Mycoplasma are a genus of bacteria that lack a cell wall. This makes them resistant to antibiotics that target cell wall synthesis. They can be parasitic or saprotrophic. Mycoplasma have no nucleus or organelles and their genetic material is naked DNA. They require specific growth factors, isotonic medium, and the absence of inhibitors for growth in culture. Samples are inoculated on agar plates and examined microscopically for the characteristic "fried-egg" mycoplasmal microcolonies after incubation.

1. Mycoplasma
Mycoplasma is a genus of bacteria that lack a cell wall around their cell membranes. This characteristic
makes them naturally resistant to antibiotics that target cell wall synthesis (like the beta-lactam
antibiotics). They can be parasitic or saprotrophic
Scientific classification
Domain: Bacteria
Phylum: Tenericutes
Class: Mollicutes
Order: Mycoplasmatales
Family: Mycoplasmataceae
Genus: Mycoplasma
Important characteristics of mycoplasma bacteria
 Cell wall is absent and plasma membrane forms the outer boundary of the cell.
 Due to the absence of cell wall these organisms can change their shape and are pleomorphic.
 Lack of nucleus and other membrane-bound organelles.
 Genetic material is a single DNA duplex and is naked.
 Ribosomes are 70S type.
 Possess a replicating disc at one end which assist replication process and also the separation of
the genetic materials.
 Heterotrophic nutrition. Some live as saprophytes but the majority are parasites of plants and
animals.
 The parasitic nature is due to the inability of mycoplasma bacteria to synthesize the required
growth factor
Culture media
Mycoplasmas are fastidious organisms and most require specific growth factors, an isotonic
medium and the absence of inhibitory substances for growth. The basic medium is a good
quality beef infusion with supplements. Mycoplasma and Ureaplasma species require
cholesterol and this is usually supplied by 20% horse serum. Serum from individual animals can
vary in growth-promoting properties; pooled serum from several animals is preferred. Other
growth factors are catered forby the addition of yeast extract, DNA and possibly nucleotides.
The water for the medium should be of a quality similar to that used for tissue culture. Penicillin
can be added to discourage the growth of Gram-positive bacteria and thallium acetate
to inhibit fungi and Gram-negative bacteria. The final pH of the medium is adjusted to between
7.2 and 7.8 for Mycoplasma species. The mycoplasmal transport medium can also make a
suitable broth medium for isolation. This can be converted to an agar medium by substituting 28
g of PPLO agar (Difco) for the 16.8 g of PPLO broth.
Sample sources
 Fluid materials such as foetal fluids and exudates
 Tissues, such as pieces of lung, a freshly cut surface on a block of the tissue can be moved
across the surface of an agar plate to inoculate it. Alternatively, the tissue can be homogenized
in broth.

2. Procedure in lab
The inoculated agar plates are incubated in a humid atmosphere at 37°C. We can incubate duplicate
plates, one aerobically and one under 5% CO2 and 95% N2. A candle jar may be satisfactory. The
plates should be examined after 48 and 96 hours’ incubation. The plates are viewed under the low-
power objective of a light microscope, for the characteristic mycoplasmal ‘fried-egg’
microcolonies. The cultures can be regarded as negative if no microcolonies are seen after 14
days’ incubation. Tubes of broth, incubated aerobically at 37°C, are checked daily for growth. When a
slight turbidity is seen (or a colour change if the broth contains phenol red), loopfuls of the broth can be
used to inoculate appropriate agar media.
Colonial morphology:
 When examined microscopically at low magnification, unstained microcolonies of Mycoplasma
species are 0.1 to 0.6 mm in diameter and have a 'fried-egg' appearance. Some species produce colonies
up to 1.5 mm in diameter which can be seen without magnification.