Anaerobic Agar, Dehydrated Culture Medium, Cat Nº: AG-1000-500g,
For the cultivation of anaerobes, especially Clostridium
Formula In g/l
|Casein Peptone||17.50||Sodium Formaldehyde Sulfoxylate||1.00|
|Soy Peptone||2.50||Methylene Blue||0.002|
|Sodium Chloride||2.50||Bacteriological Agar||15.00|
|Sodium Thioglycollate||2.00||Final pH 7.2 ± 0.2 at 25ºC|
Suspend 51 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 °C for 15 minutes. The prepared medium should be stored at 8-15ºC. The color is white with a blue tint .The medium can be incubated in an anaerobic jar or with Brewer lids for anaerobiosis.
The dehydrated medium should be homogeneous, free-flowing and clear beige in color. If there are any physical changes, discard the medium.
ANAEROBIC AGAR is used for cultivation of anaerobic microorganisms. Anaerobic bacteria are unable to use oxygen as a terminal electron acceptor.
Casein peptone and Soy peptone provide nitrogen, vitamins, minerals and amino acids essential for growth. Dextrose is the fermentable carbohydrate providing carbon and energy. Sodium thioglycollate and Sodium formaldehy de sulfoxylate act as reducing agents generating a low oxidation-reduction potential, thus securing good anaerobic conditions. Methylene blue acts as the redox indicator, the blue color indicating the presence of oxygen.
The testing procedures can be carried out using standard Petri dishes or Brewer’s Anaerobic Agar Plates, both with the medium cooled to 45 – 50°C.
The seeding of the sample (clinical or food) can be performed by surface inoculation or by pour plate method. Normally the sample should never be heated to destroy the vegetative forms of the aerobe, as the anaerobic non-spore formers will also be destroyed. Nevertheless, sometimes it could be useful to heat the sample when spore formers such as Clostridium are sought, except C. perfringens, which rarely forms spores. When heating is indicated, warm the sample suspended in a liquid diluent (peptone water, buffering phosphate solution, etc.) to 70 – 80°C for 10 minutes. Incubate at 35 ± 2°C for 18 – 48 hours.
The plates of Anaerobic Agar can also be incubated in a normal atmosphere covering the surface of the plates with a Brewer lid. When growth is observed, open the plate and pick the desired colonies. Incubate longer if necessary. If the medium has not been prepared fresh before use, it is necessary to heat and remelt to expel the dissolved oxygen.
Thioglycollate Medium (Cat. 1508) without Indicator is an excellent enrichment broth and, frequently using it previously, yields better results than direct seeding.
The following results were obtained in the performance of the medium from type cultures after incubation at a temperature of 35 ± 2°C and observed after 18 – 48 hours under anaerobic conditions
Clostridium butyricum ATCC 9690
Clostridium perfringens ATCC 12919
Clostridium sporogenes ATCC 11437
Standard Methods for the Examination of Water and Wastewater 1 5th Ed. American Public Health Association, Inc, Washington, D.C. 1980. Andrew, W.H.C.D. Diggs, and C.R. Wilson, 1975.
Evaluation of medium for the rapid recovery of Escherichia coli from shellfish. App. Microbiol. 29: 130-131
Once opened keep powdered medium closed to avoid hydration.