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2. INTRODUCTION
 Mycoplasmas are the smallest and simplest
self-replicating bacteria.
 The mycoplasma cell contains minimum set of
organelles essential for growth and replication: a
plasma membrane, ribosomes, and a genome
consisting of a double-stranded circular DNA
molecule.
 Unlike all other prokaryotes, the mycoplasmas
have no cell walls, and they are consequently
placed in a separate class Mollicutes(mollis,
soft; cutis, skin).

3. CONTD….
 Mycoplasmas have been nicknamed the
“crabgrass” of cell cultures.
 Contamination of cell cultures by mycoplasmas
presents serious problems in research laboratories
and in biotechnological industries using cell
cultures.
 The origin of contaminating mycoplasmas is in
components of the culture medium, particularly
serum, or in the flora of the technician's mouth,
spread by droplet infection.

4. GENERAL CHARACTERISTICS
 Very small(0.2-0.3 µm) .
 Can pass through bacterial filters.
 Lack a rigid cell wall.
 Bound by a single trilaminar cell membrane that
contains a sterol.
 Extremely pleomorphic varying in shape from
coccoid to filamentous to other bizzare forms.

5. CONTD…
 Cells may either divide
by binary fission or first
elongate to
multinucleate filaments,
which subsequently
breakup to coccoid
bodies.

6. CONTD….
 Membrane proteins & glycolipids exposed on the cell
surface are the major antigenic determinants in
mycoplasmas.
 Antisera containing antibodies to these components
inhibit growth and metabolism of the mycoplasmas and,
in the presence of complement, cause lysis of the
organisms.
 These properties are used in various serologic tests that
differentiate between mycoplasma species and
serotypes and detect antibodies to mycoplasmas in sera
of patients.

7. CONTD….
 Some mycoplasmas possess unique attachment organelles,
which are shaped as a tapered tip in M. pneumoniae and M.
genitalium. Mycoplasma pneumoniae is a pathogen of the
respiratory tract, adhering to the respiratory epithelium,
primarily through the attachment organelle.
 Interestingly, these two human mycoplasmas exhibit gliding
motility on liquid-covered surfaces. The tip structure always
leads, again indicating its importance in attachment.
 One of the most useful distinguishing features of
mycoplasmas is their peculiar fried-egg colony shape,
consisting of a central zone of growth embedded in the agar
and a peripheral one on the agar surface.

8. CULTURAL CHARACTERISTICS
 Aerobes and facultative anaerobes except Anaeroplasma which is
strictly anaerobic.
 For primary isolation, an atmosphere of 95% Nitrogen and 5%
Carbon dioxide is preferred.
 They can grow within a temperature range of 22-41°C, the parasitic
species growing optimally at 35-37°C.
 For fermentative organisms, the initial pH of the medium is adjusted
to 7.3-7.8, for arginine metabolizing organisms it should be around 7
and for ureaplasmas, range of pH should be 6-6.5.

9. CONTD….
 The dependence of mycoplasmas on their host for
many nutrients explains the great difficulty of
cultivation in the laboratory.
 The complex media for mycoplasma culture contain
serum, which provides fatty acids and cholesterol
for mycoplasma membrane synthesis.
 The requirement of most mycoplasmas for
cholesterol is unique among prokaryotes.
 The consensus is that only a small fraction of
mycoplasmas existing in nature have been
cultivated so far.

10. CONTD….
 Some of the cultivable mycoplasmas, including the
human pathogen M pneumoniae, grow very
slowly, particularly on primary isolation.
 Ureaplasma urealyticum, a pathogen of the
human urogenital tract, grows very poorly in vitro,
reaching maximal titers of 107organisms/ml of
culture.
 Mycoplasma genitalium, another human
pathogen, grows so poorly in vitro that only a few
successful isolations have been achieved.

11. MEDIA
 PPLO broth:
Bovine heart infusion broth to which are added 20% horse serum and
10% fresh yeast extract along with glucose and phenol red as a pH
indicator.
Growth of M.pneumoniae is detected by turbidity and colour
change(red to yellow) of phenol red indicator, due to fermentation of
glucose.
Ureaplasma and other mycoplasmas which do not ferment glucose
show only turbidity.
This medium can be solidified by the addition of agar.
Penicillin, polymyxin B and amphotericin B may be added to inhibit
contaminating bacteria and fungi respectively.

