anaeroGRO™

ANAEROBIC PEA (Phenylethyl Alcohol) AGAR

Cat. no. AG901 Anaerobic PEA*, Monoplate 1 plate/pouch
Cat. no. AG312 DuoPak B, BRU with Hemin and Vitamin K* Monoplate; BBE/PEA*, Biplate 2 plates/pouch
Cat. no. AG303 MultiPak A, BRU with Hemin and Vitamin K* Monoplate; PEA* Monoplate; BBE/LKV*, Biplate 3 plates/pouch
Cat. no. AG313 MultiPak B, BRU with Hemin and Vitamin K* Monoplate; PEA* Monoplate; LKV*, Monoplate 3 plates/pouch

* All AnaeroGRO™ plated media is provided in standard 15x100mm monoplates or biplates. Each plate or set of plates is packaged in an oxygen-free, gas-flushed foil pouch containing a desiccant and an oxygen scavenger sachet.

INTENDED USE

Hardy Diagnostics AnaeroGRO™ Anaerobic PEA Agar is recommended for use as an enriched and selective medium for the cultivation and selective isolation of gram-positive and negative obligate anaerobic bacteria. It is useful in isolating obligate anaerobes from mixed flora, by inhibiting gram-negative facultative anaerobes and the control of swarming organisms.

SUMMARY

PEA should be inoculated for purulent specimens and when mixed infections are suspected. PEA inhibits facultative gram-negative rods, preventing Enterobacteriaceae from overgrowing the anaerobes, and inhibits swarming of Proteus. It also prevents certain clostridia, such as a C. septicum, from swarming, which will facilitate the isolation of other colonies. Most gram-negative and gram-positive anaerobes will grow on primary PEA medium, especially in mixed culture, and the morphology of the colonies is similar to that on blood agar plates; however, a longer incubation time may be necessary to detect the more slowly growing and pigmented anaerobes. However, pigment develops more rapidly than on non-selective Brucella Agar with H and K (Cat. no. AG301).

The basal medium of Anaerobic PEA consists of pancreatic digest of casein and enzymatic digest of soybean meal. Both provide amino acids, carbohydrates, and vitamins. The medium is supplemented with yeast extract, sheep blood, vitamin K, hemin, and L-cystine. Yeast extract enhances growth of fastidious microorganisms; sheep blood demonstrates hemolysis; vitamin K promotes growth and enhancement of pigmented Prevotella spp.; hemin and L-cystine improve the growth of Clostridium haemolyticum, Fusobacterium necrophorum, certain strains of Actinomyces israelii, Bacteroides thetaiotaomicron, and thiol-dependent streptococci.(6) The medium is made selective with the addition of phenylethyl alcohol which inhibits facultative anaerobic gram-negative bacilli.

AnaeroGRO™ Anaerobic PEA is packaged under oxygen-free conditions to prevent the formation of toxic oxidized products that may damage obligate anaerobes and inhibit the growth of more fastidious species.

Culture media that is exposed to environmental oxygen leads to a build-up of reactive oxygen species (ROS) that initiate damaging free radical reactions, which inhibit the growth of anaerobic bacteria. Therefore, ingredients have been added to the AnaeroGRO™ media to neutralize the growth inhibiting effects of peroxide and other reactive oxygen species (ROS) that may develop during the medium's brief exposure to oxygen after it is sterilized and before it is packaged in an oxygen-free environment.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic and Enzymatic Digests of Casein 15.0gm
Papaic Digest of Soybean Meal 5.0gm
Sodium Chloride 5.0gm
Yeast Extract 5.0gm
Phenylethanol 2.5gm
Reducing Agents/Peroxide Inhibitors 1.5gm
Vitamin K 10.0mg
Hemin 5.0mg
L-Cystine 0.4mg
Sheep Blood 50.0ml
Agar 15.0gm

Final pH 7.3 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 15-30ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(1,2,4,6,9,10) It is not recommended that a swab be used for specimen collection. Swabs are prone to drying and may be easily exposed to ambient air. The preferred means of anaerobic specimen collection is aspiration with needle and syringe. The specimen should be transferred to an anaerobic transport system (Cat. no. S120D) in order to protect it from oxygen exposure. Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold.

