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 :
