CULTURE - Aerobes and facultative anaerobes
- Slow growing, highly fastidious,senstivity of isolation is 40%
- Specimen to be stored at -70 C if immediate plating not possible(>24 hrs)
- Media:1-PPLO based agar,broth or biphasic media(bovine heart infusion broth) Enriched with yeast extract,horse serum,glucose.
- Penicillin and Polymixin B is added to inhibit contaminating bacteria.
- -Phenol red added as indicator
- -Incubated at 37 C,with 5-10%CO2 ,95%N2
- Fried egg appearance of colony in 2-6 days
- Colonies visualised by Dienes staining or IF method
Fried egg appearance of colony Dienes method-Visualisation of colony - A block of agar containing the colony is cut and placed on slide.
- It is covered with a cover slip on which an alcoholic solution of methylene blue and azure has been dried.
- Observed under low power
- Subcultures are done by cutting agar blocks with colonies and rubbing them on fresh plates.
colony Further identification of colonies - Haemadsorption test:colonies growing on surface of agar are flooded with washed guinea pig RBCS
- -Colonies of M pneumoniae adsorb RBCS on their surface and show beta hemolysis
- Tetrazolium reduction test:colourless coloured compound
SEROLOGY NON SPECIFIC TESTS - 1-Steptococcus MG test:
- Serial dilutions of patients unheated sera are mixed with killed suspension of streptococcus MG
- Agglutination is observed after overnight incubation at 37 C
- A titre of>1:20 is suggestive
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