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MEMBRANE CLOSTRIDIUM PERFRINGENS (m-CP) MEDIUM

Code: CM0992

A medium for rapid isolation and presumptive identification of Clostridium perfringens from water samples.

Typical Formula*
gm/litre

Tryptose

30.0

Yeast Extract

20.0

Sucrose

5.0

L-cysteine hydrochloride

1.0

Magnesium sulphate.7H2O

0.1

Agar

15.0

Bromocresol purple

0.04

pH 7.6 ± 0.2 @ 25°C

 

* Adjusted as required to meet performance standards

m-CP SELECTIVE SUPPLEMENT

Code: SR0188

Vial contents (each vial is sufficient for 500ml of medium)

per vial
per litre

Polymyxin B sulphate

12.5mg
(105,000IU)

25.0mg
(210,000IU)

D-Cycloserine

200.0mg

400.0mg

Directions
Suspend 35.55g of m-CP Agar Base in 500ml of distilled water Mix well and sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and aseptically add the contents of one vial of m-CP Selective Supplement reconstituted as directed. Aseptically add the following sterile solutions dissolved in distilled water:

Component

Solution
Strength

Volume

Phenolphthalein biphosphate tetrasodium salt

0.5%

10.0ml

Ferric chloride hexahydrate

4.5%

1.0ml

Indoxyl b-D-glucoside

0.75%

4.0ml

equivalent to 30mg in 4ml
NB. Fresh solutions must be used. Mix well and pour into sterile Petri dishes.
To reconstitute m-CP Selective Supplement, aseptically add 2ml of sterile distilled water to 1 vial of supplement. Mix gently to dissolve.

Description
Membrane Clostridium Perfringens (m-CP) Medium is a selective and chromogenic medium for the presumptive identification of Clostridium perfringens from water samples.

m-CP Medium was first described by Bisson and Cabelli1 for the rapid quantitation of Clostridium perfringens from a variety of water samples (seawater, potable water and sewage). The medium was shown to give better recovery of Clostridium perfringens from water and sewage samples than the Bonde pour tube method1.

m-CP Medium has now been recommended in European Council Directive 98/83/EC for testing the quality of water intended for human consumption2.

In m-CP Medium lack of b-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 b-D glucosidase activity means that Clostridium perfringens does not cleave the chromogen, indoxyl b-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 b-D glucoside and also fermenting sucrose (see table).

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 posses acid phosphatase. It is important 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.

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

Technique
Filter the water sample using a 0.45mm cellulose acetate filter,(cellulose acetate has been found by Oxoid to be the best performing filter type, however the filter quality may vary from brand to brand. It is advised to validate the filter type to be used, according to ISO 7706). Place the filter onto the m-CP Medium. Incubate anaerobically for 21 ± 3 hours at 44 ± 1°C. Examine the plates for presumptive positive opaque yellow colonies that turn pink or red after exposure to ammonium hydroxide vapours for 20-30 seconds.
On m-CP Medium typical colonies will appear as follows:

Organism

Typical Colony Colour

Clostridium perfringens

Opaque Yellow
Sucrose positive/Glucosidase negative then pink/red after exposure to NH4OH

Other Clostridium spp.

Blue/Green
Sucrose positve/Glucosidase positive(e.g. Cl. baratii, Cl. paraputrificum, Cl. tertium)

Purple
Sucrose negative/Glucosidase positive or negative (e.g. Cl. biferentans, Cl. difficle, Cl. sporogenes)

Opaque Yellow
Sucrose positive/Glucosidase negative remain yellow after exposure to NH4OH

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
m-CP Selective Supplement SR0188 should be stored at 2-8°C.
Prepared medium may be stored for up to 5 days at 2-8°C in the dark.

Quality control

Positive control:

Expected results

Clostridium perfringens ATCC® 13124Yellow, then Pink/Red
Negative controls:  

Escherichia coli ATCC® 25922 *

Inhibited

Clostridium sporogenes ATCC® 19404 *

Purple

* This organism is available as a Culti-Loop®

References
1.
Bisson, J. W. and Cabelli, V. J. (1979) Applied and Environmental Microbiology, Vol. 37, No. 1, pp 55-88.
2. E.U. (1998) 98/83/EC of Council of 3rd of November 1998 on the quality of water intended for human consumption. Off. J. Eur. Commun., L330, 32-54

 
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