Method of Use: Open pouch and immediately inoculate and streak the specimen for isolated colonies. A large inoculum should be used in order to minimize the toxic effects of oxygen.(8) Incubate plates at 35-37ºC. for 18-48 hours in an anaerobic atmosphere. Examine for typical colonial morphology and characteristics.

A non-selective medium such as Brucella with H and K (Cat. no. A30) should be inoculated in parallel to the selective medium for enhanced recovery of anaerobic microorganisms.

Aerotolerance Testing: Confirmation of obligate anaerobic microorganisms should be performed. A Chocolate Agar plate (Cat. no. E14) incubated in 5-10% CO2 is required for aerotolerance testing to detect isolates that require CO2, especially slow-growing, fastidious, facultative or microaerophilic species that do not grow alone on media containing blood (such as Haemophilus and Actinobacillus spp.). Use of traditional blood agar media alone for CO2 incubation may yield false-negative results. An additional Blood Agar plate (Cat. no. A10) incubated in air will further detail the atmospheric requirements and hemolytic properties of facultatively anaerobic microorganisms.

INTERPRETATION OF RESULTS

Refer to the Wadsworth-KTL Anaerobic Bacteriology Manual or other texts for more information on identification of anaerobic bacteria.(10)

LIMITATIONS

The plates must be inoculated immediately after opening the AnaeroGRO™ pouch. After inoculation, the plates must be placed immediately into an anaerobic atmosphere (pouch, jar, or chamber) to ensure optimal growth of anaerobic bacteria.

Many anaerobes are more sensitive to oxygen during the log phase of growth; therefore, it may be necessary to incubate inoculated media for a full 48 hours prior to examination and exposure of the culture to ambient air.

Some organisms for which the medium is designed to isolate may be inhibited by the selective nature of phenylethyl alcohol.

Large numbers of anaerobic bacteria are normally present in the following sites: throat, gingiva, sputum, gastric contents, small bowel, feces, rectal swabs, surfaces of decubitus ulcers, encrusted walls of abscesses, mucosal lining, eschar, voided urine, vagina or cervix, skin and adjacent mucous. Specimens for anaerobic culture, therefore, should not be collected from these sites.

Failure to cultivate and/or isolate obligate anaerobes may be due to the following:

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, slides, staining supplies, other culture media, microscopes, incinerators, anaerobic holding jars (Cat. no. 16000), gas generators (Cat. no. AN25US), anaerobic transports (Cat. no. S120D), indicators (Cat. no. BR1255), etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bacteroides fragilis
ATCC® 25285**
A 18-24hr 35°C Anaerobic Growth
Prevotella melaninogenica
ATCC® 25845
A 18-24hrs 35°C Anaerobic Growth
Proteus mirabilis
ATCC® 12453**
B 18-24hr 35°C Aerobic Partial inhibition; slight growth without swarming

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

PHYSICAL APPEARANCE

AnaeroGRO™ Anaerobic PEA should appear opaque, and dark red in color.

B. fragilis growing on AnaeroGRO™ Anaerobic PEA Agar

Bacteroides fragilis (ATCC® 25285) colonies growing on AnaeroGRO™ Anaerobic PEA Agar (Cat. no. AG901). Incubated anaerobically for 24 hours at 35°C.

P. mirabilis partial to complete inhibition on AnaeroGRO™ Anaerobic PEA Agar

Proteus mirabilis (ATCC® 12453) partial to complete inhibition on AnaeroGRO™ Anaerobic PEA Agar (Cat. no. AG901). Incubated aerobically for 24 hours at 35°C.

C. perfringens growing on AnaeroGRO™ Anaerobic PEA Agar

Clostridium perfringens (ATCC® 13124) colonies growing on AnaeroGRO™ Anaerobic PEA Agar (Cat. no. AG901). Incubated anaerobically for 48 hours at 35°C.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Ellner, et al. 1966. Am. Journ. Clin. Path.; 45:502.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

6. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

9. Jousimies-Somer, H., et al. 2002. Wadsworth Anaerobic Bacteriology Manual, 6th ed. Star Publishing Company, Belmont, CA.

10. Jousimies-Somer, H.R., S.P. Citron, D. Baron, E.J. Wexler, and H.M. Finegold. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed. Star Publishing Company, New York, N.Y.

ATCC is a registered trademark of the American Type Culture Collection.

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