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Anaerobic agar

Catalog Number: 1001117
Anaerobic agar

For the cultivation of anaerobes and conduct of sensitivity tests on anaerobic organisms.

Anaerobic Agar is a medium developed by Brewer to grow anaerobic microorganisms such as clostridia without the need for an anaerobic jar or container by using a specially designed Brewer dish.

Approximate formula in g/l:
Casein Peptone 17.5Soy Peptone 2.5
Sodium Chloride 2.5L-Cystine 0.4
Dextrose 10.0Agar15.0
Sodium Thioglycollate 2.0Sodium Sulfoxyl Formaldehyde 1.0
Methylene Blue 0.002
Final pH 7.2 + 0.2

Preparation: Suspend 51 g of the medium in a liter of deionized or distilled water. Mix well and heat with agitation. Boil for one minute or until the medium is completely dissolved. Sterilize in the autoclave at 15 lbs psi (121oC) for 15 minutes. Pour into Petri dishes about 3/4 full. Once the medium is solidified, place the special Brewer lid on top. This lid creates a seal which does not allow oxygen to penetrate the dish. The medium contains three reducing agents which lowers the oxidation-reduction potential and generates the anaerobic atmosphere necessary for growth. Methylene blue acts as the redox indicator.

Uses: The seeding of the sample (clinical or food) can be performed by surface inoculation or in poured tubes which have been melted and cooled to 45-50oC. Normally the sample should never be heated as this treatment would destroy the vegetative forms of the anaerobe. Nevertheless, at times it is proper to heat the sample when sporeformers such as clostridia are sought. When heating is indicated warm the sample suspended in a liquid diluent (peptone water, weak phosphate solution, etc.) for 10 minutes between 70oC-80oC. Once the suspension cools, inoculate 2 plates of Anaerobic Agar and incubate one in an anaerobic environment with CO2 at 35oC for 18-48 hours and the other plate in an anaerobic atmosphere without CO2. Better results are usually obtained using anaerobic jars with CO2 than with anaerobiosis alone.

The plates of Anaerobic Agar can also be incubated in air if the surface of the plates are covered by a Brewer lid. In this case, leave about 1.5 cm on the outer edge of the plate uninoculated. With care place the Brewer lid on the plate to obtain a hermetic seal. The central part of the lid should not touch the surface of the plate but form a chamber of 2-5 mm above the surface. When growth is observed, open the plate and pick the desired colonies. Incubate longer if necessary.

If for some reason the sample can not be streaked on the Anaerobic Agar plate, place the sample in Thioglycollate Medium without indicator previously heated and cooled. Incubate until the next day and seed the Anaerobic Agar plate. Thioglycollate Medium without Indicator is an excellent enrichment broth and frequently this method gives better results than direct seeding.

If the sample contains a mixed flora, place a disc of filter paper impregnated with 30 mcg of kanamycin in the area of heavy inoculum on an Anaerobic Agar with 5% sterile, defibrinated sheep or rabbit blood. Place a disc containing 10 units of bacitracin in the same area of a Blood Agar plate as a control. Both antibiotics will inhibit aerobes in the immediate area but not anaerobes.

For sensitivity tests of anaerobes, proceed with the following steps:
  1. Inoculate a pure culture of the anaerobe on the surface of the agar.
  2. Deposit 4-5 discs of paper impregnated antibiotics of choice. Do not put more than this number of discs on the plate as the zones of inhibition can be large and may interfere with each other.
  3. Carefully cover the inoculum and discs with a layer of the same medium.
  4. Once the upper layer has solidified, place the Brewer lid on top and incubate aerobically, or anaerobically if a regular Petri dish is used.

Typical Microbiology Testing:

The following typical results were obtained in the performance of the medium from type cultures after incubation at a temperature of 37°C and observed after 24 hours.

Clostridium butyricum ATCC 9690
Clostridium perfringens ATCC 12919
Clostridium sporogenes ATCC 11437

Availability: Catalog #1001117, Anaerobic Agar, 500 g size