SPS Agar (Sulfite Polymyxin Sulfadiazine)

Description

The nutritional substrates constituted by the tryptone and the yeast extract are complemented by the polysorbate, which also allows the recovery of the most delicate cells. The anaerobic conditions are improved by the thioglycolate, which permits the use of the medium on the plates.

The differential system consists of sodium sulfite and ferric citrate which allows the detection of sulfite reducing organisms, which form black colonies due to ferrous sulfide precipitate.

Technique

Collect, dilute and prepare samples and volumes as required according to specifications, directives, official standard regulations and/or expected results.

Melt the medium contained in the tubes in a water bath or in a microwave oven, avoiding overheating.

Add the sample directly into the tube after making a dilution bank, or by needle stab.

Incubate the tube in anaerobic conditions at 44 ± 1 °C for 24-48 h.

(Incubation times longer than those mentioned above or different incubation temperatures may be required depending on the sample, on the specifications, etc.)

After incubation, enumerate all the colonies that have appeared in the agar, with a black precipitate.

Each laboratory must evaluate the results according to their specifications.

Presumptive isolation of Clostridium sp. must be confirmed by further microbiological and biochemical tests.

Calculate total microbial count per mL of sample by multiplying the average number of colonies per plate by the inverse dilution factor if a diluted sample was streaked. Report results as colony forming units (CFU) per mL or g along with incubation time and temperature.

Since the medium is not extremely selective, it is advisable to verify black colonies by checking that they are Gram-positive sporulated non-motile organisms incapable of reducing nitrates to nitrites.

Most clostridia are sulfite reducers. Among them are C. perfringens and C. botulinum which along with C. bifermentans are the species most frequently involved in food poisoning.

LC-4712, 55 mm plates for filtration

Solid medium for the detection of Clostridium perfringens in water and food samples.

The usual technique for the use of this medium is as follows:

Collect, dilute and prepare samples and volumes to be filtered as required according to specifications, directives, official standard regulations and/or expected results.

Filter the sample through a 0.45 µm pore membrane and apply it onto the surface of the SPS agar.

Incubate the plates anaerobically at 35 ± 2 °C for 24-48 h. If a more selective culture medium is desired, incubate at 44 ± 1 °C.

(Incubation times greater than those mentioned above or different incubation temperatures may be required depending on the sample, on the specifications, etc.)

After incubation, enumerate all the colonies that have appeared onto the surface of the membrane.

After incubation, enumerate the colonies with a black iron sulfide precipitate.

90% of the black colonies which are formed can usually be attributed to Clostridium perfringens.

Since the medium is not extremely selective, it is advisable to verify black colonies by checking that they are Gram-positive sporulated non-motile organisms incapable of reducing nitrates to nitrites.

Most clostridia are sulfite reducers. Among them are C. perfringens and C. botulinum which along with C. bifermentans are the species most frequently involved in food poisoning.

LC-4712, 55 mm plates for filtration

Physical/chemical control

Microbiological control

LC-4255: inoculate by stabbing.

LC-4712: membrane filtration / practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10⁴-10⁶ CFU for selectivity.

Anaerobiosis. Incubation at 35 ± 2.5 °C, reading after 24-48 hours.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• ANGELOTTI, HALL, FOSTER & LEWIS (1962) Quantisation of Clostridium perfringens in foods. Appl. Microbiol., 10:193.
• DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 4th ed., APHA. Washington.
• F.D.A. (1998) Bacteriological Analytical Manual. 8th ed. Rev. A., AOAC International. Gaithersburg. MD.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• MOSSEL, D.A.A. (1959) Enumeration of sulfite-reducing bacteria occurring in foods. J. Sci. Food Agric. 19:662.