If you have an experience with this bacteria please give an advice which medium is the best choice for cultivation. What cultivation conditions are better - aerobic or anaerobic?
Dear dr. Dimitar Parvanov!
Here is my answer about sutiable culture medium for Gardanella sp.:
GARDNERELLA SELECTIVE AGAR
Cat. no. G292Gardnerella Selective Agar, 15x100mm Plate, 18ml10 plates/bag
INTENDED USE
Hardy Diagnostics Gardnerella Selective Agar is recommended for the selective isolation and differentiation ofGardnerella vaginalis from clinical specimens.
SUMMARY
Gardnerella vaginalis is considered one of the leading causes of bacterial vaginitis. Conflicting interpretations on the clinical significance of this organism have varied over the years, along with its taxonomic classification.(2,5,10)Consequently, the organism has been suggested as the etiologic agent in a variety of other urogenital and reproductive diseases, including preterm birth, chorioamnionitis, urinary tract infections, newborn infections, and septicemia.(4)Because G. vaginalis may be isolated from patients with nonspecific vaginitis and asymptomatic patients, use of selective and differential media to detect the organism in mixed cultures significantly improve isolation rates.(3,5)
Detection of G. vaginalis on standard blood-containing media in routine clinical practice may be difficult, as normal flora such as lactobacilli and streptococci grow and produce similar alpha hemolysis.(2) Mickelsen et al. developed a semi-selective medium containing 1% corn starch to distinguish G. vaginalis colonies by their hydrolytic clearing on opaque starch medium.(5) Gardnerella Selective Agar is based on this formulation and contains selective agents to inhibit the growth of normal competing flora, including yeast.
Gardnerella Selective Agar contains animal peptones, peptic digest of casein, yeast and beef extracts, which provide carbon, nitrogen, amino acids, and essential minerals to support microbial growth. Sodium chloride is added to maintain osmotic equilibrium. The medium is supplemented with corn starch to aid in the detection of hydrolytic colonies. Bromcresol purple is the pH indicator and agar is the solidifying agent. Colistin, nalidixic acid and amphotericin B are selective agents used to inhibit the growth of competing flora present in the sample. Organisms, such as G. vaginalis, capable of hydrolyzing starch create acid byproducts that change the surrounding medium yellow.
FORMULA
Ingredients per liter of deionized water:*
Pancreatic Digest of Casein12.0gm
Proteose Peptone10.0gm
Peptic Digest of Animal Tissue5.0gm
Sodium Chloride5.0gm
Beef Extract3.0gm
Yeast Extract3.0gm
Corn Starch1.0gm
Colistin0.01gm
Nalidixic Acid0.01gm
Amphotericin B0.004gm
Bromcresol Purple1.2mlAgar13.5gm
Final pH 7.0 +/- 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), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.
The expiration dating on the product label applies to the product in its intact packaging when stored as directed.
Refer to the document "Storage" on the Hardy Diagnostics Technical Document website for more information.
PRECAUTIONS
This product may contain components of animal origin. Certified knowledge of the origin and/or sanitary state of the animals does not guarantee the absence of transmissible pathogenic agents. Therefore, it is recommended that these products be treated as potentially infectious, and handle observing the usual universal blood precautions. Do not ingest, inhale, or allow to come into contact with skin.
This product is for in vitro diagnostic use only. It is to be used only by adequately trained and qualified laboratory personnel. Observe approved biohazard precautions and aseptic techniques. All laboratory specimens should be considered infectious and handled according to "standard precautions." The "Guidelines for Isolation Precautions" is available from the Centers for Disease Control and Prevention at www.cdc.gov/ncidod/dhqp/gl_isolation.html.
For additional information regarding specific precautions for the prevention of the transmission of all infectious agents from laboratory instruments and materials, and for recommendations for the management of exposure to infectious disease, refer to CLSI document M-29: Protection of Laboratory Workers from Occupationally Acquired Infections: Approved Guideline.
Sterilize all biohazard waste before disposal.
Refer to the document "Precautions When Using Media" on the Hardy Diagnostics Technical Document website for more information.
Refer to the document SDS Search instructions on the Hardy Diagnostics website for more information.
PROCEDURE
Specimen Collection: Specimens should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is a delay in processing, specimens should be inoculated into an appropriate transport medium and refrigerated until inoculation. Consult listed references for more information on specimen collection.(2,6-10)
Method of Use:
1. Bring medium to room temperature prior to use.
2. Inoculate the surface of the medium and streak for isolation.
3. Incubate plates at 35ºC. for 24-48 hours using an atmosphere containing 5-10% CO2: a candle jar, CO2 incubator or CO2 Gen Compact (Cat. no. CD020C) may be used.
