Bryant-Burkey Broth Base (modified with resazurin)

During the milking process, small numbers of butyric acid fermenting bacteria are introduced into the raw milk. When the contaminated milk is used for producing cheese, the brines become contaminated with heat-resistant Clostridia spores. During the ripening of salt-brined semi-hard and hard cheeses (for example, Gouda, Edam, Emmental, Gruyère and Parmesan), late blowing gasogenic Clostridia ferment lactate into butyric acid, acetic acid and gas (CO₂ and H₂). The gas expands the cheese and causes a defect known as "late blowing" or butyric swelling.

The medium does not contain lactate, so it must be added when the medium is prepared. Sodium lactate is fermented under anaerobic conditions by C. tyrobutyricum and other lactate-fermenting Clostridia, which use it as a source of carbon and energy, producing hydrogen and CO₂. Tryptone and beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group, essential for bacterial growth. Sodium acetate is the selective agent inhibiting Gram-negative bacteria and also promotes the growth of C. tyrobutyricum. L-cysteine is the reducing agent and resazurin is an oxidation indicator, turning from pink (aerobic) to colorless (under anaerobic conditions).

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 33 grams of the medium in one liter of distilled water. Add 10 mL of 50% sodium lactate. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into tubes of 10 mL and sterilize in autoclave at 121 °C for 15 minutes.

Instructions for use

1. Before use, heat the tubes and boil for 10 minutes to regenerate anaerobic conditions.
2. Allow the tubes to cool to a temperature of 75 °C. At this temperature, the vegetative cells die and the spores are activated.
3. Prepare decimal dilutions of the sample, inoculate into 10 mL of medium in tubes and allow them to cool at room temperature.
4. Pour 2 mL of melted paraffin (60-65 °C) into each tube, previously autoclaved at 121 °C for 20 minutes.
5. Read results after incubation at 37 ± 2 °C for up to 7 days, considering the tubes with growth and gas production as positive.
6. To count the spores, use the most probable number method (MPN).

• BRYANT M.P. and BURKEY L.A. 1956. The characteristics of lactate fermenting spore forming anaerobes from silage. J. Bact., 43-46.
• CERF O. et BERGERE J.L. 1968. Numeration des spores de Clostridium et son application au lait et aux produits laitiers. Numeration des différents groupes de Clostridium. Le lait, 48, 501-519.