflavine can be photo-oxidized to form inhibitory compounds).
Note: Since some ingredients have low solubility, it is important to mix well to ensure a complete
dissolution.

End of Document