Culture
The culture method is slow and time-consuming, requiring specialized media. Culture is, however, used by specialized laboratories to type organisms. Some of these Mollicutes take as much as 8 weeks to grow.
- Media must contain beef or soybean protein, serum, fresh yeast extract, and other factors to help growth occur.
- The medium must contain a selective agent to prevent the overgrowth of other bacteria during this slow growth phase.
Specialized buffered commercial media is available for M. pneumoniae. A7 or A8 agar medium may be used for isolation of U. urealyticum, U. parvum, or M. hominis. Broths, such as SP-4 glucose with arginine or urea may be added to the procedure. (Broths may contain pH and/or urea as indicators, helping the technologist determine if growth is present and when to subculture the broth to agar.) In general, agar is incubated at 37°C in 5-10% CO2 or anaerobically in 95% N2 plus 5% CO2.
(Note: Hayflick agar - as seen in the image - is useful in the detection of mycoplasmas contaminating normally sterile tissue culture media or pharmaceutical products during the production process.)
Appearance on special culture media
Colonies are usually quite small. The term, "T-strain" has been used to describe the tiny strain colonies of Ureaplasmas and Mycoplasmas. (Colonies are a mass of bacterial microorganisms originating from a single mother cell.)
Colonial growth may be anywhere from 10 to 300 μm (depending on the genus and species) - and may be viewed using a stereoscopic examination of the agar plates17:
- Mycoplasma pneumoniae colonies have a characteristic spherical (and sometimes, "fried egg") appearance on buffered agar. Typical colonies are 10 to 100 μm.
- Colonies of M. genitalium often appear more spherical, with a "fried egg" look.
- M. hominis colonies appear larger (200-300 μm), with a "fried egg" look.
- The colonies of Ureaplasma species are often smaller (15 to 60 μm), appearing as "sea urchin" shapes.
Rate of growthUreaplasma species and
Mycoplasma hominis cultures are usually held at least 10 days before reporting as negative, while
Mycoplasma pneumoniae cultures may be held for as long as 21 days.
Mycoplasma genitalium cultures can take months to grow (if they grow at all). This slow growth coupled with the complexity of culture is precisely why other test methods, such as direct specimen PCR testing (see next page) have become so popular.
Colonial growth is enumerated, and PCR may be used to provide definitive species identification.
Metabolic activity
After a 24-hour growth of the pure organism in a liquid medium (method-dependent), testing by substrates is necessary. Substrates may be used to differentiate the major Mycoplasmas and Ureaplasma. A few notable reactions are:
- M. pneumoniae requires glucose;
- U. urealyticum and U. parvum require urea; and
- M. hominis requires arginine.
17. Leber A, Burnham CD, eds. Clinical Microbiology Procedures Handbook. 5th ed. Washington DC: ASM Press; 2023.
18. CDC/Forrester. Image #10930. "Under a magnification of 125X, this photograph revealed some of the morphologic features exhibited by a number of medium-sized, Ureaplasma urealyticum colonies, otherwise known as T-strain mycoplasma. These organisms produced heavy growth on 1ml agar." PHIL public domain. Created 1973. Accessed December 29 2023. https://phil.cdc.gov/Details.aspx?pid=10930
