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Practical seminar 4 • Acid fast staining - Mycobacterium tuberculosis • Corynebacteriuôm diphtheriae – Albert´s staining of metachromatic granules Acid fast staining • Bacterial cell resistant to colorisation with some staining (Gram) • Their cell wall is relatively not permeable, containig lipids, fatty acids...(ex. Mycobacterium tbc, Corynebacteria...) • If stained (with strong basic stains (basic fuchsin in 5% fenole – they resist to decolorisation with strong acids (20% H2SO4) Ziehl Neelsen staining • Heat fixed smear • Apply carbol fuchsin (basic fuchsin + phenol) • Warm untill evaporation • Rinse with water • Apply 3% HCl (or 95% alcohol • Counterstain with malachit green Mycobacterium tbc – farbenie podľa Ziehl Neelsena Metachromatic granules staining • • • • • Volutin storage granules in cytoplasma Stained with basic stain Toluidin blue, methylen blue They are acidoresistent Differentiation of C. difteriae from saprophytic corynebacteria • Albert´s staining Metachromatic staining principals • Stain aplied on fixed cell or tissue is of different color as is the result color • Ernst Babes inclusions – metachromatic granuls – volutine granuls – phosphate molecules - Corynebacterium diphteriae • Volutin – nucleoprotein complex – in cytoplasm of some bacteria, molds and parasits in the forme of storage granules Albert´s staining • • • • Fix smear Apply Albert´s solution Add Lugol solution Bacteria are green or blue, (malachit green or methylen blue), granuls are red
