Schaedler Agar

Intended Use

Schaedler Agar is a base for several media formulations used for the recovery of anaerobic microorganisms.

Schaedler Agar with Vitamin K₁ and 5% Sheep Blood is used for the isolation and cultivation of fastidious aerobes and anaerobes from a variety of clinical and nonclinical specimens.

It is especially useful for the recovery of the fastidious anaerobic bacteria such as Bacteroides, Prevotella and Porphyromonas species. Schaedler K-V Agar with 5% Sheep Blood, containing kanamycin and vancomycin, is especially useful in the selective isolation of Bacteroides and Prevotella species.

Summary and Explanation

In 1965, Schaedler, Dubos and Costello¹ reported on the bacterial flora of the gastrointestinal tract of mice. In these studies, several new media formulations were introduced. The majority of these contained inhibitors of specific bacterial species or groups since the authors indicated the need for selective media when processing specimens which contain large numbers of a heterogeneous bacterial population. The basal medium, without inhibitors, is the original version of the medium designated as Schaedler Agar. It was formulated to support the growth of fastidious anaerobic microorganisms such as lactobacilli, streptococci, clostridia and Bacteroides.

Mata and coworkers,² studying the fecal microflora in healthy persons in Central America, modified Schaedler Agar to produce a number of new formulations. The modifications in the basal medium of Schaedler included adjustments in the peptone content, since Trypticase™ Soy Broth was substituted for the Trypticase peptone component of the original formulation, and an increase in the sodium chloride content. Additionally, the dextrose concentration was reduced to avoid interference with hemolytic reactions and the yeast extract level lowered to avoid darkening of the medium.³

Principles of the Procedure

The combination of three peptones derived from both animal and vegetable sources, dextrose and yeast extract render the basic formulation highly nutritious by providing nitrogenous growth factors, carbohydrates as energy sources and vitamins. The sheep blood and hemin also are important in stimulating the growth of fastidious microorganisms. As discussed above, the vitamin K₁ additive is crucial for the recovery of certain anaerobes.

The addition of the antimicrobial agents kanamycin and vancomycin in the agar medium renders the medium selective for gram-negative microorganisms. The kanamycin inhibits protein synthesis in susceptible organisms, whereas the vancomycin inhibits gram-positive bacteria by interfering with cell wall synthesis.⁸

Using Schaedler media, fastidious aerobes and anaerobes grow well; however, the type of organisms recovered is dependent on the environment utilized in the incubation process (aerobic, aerobic supplemented with carbon dioxide or anaerobic conditions).

User Quality Control

Identity Specifications
Schaedler Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       4.19% solution, soluble in purified water
                                    upon boiling. Solution is medium, tan to
                                    yellow, clear to slightly hazy.
Prepared Appearance:     Medium, tan to yellow, clear to slightly hazy.
Reaction of 4.19%
Solution at 25°C:            pH 7.6 ± 0.2
Cultural Response

Schaedler Agar
Prepare the medium per label directions without (plain) and with added vitamin K₁ and 5% sheep blood

Formula

Schaedler Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein.......................................... 8.2 g
Peptic Digest of Animal Tissue...................................... 2.5 g
Papaic Digest of Soybean Meal..................................... 1.0 g
Dextrose.................................................................... 5.8 g
Yeast Extract............................................................... 5.0 g
Sodium Chloride.......................................................... 1.7 g
Dipotassium Phosphate................................................ 0.8 g
L-Cystine.................................................................... 0.4 g
Hemin...................................................................... 0.01 g
Tris (hydroxymethyl) aminomethane............................. 3.0 g
Agar......................................................................... 13.5 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 1 L of purified water:
    Schaedler Agar – 41.9 g;
    Mix thoroughly.
2. If desired, add 1 mL of a 1% vitamin K₁ solution in absolute ethanol.
3. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
4. Autoclave at 121°C for 15 minutes.
5. For the agar medium, cool to approximately 45°C and add 5% sterile defibrinated sheep blood when required.
6. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Agars
These media should be reduced immediately prior to inoculation by placing them under anaerobic conditions for 18-24 hours.⁹ Use standard procedures to obtain isolated colonies from specimens. Inoculate an enrichment broth, such as Enriched Thioglycollate Medium, at the same time as the primary plates to detect small numbers of anaerobes.

Incubate plates and tubes immediately after inoculation, with plates in an inverted position (agar side up) under anaerobic conditions at 35°C, or place the media in a holding jar flushed with oxygen-free gas(es) until a sufficient number of plates and tubes is accumulated (no longer than 3 hours). Incubate for at least 48 hours and, if no growth occurs, continue incubation for up to 7 days. It is recommended that an indicator of anaerobiosis be used.

Examine the selective medium for growth after 48 hours of incubation. Cultures should not be regarded as negative until after 7 days incubation. Since some anaerobes may be inhibited due to the selective nature of the medium, it is advisable to include a nonselective medium such as Schaedler Agar with Vitamin K₁ and 5% Sheep Blood.

Expected Results

Agars
In order to determine the relationship to oxygen of each colony type present on the medium, follow established procedures.10 The colony types that prove to contain obligate anaerobes can be further studied.¹¹

*Store at 2-8° C.

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