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The use of animal tissue for culturing anaerobic organisms was first employed by Theobald Smith in 1890. Von Hibler later used brain tissue for cultivating and classifying anaerobic bacilli. Robertson replaced brain tissue with beef heart and used this medium to differentiate putrefactive and saccharolytic species. The formulation presently used is a modified version of Robertson's formulation. This medium is also referred to as Chopped Meat Medium.  Nutritional requirements needed by most bacteria are provided by beef heart, peptone and dextrose. Dextrose, yeast extract, hemin and vitamin K are added to enhance the growth of anaerobic microorganisms. Amino acids and other nutrients are supplied by the muscle protein in the heart tissue granules. Reducing substances, which permit the growth of strict anaerobes, are supplied by the muscle tissue and the iron filings. It is thought that the meat particles act as a reducing and detoxifying substance, thereby disabling harmful by products that may be produced by the replicating organism. Because reducing substances are more available in denatured protein, the meat particles are cooked before use in the medium. Growth of spore-forming and non-spore-forming obligate anaerobes is supported by this medium. Cooked Meat Medium is also useful as an enrichment broth for cultivating organisms from a very small inoculum. Additionally, researchers have found that Cooked Meat Medium preserves viability of organisms over a long period of time and is useful in maintaining anaerobic stock organisms. The Food and Drug Administration recommends its use in the enumeration and identification of Clostridium perfringens from food.

A sterile selective supplement used for isolation and presumptive identification of Clostridium perfringens, according to ISO 7937 and ISO 14189, and other regulations.

Compositon (g/vial) D-Cycloserine....................................................... 0.200 Reconstitute the original freeze-dried vial by adding Sterile Distilled Water...........................................6 ml

Note: each vial is sufficient to supplement 500 ml of medium. TSC Agar Base.

D-cycloserine selective supplement is added to TSC Agar in order to obtain a final selective medium which has the advantage to simplify the counting of plates with high numbers of colonies because smaller colonies of C.perfringrens are formed. Sodium metabisulphite and ferric ammonium citrate are used as an indicator of sulphite reduction made by Clostridium perfringens spp. that produce black colonies in TSC agar.

Principles and uses
T.S.C. Agar Base (Tryptose Sulfite Cycloserine) was originally formulated by Harmon for presumptive identification and enumeration of Clostridium perfringens from food. This medium has been documented as one of the most useful media for the quantitative recovery of C. perfringens, while suppressing growth of other facultative anaerobes. This media is recommended by ISO Normative Committee. Depending on the formula, supplements are added to increase the selectivity of the medium. Egg Yolk Emulsion supplement is added for the demonstration of lecithinase activity, and after incubation, lecithinase-producers produce an opaque area in the colony surroundings. The superior nutrient base provides optimal conditions for the development of Clostridia. Tryptose and soy peptone provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is source of vitamins, particularly the B-group, essential for bacterial growth. Ferric ammonium citrate and disodium disulfite are H2S indicators. Bacteriological agar is the solidifying agent. Cycloserine inhibits the accompanying bacterial flora and may cause the colonies which develop to remain smaller.
Colonies producing hydrogen sulfide are characterized by a blackening due to the reaction with the ferric salt. The degradation of the egg yolk lecithin produces insoluble products which accumulate around the colonies, forming a white precipitate. After 24 hours incubation, all black colonies, lecithinase positive as well as the lecithinase negative ones, have to be considered as positive presumptive C. perfringens and the corresponding confirmation tests have to be made.

Preparation
Suspend 42 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121 ºC for 15 minutes. Cool the medium according to the applicable normative and aseptically add two vials of Clostridium perfringens Selective Supplement. If desired, 25 ml of Egg Yolk Emulsion Supplement can be added (Not indicated in ISO Normative). Homogenize gently and dispense into Petri dishes.

Principles and uses
Reinforced Clostridial Medium is a semisolid medium. It is recommended for the cultivation and enumeration of anaerobes, particularly Clostridium and other microorganisms, in foods and clinical specimens.
It was formulated by Hirsch and Grinstead in 1954.Their work demonstrated that the medium outperformed other media in supporting the growth of Clostridium from small inoculum and produced higher viable cell counts.
Peptone and beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. yeast extract is the source of vitamins, particularly of the B-group. Dextrose is the fermentable carbohydrate providing carbon and energy. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Starch in the medium acts as a growth factor, probably functioning like a colloid protector, and neutralizes toxic products that form during the development of the organisms. L Cysteine hydrochloride is the reducing agent and sodium acetate is the buffer.
Since the medium is a non-selective enrichment one, it allows the growth of various anaerobic microorganisms and facultative bacteria when incubated under anaerobic conditions.
The European Pharmacopoeia, USP, recommends the Reinforced Clostridial Medium in the Paragraph 2.6.13 “Microbiological examination of non-Sterile products: test for specified microorganisms” for the testing of Clostridia in products.

Preparation
Suspend 38 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into appropriate containers and sterilize in autoclave at 121°C for 15 minutes. Cool to 45-50 ºC and, if desired, add 0,02 g/l of polymyxin B in a sterile filtered solution.

Thioglycolate Medium with Resazurin is used for sterility tests of biological products

Preparation

# Suspend 30.0 g of the powder in 1 liter of distilled or deionized water.

# Mix well. Heat to boil and shake until completely dissolved. Dispense into tubes.

# Sterilize in autoclave at 121°C for 15 minutes.

Quality control (35 ± 2°C / 18-24 h)

Storage

Store product in the original container with the lid tightly closed at between 10 and 30°C in low humidity conditions away from direct sunlight. Kept under these conditions, the product may be used up to the date of expiry shown on the product label.

Ordering information

The Iron Sulfite Agar (ISA) (ISO 15213-1) is recommended for the enumeration of sulfite-reducing Clostridium spp.

Meat-Liver Glucose 0.6% Agar is suitable for sterility tests of pharmaceutical products and canned foods

Tryptone Sulfite Cycloserine Agar was described by Harmon for the selective isolation and enumeration of Clostridium perfringens in water and food samples. The medium was recommended for the enumeration of sulfur-reducing anaerobes from foods of animal origin.

Lactose-Sulfite Broth is a confirmation medium allowing the selective detection of both the vegetative cells and spores of Clostridium perfringens in food products and biological samples of animal origin, without the usual confirmatory tests.

Bryant and Burkey Broth with Resazurin (modified by Bergère) is used to enumerate the spores of lactate fermenting Clostridium in milk and dairy products. In particular, it is used to detect Clostridium tyrobutyricum, responsible for the late blowing of cheeses such as gruyère, emmental, gouda, edam, cheddar or parmesan. The phenomenon arises from a high number of spores in the milk used to prepare the cheese. This number depends above all on the type of food given to the animals, since the main source of contamination is silage. 

Reinforced Clostridial Agar is a non-selective medium used for the isolation and enumeration of Clostridia, other anaerobic bacteria and lactobacilli in biological samples, dairy products, and other food products.