Structural staining
* Structural staining is used observe certain structures on bacteria.
* This is important because certain structures on bacteria can be antigenic or acts as an endotoxin.
* Structural stains are more complex than simples ones and use more than one stain to differentiate cellular components.
* They are used to examine structural differences between bacterial groups or to provide contrast to different structures within the same organisms.
* Examples of structural staining methods are cell wall staining, PHB staining, endospore staining etc.
cell wall staining
* Bacterial cell wall is present beneath the capsule and surrounding the cytoplasm.
* It is a rigid structure that gives definite shape to the bacterial.
* It is made made up of peptidoglycan.
* And because of its thickness and chemical nature, it is resistant to the staining.
* Thus, it is normally difficult to separately stain the cell wall of the cells.
* There are various staining techniques used to stain cell wall like chance's method, Ringer's method and Dyar's method.
Chance's Method
Principle :
"Bacterial cell is stained pink with new fuschin and then only cytoplasm is decolorized with congored solution while cell wall remains pink"
Mechanism :
* As we know cell wall and cytoplasm are acidic in nature where cell wall is more acidic than cytoplasm.
* So when we apply the first stain that is 0.5% new fuchsine which is the basic stain it stains the cytoplasm as well as the cell wall.
* But here we want to stain the cell wall so the smear is treated with the second stain that is 0.5% congo red solution.
* As we know congo red is a selective decolorizing agent it selectively decolorizes the less acidic portion that is cytoplasm.
* Hence cell wall remains stained by 0.5% new fuchsine stain.
Requirements:
* Clean grease free slide.
* Nichrome wire loop.
* Bacterial suspension.
* New fuchsine solution.
(Basic fuschin - 0.5g; Distilled water - 100ml)
* 0.5% congored solution.
Procedure:
* Take a clean, grease free slide.
* Prepare the smear of the suspension on the clean slide with a loopful of sample.
* Air dry and do not heat fix.
* New fuchsine is poured on smear and kept for about 3-4 minutes and rinse with water.
* Prepare a thin film of congored on dried smear with the help of another slide.
* Air dry, Blot dry and observe under microscope.
Observation:
Ringer et al. method
* Here mordant is used.
* In this method, tannic acid acts as a mordant that accelerates the reaction between cell and stain.
* Congored acts as decolorizing agent that decolorizes cytoplasm.
* And crystal violet (basic stain) stains the cell wall.
Principle :
" Bacterial cell wall is made positively charged by treatment with cationic surface agents e.g Tannic acid, so that it can take up anionic stain. But cytoplasm retains negative charge and thus, can be stained with contrasting basic stain".
Requirements:
* Clean grease free slide.
* Nichrome wire loop.
* Bacterial suspension.
* 4% tannic acid.
* 0.5% crystal violet solution.
* 0.5% congored solution.
Procedure :
* Take a clean, grease free slide.
* Prepare the smear of suspension on the clean slide with loopful of sample.
* Air dry and do not heat fix.
* Tannic acid solution is poured on smear and allow it to react for 30 minutes.
* Pour crystal violet and keep it for 1-2 minutes.
* Gently wash the slide with water.
* Prepare a thin film of congored on smear and allow it to react for 3 minutes.
* Air dry, blot dry and observe under microscope.
Observation :
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 :
