CRITERION™ Mycoplasma Broth Base

Cat. no. C8000 CRITERION™ Mycoplasma Broth Base 50gm
Cat. no. C8001 CRITERION™ Mycoplasma Broth Base 500gm
Cat. no. C8002 CRITERION™ Mycoplasma Broth Base 2kg
Cat. no. C8003 CRITERION™ Mycoplasma Broth Base 10kg
Cat. no. C8004 CRITERION™ Mycoplasma Broth Base 50kg

INTENDED USE

Hardy Diagnostics CRITERION™ Mycoplasma Broth Base is recommended for use as an enrichment medium for cultivating Mycoplasma spp. prior to subculturing specimens to plated culture media.

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.

SUMMARY

Mycoplasma belong to the class of Mollicutes, or "soft skin," and were first isolated from a pleuropneumonia infection in cattle.(1) Mycoplasma spp. are believed to be the smallest, free-living organisms; they are pleomorphic and vary in size from 0.2 to 0.3 nanometers.(2)

Since their initial discovery, several species of Mycoplasma have been isolated from human respiratory and genital tracts. M. pneumoniae is known to cause primary atypical pneumonia or "walking pneumonia", whereas M. hominis, M. genitalium and Ureaplasma urealyticum are important colonizers and potential pathogens of the human genital
tract.(1,3,6) Mycoplasma pneumoniae usually infects people younger than 40 years of age, causing between 15 and 50 percent of all pneumonia cases in adults, and causes an even higher percentage of pneumonia in school-aged
children.(2) The resulting symptoms from infection by M. pneumoniae include headache, fever, cough, chest pain, and sore throat.

Morton, Smith and Leberman first described the use of pleuropneumonia-like organism, or PPLO, media for identifying and cultivating Mycoplasma.(3) Most species of Mycoplasma use either glucose or arginine as their major source of energy and require cholesterol or related sterols for growth. Therefore, special or complex media are required for the successful isolation of Mycoplasma spp.(4,6)

Hardy Diagnostics CRITERION™ Mycoplasma Broth Base, when supplemented, contains horse serum to provide cholesterol and a source of protein and yeast extract for essential vitamins and amino acids. If desired, thallium acetate can be added, in addition to penicillin or ampicillin, to inhibit accompanying microbial flora. Alternatively, cefoperazone may be added when researching Mycoplasma spp. from human patient specimens. CRITERION™ Mycoplasma Broth Base is available for use as an enrichment medium prior to subculturing specimens onto a suitable plated medium for the culture of isolated colonies.

FORMULA*

Gram weight per liter: 25gm/L
Casein Peptone 7.0gm
Sodium Chloride 5.0gm
Yeast Extract

3.0gm
Beef Extract 3.0gm
Disodium Phosphate 2.2gm
Monopotassium Phosphate 2.0gm
Beef Heart Infusion 2.0gm

Final pH 7.8 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original light beige.

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

1. Suspend 25gm of the dehydrated culture media in 750ml of distilled or deionized water. Stir to mix thoroughly.

2. Heat as necessary to dissolve completely.

3. Sterilize in the autoclave at 121ºC. for 10 minutes.

4. Cool media to 45-50ºC.

5. Aseptically add 200ml of horse serum and 50ml of filter sterilized yeast extract to the cooled medium. Alternatively, Mycoplasma Supplement (Cat. no. 283610) can be used. Refer to the manufacturer's package insert for supplement preparation instructions. Also, add antibiotic supplements as desired.

6. Aseptically pour desired volume into sterile tubes.

PROCEDURE AND INTERPRETATION OF RESULTS

For information on procedures and interpretation of results, consult listed references.

LIMITATIONS

Due to nutritional variation, some strains may grow poorly or fail to grow at all on this medium.

There is little research for the comparison of direct culture versus enrichment culture. It can be assumed that enrichment culture will detect Mycoplasma spp. in smaller numbers more effectively than direct culture. However, enrichment culture may also detect nonpathogenic Mycoplasma in small numbers.(4)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycoplasma pneumoniae***
ATCC ® 29085
10 to 100 CFU 7-14days 35°C CO 2 ** Growth upon subculture to SP4 Agar w/Glucose
Escherichia coli****
ATCC ® 25922
B 24-48hrs 35°C Aerobic Partial to complete inhibition

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

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

**** Recommended negative QC strain for User Quality Control according to the CLSI document M22 if medium is prepared with selective agents.

User Quality Control

Physical Appearance

CRITERION™ Mycoplasma Broth Base powder should appear homogeneous, free-flowing, and light beige in color. The prepared media should appear clear, and light amber in color.

REFERENCES

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

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

3. Morton, Smith and Leberman. 1951. Venereal diseases. Am. J. Syphylis Gonorrh.; 35:361.

4. UC Davis Veterinary Medicine Extension, Culturing for Mycoplasma.

5. 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.

6. Cumitech 19; Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections. American Society for Microbiology, Washington D.C., August, 1984.

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

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

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


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061416gr