SPS Agar

Intended Use

SPS Agar is used for detecting and enumerating Clostridium perfringens in food.

Summary and Explanation

In the 1950s, Mossel1 and Mossel et al.2 proposed media for enumerating anaerobic sulfite-reducing clostridia in foods. Angelotti et al.3 modified the formula as Sulfite Polymyxin
Sulfadiazine (SPS) Agar and used it to quantitate C. perfringens in foods.
C. perfringens is found in raw meats, poultry, dehydrated soups and sauces, raw vegetables and other foods and food ingredients. Occurrences of foodborne illness from C. perfringens are usually associated with cooked meat or poultry products.4 Spores of some strains that may resist heat during cooking germinate and grow in foods that are not adequately refrigerated.5 Enumerating the microorganism in food samples plays a role in epidemiological investigation of outbreaks of foodborne illness.4

Principles of the Procedure

SPS Agar contains peptone as a source of carbon, nitrogen, vitamins and minerals. Yeast extract supplies B-complex vitamins which stimulate bacterial growth. Ferric citrate and sodium
sulfite are H2S indicators. Clostridia reduce the sulfite to sulfide, which reacts with the iron from ferric citrate to form a black iron sulfide precipitate. Polysorbate 80 is a dispersing agent.
Polymyxin B sulfate and sulfadiazine are inhibitors to organisms other than Clostridium spp. Sodium thioglycollate is a reducing agent. Agar is the solidifying agent.

User Quality Control

Identity Specifications
SPS Agar
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:                       4.1% solution, soluble in purified water upon
                                   boiling. Solution is light to medium amber, very
                                   slightly to slightly opalescent.
Prepared Appearance:    Light to medium amber, slightly opalescent.
Reaction of 4.1%
Solution at 25°C:           pH 7.0 ± 0.2
Cultural Response
SPS Agar
Prepare the medium per label directions. Inoculate using the pour plate technique and incubate anaerobically at 35 ± 2°C for 24-48 hours.

Formula

SPS Agar
Approximate Formula* Per Liter
Tryptone ................................................................... 15.0 g
Yeast Extract.............................................................. 10.0 g
Ferric Citrate................................................................ 0.5 g
Sodium Sulfite............................................................. 0.5 g
Sodium Thioglycollate................................................... 0.1 g
Polysorbate 80........................................................... 0.05 g
Sulfadiazine............................................................... 0.12 g
Polymyxin B Sulfate.................................................... 0.01 g
Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.<

Directions for Preparation from Dehydrated Product

1. Suspend 41 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

1. Dispense inoculum into sterile Petri dish.
2. Pour medium cooled to 50-55°C over the inoculum.
3. Gently but thoroughly mix the inoculum and medium. Allow to solidify on a flat surface.
4. Incubate anaerobically at 35 ± 2°C for 24-48 hours.

Expected Results

Clostridium perfringens will grow as black colonies with good growth.

Limitation of the Procedure

The high degree of selectivity of SPS Agar may inhibit some strains of C. perfringens while other strains that grow may fail to produce distinguishing black colonies.4

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