Rapid Perfringens Medium (RPM)

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Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.) 9 1995 Elsevier Science B.V. All rights reserved

Rapid Perfringens Medium (RPM) Description and h&tory This medium was developed by Erickson and Deibel (1978) for the detection and enumeration of Clostridium perfringens in foods. RPM is a liquid medium with a litmus milk base and is prepared in tubes. Selectivity is provided by the antibiotics polymyxin B sulphate and neomycin sulphate, coupled with an incubation temperature of 46~ Detection of Clostridium perfringens is based on production of a stormy fermentation within 24 h. Gelatin hydrolysis, a characteristic common to strains of Clostridium perfringens, can also be detected in RPM (De Boer and Boot, 1983). A 3-tube most probable number (MPN) procedure can b e used to enumerate Clostridium perfringens in food samples and other products (Smith and Mood, 1983; Smith, 1985).

Composition (grams) Solution A Litmus milk powder (Difco) Neomycin sulphate Polymyxin B sulphate Distilled or deionised water

140.0 0.150 0.025 1000.0

Solution B Thioglycollate medium, fluid (Difco) Gelatin Peptone Glucose Di-potassium hydrogen o r t h o p h o s p h a t e Yeast extract Sodium chloride Iron (II) sulphate Distilled or deionized water

60.0 120.0 10.0 10.0 10.0 6.0 3.0 1.0 1000.0

Preparation Prepare solution A by suspending the litmus milk powder in the water and bring to the boil. Adjust pH to 6.8. Sterilize by autoclaving at 121~ for 5 min. Cool to 50~

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and aseptically add neomycin sulphate and polymyxin B sulphate. Prepare solution B by suspending the ingredients in the water and bring to the boil. Adjust pH to 7.1. Distribute 5 ml amounts in sterile screw-capped glass tubes. Sterilize by autoclaving at 121~ for 5 min. Cool to 50~ Prepare solution at 4~ 45-50~

the final medium by aseptically adding 5 ml of solution A to each tube of B. Cap the tubes tightly and invert several times to facilitate mixing. Store Before use liquify the medium by placing the tubes in a waterbath at for 30 min.

Physical properties Appearance pH

Light brown, opaque. 7.0 + 0.2

Shelf life Ready to use medium

4 weeks at 4 + 2~

Inoculation method for samples Inoculate RPM tubes with 1 ml of macerates of foods and mix well.

Incubation method At 46~ for 18-20 h in air.

Reading of results and interpretation Detection of Clostridium perfringens is based on production of a stormy fermentation reaction. As stormy fermentation is a presumptive test, the identity of isolates should be confirmed by additional tests.

Quality assessment (i) Productivity Test strains

Clostridium perfringens (haemolytic) 50027 Clostridium perfringens (non haemolytic) 50028

Inoculation method

Inoculate medium with 100-1000 cells of the test strains.

Criteria

Stormy fermentation within 24 h at 46~

415 (ii) Selectivity T e s t strains

Clostridium bifermentans 50026 Proteus mirabilis ( A T C C 2 9 9 0 6 / C E C T

4168/NCTC

11938) Inoculation method

D i l u t i o n to extinction.

Criteria

D i f f e r e n c e in g r o w t h s h o u l d b e e q u a l to o r less t h a n 5 titre u n i t s of t h e g r o w t h in t r y p t o n e soya b r o t h . N o s t o r m y f e r m e n t a t i o n w i t h i n 24 h at 46~

References De Boer, E. and Boot, E. (1983) Comparison of methods for the isolation and confirmation of Clostridium perfringens from spices and herbs. J. Food Protect. 46, 533-536. Erickson, J.E. and Deibel, R.H. (1978) New medium for rapid screening and enumeration of Clostridium perfringens in foods. Appl. Environ. Microbiol. 36, 567-571. Smith, M. and Mood, T.J. (1983) Direct testing of gelatin hydrolysis in Rapid Perfringens Medium. J. Assoc. Off. Anal. Chem. 66, 1045-1046. Smith, M. (1985) Comparison of Rapid Perfringens Medium and Lactose Sulfite Medium for detection of Clostridium perfringens. J. Assoc. Off. Anal. Chem. 68, 807-808.