12. COLONIES
 Incubation: 2-6 days. Media for isolation of genital
mycoplasmas and M. pneumoniae should be incubated
for 1 and 4 weeks respectively, before a final culture
report is made.
 Size: 200-500µm for mycoplasmas and 15-60µm for
ureaplasmas. The colonies of Ureaplasma are extremely
small and thus Ureaplasma are also called T-strains
(tiny strains).
 Platinum loops: Can’t be picked up.
 Subculture is done by cutting out an agar block with
colonies and rubbing it on fresh plates.

13. COLONY MORPHOLOGY

14. CONTD….
 Mycoplasmas with the FRIED EGG COLONY
morphology appear highly granular and stain with a
dark blue centre and a light blue periphery.
 The agar background appears clear or slightly
violet.
 Mycoplasmas other than M. pneumoniae remain
stained, but M.pneumoniae reduces the methylene
blue after a period and becomes colourless.

16. RESISTANCE:
 Destroyed at 450C in 15 min.
 Resistant to lysis by Osmotic shock, penicillin and
Cephalosporins.
 Sensitive: To Surface acting agents, lipolytic
agents(taurocholate, digitonin), Tetracyclines,
Erythromycin.
 Susceptibility to Macrolide antibiotics and erythromycin is
used for species differentiation.
 Growth is inhibited by Gold salts.
 They( M. pneumoniae) can grow in presence of 0.002%
methylene blue in agar while other species are inhibited.

17. PATHOGENESIS

18. CONTD….
 Most mycoplasmas that infect humans and other
animals are surface parasites, adhering to the
epithelial linings of the respiratory and urogenital
tracts.
 Adherence is firm enough to prevent the elimination
of the parasites by mucous secretions or urine.
 The intimate association between the adhering
mycoplasmas and their host cells provides an
environment in which local concentrations of toxic
metabolites excreted by the parasite build up and cause
tissue damage .

19. CONTD….
 Moreover, because mycoplasmas lack cell walls, fusion
between the membranes of the parasite and host has been
suggested, and some experimental evidence for it has
recently been obtained.
 Membrane fusion would alter the composition and
permeability of the host cell membrane and enable the
introduction of the parasite's hydrolytic enzymes into the
host cell, events expected to cause serious damage.
 Recent studies have indicated the presence in mycoplasmas
of antigenic variability systems. These systems, some of
which are already defined in molecular genetic terms, are
responsible for rapid changes in major surface protein
antigens. The change in the antigenic coat of the parasite
helps it to escape recognition by the immune
mechanisms of the host.

20. EPIDEMIOLOGY
 All mycoplasmas cultivated and identified thus far
are parasites of humans, animals, plants, or
arthropods.
 The primary habitats of human and animal
mycoplasmas are the mucous surfaces of the
respiratory and urogenital tracts and the joints in
some animals.
 Although some mycoplasmas belong to the normal
flora, many species are pathogens, causing various
diseases that tend to run a chronic course.

21. LABORATORY DIAGNOSIS
 SPECIMENS COLLECTED:
Throat swabs, nasopharyngeal swabs, sputum, throat washings, bronchoalveolar lavage, tracheal aspirates and lung tissue
specimens.
Genital mycoplasmas may be isolated from urethral, vaginal and cervical swabs, semen, prostatic secretions, urine, blood,
CSF, amniotic fluid and biopsy specimens from endometrium, fallopian tubes, placentae and aborted foetus.
 CULTURE:
(1) PPLO broth medium dispensed into small vials may be used for transport of swab specimens, while other specimens may
be transported in sterile screw capped containers.
(2) The culture media should be inoculated as soon as possible.
(3) If delay in inoculation is expected, then specimens my be kept at 4°C for 24 hours and at -70°C for more than 24 hours.
(4) Urine specimens should be centrifuged and deposit inoculated into the medium. If inoculation is not immediately possible,
then urine deposit should be diluted with equal volume of transport medium and frozen.
(5) Later the specimens are processed as mentioned earlier.