4. Proceed with confirmatory testing on suspect colonies that produce a yellowing of the medium.
INTERPRETATION OF RESULTS
Gardnerella vaginalis will demonstrate growth and a yellowing of the medium surrounding colonies.
Enterococcus faecalis may grow, but will not produce a yellow color change.
LIMITATIONS
It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
G. vaginalis isolates should be incubated in a 5-10% CO2 enriched atmosphere for best results.(2,6-9)
Refer to the document "Limitations of Procedures and Warranty" on the Hardy Diagnostics Technical Documentwebsite for more information.
MATERIALS REQUIRED BUT NOT PROVIDED
Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, atmospheric chambers, CO2 Gen (Cat. no. CD020C), etc., as well as serological and biochemical reagents, are not provided.
QUALITY CONTROL
Hardy Diagnostics tests each lot of commercially manufactured media using appropriate quality control microorganisms and quality specifications as outlined on the Certificates of Analysis (CofA). The following organisms are routinely used for testing at Hardy Diagnostics:
Test OrganismsInoculation Method*IncubationResultsTimeTemperatureAtmosphereGardnerella vaginalis ATCC® 14018***A24-48hr35°CCO2**Growth; yellow color change in medium surrounding coloniesEnterococcus faecalis ATCC® 29212A24-48hr35°CCO2**Growth; no color changePseudomonas aeruginosa ATCC® 27853B24hr35°CAerobicPartial to complete inhibitionProteus mirabilis ATCC® 12453B24hr35°CAerobicPartial to complete inhibitionEscherichia coli ATCC® 25922***B24hr35°CAerobicPartial to complete inhibitionCandida albicans ATCC® 10231B24hr35°CAerobicPartial to complete inhibition
* Refer to the document "Inoculation Procedures for Media QC" on the Hardy Diagnostics Technical Documentwebsite for more information.
** Atmosphere of incubation is enriched with 5-10% CO2.
*** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.
USER QUALITY CONTROL
End users of commercially prepared culture media should perform QC testing in accordance with applicable government regulatory agencies, and in compliance with accreditation requirements. Hardy Diagnostics recommends end users check for signs of contamination and deterioration and, if dictated by laboratory quality control procedures or regulation, perform quality control testing to demonstrate growth or a positive reaction and to demonstrate inhibition or a negative reaction, if applicable. Hardy Diagnostics quality control testing is documented on the certificates of analysis (CofA) available from Hardy Diagnostics Certificates of Analysis website. In addition, refer to the following documents on the Hardy Diagnostics Technical Document website for more information on QC: "Introduction to Quality Control" and "Finished Product Quality Control Procedures," or see reference(s) for more specific information.
PHYSICAL APPEARANCE
Gardnerella Selective Agar should appear slightly opalescent, and purple in color.
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Gardneralla vaginalis (ATCC® 14018) colonies growing on Gardnerella Selective Agar (Cat. no. G292). Incubated in CO2 for 24 hours at 35ºC.
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Enterococcus faecalis (ATCC® 29212) colonies growing on Gardnerella Selective Agar (Cat. no. G292). Incubated in CO2 for 24 hours at 35ºC.
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. Catlin, B.W. 1992. Gardnerella vaginalis: Characteristics, Clinical Considerations, and Controversies. Clin. Microbio. Rev.; 5(3):213-237.
3. Hammann, R., A. Kronibus, N. Lang, and H. Werner. 1987. Quantitative studies on the vaginal flora of asymptomatic women and patients with vaginitis and vaginosis. Zbl. Bakt. A.; 265:451-461.
4. Martius, J. and D.A. Eschenbach. 1990. The role of bacterial vaginosis as a cause of amniotic fluid infection, chorioamnionitis and prematurity. Arch. Gynecol. Obstet.; 247:1-13.
5. Mickelsen, P.A., L.R. McCarthy and M.E. Mangum. 1977. New differential medium for the isolation of Corynebacterium vaginale. J. Clin. Microbiol.; 5:488-489.
6. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.
7. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.
8. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
9. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.
10. Piot, P. et al. 1982. Identification of Gardnerella (Haemophilus) vaginalis. J. Clin. Microbiol.; 15:19-24.
11. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.
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HDQA 2207F Rev. 012816hh
Finnaly, I am added also figures of colonies for better understanding.
Do its, make experiments and please report to me, because I am interested for the outcam!
Sincerely yours!
Dr. Bratko Filipič
Email: [email protected]