m-CP Supplement

Description

In m-CP Medium, lack of beta-D-glucosidase activity (an enzyme involved in cellobiose fermentation), fermentation of sucrose and production of acid phosphatase are used to differentiate presumptive Clostridium perfringens colonies from other Clostridium spp.

Lack of beta-D-glucosidase activity means that Clostridium perfringens does not cleave the chromogen, indoxyl beta-D-glucoside, in the medium. Furthermore, as the organisms ferment the sucrose in the medium, reducing the pH, bromocresol purple changes from purple to yellow. This results in characteristic opaque yellow Clostridium perfringens colonies.

Most other Clostridium spp. will appear as either purple colonies, due to the lack of sucrose fermentation, or blue/green colonies where the organism is still cleaving indoxyl beta-D-glucoside and also fermenting sucrose.

D-cycloserine, polymyxin B and incubation at 44 °C inhibit the growth of background flora such as Gram-negative organisms and staphylococci.

m-CP Medium has now been recommended in European Council Directive 98/83/EC for testing the quality of water intended for human consumption.

Technique

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

Reconstitute the vial with 10 mL of the sterile diluent in aseptic conditions, shake a few seconds, and add it to 500 mL of the medium base cooled to 50 °C.

Do not overheat once supplemented.

Pour the complete medium into Petri dishes and, once solidified on a flat surface, inoculate it with the 0.45 µm pore membrane through which the sample was filtered.

Incubate the plates anaerobically at 44 °C for 20-24 h.

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

Each laboratory must evaluate the results according to their specifications.

Presumptive positive Clostridium perfringens colonies can be further tested for acid phosphatase activity by exposure to ammonium hydroxide vapour for 20 to 30 seconds. Clostridium perfringens colonies turn pink or red as phenolphthalein diphosphate is cleaved by acid phosphatase. No colour change will be seen with colonies of organisms that do not possess acid phosphatase. It is important that this further test is carried out as there are a very small number of non-perfringens clostridia that produce yellow colonies. However, these colonies will remain yellow after exposure to ammonium hydroxide as they are acid phosphatase negative.

Reconstitute the original freeze-dried vial by adding 10 mL of sterile distilled water.

Note: each vial is sufficient to supplement 500 mL of m-CP Agar Base.

Physical/chemical control

Microbiological control

Add 1 vial to 500 mL of medium base. DO NOT HEAT once supplemented.

Inoculate: practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ (selectivity).

Analytical methodology according to ISO 11133:2014/Amd 1:2018; A2:2020.

Anaerobiosis. Incubation at 44 ± 1 °C for 21 ± 3 h.

Sterility control

Add 5 mL of the sample to 100 mL of TSB and to 100 mL of Thioglycollate.

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

• European Council (1997) Directive 12767/97 on the quality of water destined for human consumption. EC Bull. 16-12-1997.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.