A8 AGAR

Cat. no. G02 A8 Agar, 15x60mm Plate, 11ml 10 plates/bag

INTENDED USE

Hardy Diagnostics A8 Agar is used for isolation and differentiation of genital mycoplasmas, i.e. Ureaplasma urealyticum and Mycoplasma hominis.

SUMMARY

A8 Agar is highly nutritious due to peptones supplemented with yeast extract and inactivated horse serum present in the medium. The yeast extract provides diphosphopyridine nucleotides and the serum provides cholesterol and a source of protein. Amphotericin B and penicillin G are added to inhibit faster growing contaminants.

The discovery of a urease enzyme system in Ureaplasma urealyticum makes methods of identification possible. Ureaplasma urealyticum can be differentiated from other genital mycoplasmas on A8 due to manganous sulfate in the medium which combines with the urease to form a golden brown pigment.

FORMULA

Ingredients per 766ml of deionized water:*

Putrescine, 2HCl 34.0gm
Tryptic Soy Broth 24.0gm
Dipotassium Phosphate 2.5gm
Dextrose 2.5gm
Manganous Sulfate 0.2gm
L-Cysteine, HCl 0.1gm
Amphotericin B 2.5mg
GHL Tripeptide 2.0mg
Horse Serum 188.0ml
Yeast Extract 35.0ml
Urea Solution, 10% 10.0ml
Koenzyme Enrichments 1.0ml
Penicillin G 1,000,000U
Agar 11.0gm

Final pH 6.0 +/- 0.2 at 25ºC

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC 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: Specimen/swab should be placed in a tightly sealed transport container with suitable transport medium to prevent drying and taken directly to the laboratory. Dacron, polyester, or calcium alginate swabs are acceptable. Samples should be processed as soon as possible after arrival to the laboratory. If there is to be a delay in culturing, specimens should be refrigerated at 2-8ºC. For long-term storage, or if specimens cannot be cultured within 24 hours, freeze specimens at -70ºC. Do not freeze at temperatures greater than -70ºC.

Method of Use: Vaginal swabs, urethral swabs, cervical swabs and urine are suitable for culturing. Urethral exudates are streaked with a loop or rolled with a swab on culture plates immediately. Urine specimens are centrifuged, the supernatant removed, and the sediment streaked on culture plates. Increased recovery may be enhanced by diluting and plating the specimen serially up to 10-3. Diluting the specimen minimizes the effect of bacterial inhibitors on the growing mycoplasma.(1) Agar plates should be taped to restrict dehydration. Incubate plates in 5-10% CO2 at 35ºC for up to 10 days.

INTERPRETATION OF RESULTS

Examine inverted plates microscopically at 40X-100X. Mycoplasma hominis is recognized by typical "fried-egg" colonies or finely granular colonies with a berry-like appearance that penetrate the agar surface. M. hominis colonies range from 20-300µm. Ureaplasma urealyticum on A8 Agar is characterized by golden brown, small granular colonies that penetrate the agar surface. U. urealyticum colonies range from 15-60µm. Consult listed references for more information regarding cultivation and isolation of mycoplasmas.(4,5)

LIMITATIONS

Occasional breakthrough of bacterial growth may occur on medium. Similarities of L-form bacteria and mycoplasma organisms on the agar may cause some confusion because they both exhibit "fried-egg" colonies that penetrate the agar surface. L-form colonies tend to be larger and demonstrate a rougher surface. Many L-form will revert back to the bacterial form if passed to a penicillin-free medium.

Increased recovery may be enhanced by diluting and plating the specimen serially up to 10-3. Diluting the specimen minimizes the effect of bacterial inhibitors on the growing mycoplasma.(1)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycoplasma hominis
ATCC ® 23114
K 72-96hr 35°C CO 2 ** Growth observed microscopically at 1-4 days with a "fried-egg" or granular appearance
Ureaplasma urealyticum
ATCC ® 27618
K 72-96hr 35°C CO 2 ** Growth observed microscopically at 1-4 days; golden brown colonies, coarse, granular with irregular edges
Staphylococcus epidermidis
ATCC ® 12228
B 24hr 35°C Aerobic Inhibited
Candida albicans
ATCC ® 10231
B 24hr 35°C Aerobic Inhibited

** Atmosphere of incubation is enriched with 5-10% CO2.

User Quality Control

PHYSICAL APPEARANCE

A8 Agar should appear slighlty opalescent, and whitish to light amber in color.

M. hominis and U. urealyticum growing on A8 Agar

Microscopic image of Mycoplasma hominis (ATCC® 23114) and Ureaplasma urealyticum (ATCC® 27618) colonies growing on A8 Agar (Cat. no. G02). Incubated in CO2 for 72 hours at 35°C. All colonies are mycoplasma except for the dark one, which is ureaplasma.

REFERENCES

1. Versalovic, J., et al. Manual of Clinical Microbiology. 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. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

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

5. Clyde, W.A., et al. 1984. Cumitech 19; Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections, Coordinating ed., W.L. Drew. American Society for Microbiology, Washington, D.C.


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080216gr