CLOSTRIDIUM BROTH

 

Used for the detection and enumeration of the spores of Clostridia.

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Code: CB1/5442

 

Typical formula g/l

 

Yeast extract	3.0
Beef extract	10.0
Tryptone	10.0
Glucose	5.0
Soluble starch	1.0
Sodium chloride	5.0
Sodium acetate	3.0
Cysteine HCl	0.5
Agar	0.5

 

Final pH 6.8 +/- 0.2

   

Directions

Suspend 38g of Clostridium Broth in 1000 ml of cold distil­led water. Heat to boiling, distribute 25ml into screw-capped bottles with a capacity of 25 ml, and sterili­se by autoclaving at 115_{^{°C for 20 minutes. For the examination of water samples, prepare the Clostridium Broth at double strength reducing the water volume by half and transfer 10ml and 50ml aliquots into screw-capped bottles with a capacity respectively of 25 and 100ml. Prepare a 4% solution of sodium sulphite and a 7% solution of ferric citrate; if necessary, heat the ferric citrate solution for 5 minu­tes to dissolve completely. Sterilise the two solutions by filtration, and store at 2-5°C in closed bottles; the two solutions are stable for two weeks. The day of analysis, mix equal volumes of the two solutions and, under steri­le conditions, add 0.5ml of reagent to each 25ml of medium. For the preparation of double strength Clostridium Broth add 0.4ml of the mixture to each 10ml and 2ml to each 50ml.}}

 

_^Description

_{^{Clostridium Broth, which corresponds in the formulation to Differential Reinforced Clostridial Medium reported in ISO 6461/1 and to Reinforced medium for clostridia recommended by EP, is a non-selective medium used for the detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) in water and foodstuffs by the MPN method.}}

_{^{The medium is very rich and permits the growth of most clostri­dia, and many other anaerobes and facultative anaerobes. To demonstrate the growth of clostridia, sodium sulphite and iron citrate may be added to the medium. However, blackening of the medium, due to the reduction of sulphite to sulphur, and of its pre­cipitation in the form of iron salts, is not specific for the Clostri­dium genus, because other bacteria, such as Salmonella, Proteus, and some strains of Escherichia coli give the same reaction.}}

_{^{However, because these latter microorganisms are asporigens, pasteurisation and ethyl alcohol treatment of the specimen elimi­nates them completely.}}

 

_{^{In use technique}}

_{^{For the presumptive enumeration of the clostridia in water ISO 6461/1 reports the following technique: Heat the sample in a water batch at 75 +/- 5°C for 15 minutes recording the temperature with a thermometer. Add 50ml of sample to a screw-cap bottle containing 50ml of double strength complete Clostridium Broth. Add 10ml of sample to a series of five screw-cap bottle containing 10ml of double strength complete Clostridium Broth. Add 1ml of sample to a series of five screw-cap bottle containing 25ml of single strength complete Clostridium Broth}}

_{^{If necessary add 1ml of 10-1 dilution of the sample to a series of five screw-cap bottle containing 25ml of single strength complete Clostridium Broth. In order to carry out a qualitative examination of 100ml of water without making an MPN count, use a 200ml vial filled with a mixture of 100ml of double strength complete Clostridium Broth and 100ml of sample. If necessary top up all the bottles with a single strength complete Clostridium Broth to bring the volume of liquid level with the neck and to ensure that only a very small volume of air remains, then seal the bottles hermetically, or incubate under anaerobic conditions.}}

_{^{Incubate the inoculated bottles at 37°C for 44 hours. Large volume of culture in hermetically sealed glass bottles may explode due to gas production. The addition of iron wire, heated to redness and placed into the medium before inoculation, may aid anaerobic conditions. Bottles, in which a blackening effect occurs as a result of the reduction of sulphite, shall be regarded as positive. Express the results in accordance to ISO 8199.}}

 

_{^{Clostridium Agar has the same characteristics as Clostridium Broth, and it is recommended for the isolation of clostridia in pu­re culture, starting from their growth in bottles of Clostridium Broth. For this, Freame and Fitzpatrick recommend the use of open-en­ded test tubes (120x13 mm) and plugged at one end with a cot­ton wad and at the other with a rubber plug. 1ml of a blacke­ned culture broth of Clostridium Broth is introduced into these test tubes, which are then filled with Clostridium Agar. They are incu­bated at 30°C until discrete white (non-clostridial types) or black colonies are visible in the agar. For the isolation of clostridia in pure culture, choose the colonies to be subcultured, open the test tube at the rubber plug end, push the agar column outwards by means of the cotton wad until the desired colony is clear of the test tube. Remove the agar un­der sterile conditions and transfer the colony into Clostridium Broth with a Pasteur pipette. The culture broth derived from it is tested for purity on a plate under anaerobic conditions or, if necessary, with a new subculture in Clostridium Agar test tubes. Identification of the pure culture can be carried out using the Willis and st1:City>Hobbs, or the Smith and Holdeman procedure.}}

 

_{^{User quality assurance (37°C-48 hrs, anaerobic incubation)}}

_{^{Productivity control}}

_{^{C.perfringens ATCC 13124: black growth}}

_{^{C.sporogenes  ATCC 19404: black growth}}

_^Storage

_{^{Dehydrated media: 15-30°C}}

_{^{User prepared flasks and tubes: 1month at 2-8°C}}

_^References  

_{^{Freame, B. & Fitzpatrick, B.W.F. (1967) - The use of Differential Reinforced Clostridial Medium for the isolation and enumeration of Clostridial from foods. In «The Society for Applied Microbiology Technical series» n° 5: Isolation of Anaerobes, ed. Shapton, D.A. & Board, R. G. Vol. 5, London: Academic Press., pag. 49-55.}}

_{^{Gibbs, M.B. & Freame B. (1965) - J. Appl. Bact., 28, 95-111.}}

_{^{Eurpean Pharmacopoeia, 3rd ed. 2001 Supplement}}

_{^{ISO 6461/1 (1986) Water quality – Detection and enumeration of the spores of sulfite reducing anaerobes (clostridia) – Part 1: Method by enrichment in a liquid medium}}

_{^{Smith, L.D.S. & Holdeman, L.V. (1968) - The Pathogenic Anaerobic Bacteria. Springfield: Charles C. Thomas}}

_{^{Willis A.T. Hobbs, G. (1959) J. Path. Bact. 77, 511}}