PHB staining
* Bacterial cell inclusion bodies are also known as granules.
* These granules function as energy storage and these are also involved in reducing osmotic pressure.
* Some of these granules are polyphosphate granules, sulphur granules, glycogen granules and PHB granules etc.
* Poly (3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes.
* These granules serve as lipid reserve materials in bacteria.
* PHB granules are produced by microorganisms e.g. Bacillus megaterium apparently in respnse to conditions of physiological stress; mainly conditions in which nutrients are limited.
* PHB granules allow survival of the cells in the absence of suitable carbon sources.
* The PHB polymer is primarily a product of carbon assimilation (from glucose or starch) and is empolyed as a form of energy storage molecules to be metabolized when other common energy sources are not available.
* These granules are visible under a light microscope by staining with sudan black.
Principle :
" Sudan Black B is a slightly basic dye that combines with the acidic groups in the pilid compounds, hence staining the PHB granules".
Mechanism :
* In Burdon's method of PHB staining, Sudan black B stain is used.
* It is a fat soluble stain which stains lipid layer by getting dissolved in it.
* Safranin is the counter stain which stains the cytoplasm of bacterial cells.
Requirements :
* Clean grease free slide.
* Nichrome sire loop.
* Bacterial suspension.
* Sudan black B stain.
* Xylene
* 0.5% safranin solution.
Procedure :
* Take a clean, grease free slide.
* Prepare a thin smear of suspension on the clean slide with a loopful of sample.
* Air dry and heat fix.
* Sudan black B stain is pored on smear.
* Remove excess stain and dry it.
* Rinse the smear with xylene and blot dry.
* Flood the dried smear with 0.5% safranin.
* Allow to react for 5-10 minutes.
* Air dry, Blot dry and observe under Microscope.
Observation :
