Bacteriological STAIN *Stains is any colored, organic compound, that used to stain tissues, cells, cell components, cell contents, or microorganisms. *The stain may be natural or synthetic. *The object stained is called the substrate. *Staining facilitates the observation of a substrate by introducing differences in optical density or in light absorption between the substrate and its surroundings or between different parts of the same substrate

According to nature of stain ACIDIC: Have negative charge so they bind to positively charged cell structures like some proteins. Ex: Nigrosine, Picric acid, Eosin, Acid fuschin, India ink BASIC: Have Positive charge so bind to negatively charged molecules in cell like cell wall. Ex: Crystal Violet, Methylene Blue, Safranin , basic fuschin. NEUTRAL: Both positively and negatively charged imparts different colors to different components. Ex: Geimsa’s stain, Leishman’s stain, Wright’s stain.

Bacterial smear

Crystal violet, Methylene blue and Carbolfuchsin. Classification of stains according to the purpose of use 1-Simple stains: Crystal violet, Methylene blue and Carbolfuchsin.

Results: All cells have the same color Morphology – cocci/spherical / rod. Arrangement – clusters/chains.

Gram staining 2-Differential stains: *more than one stains are involved in the staining proses and stains react differently with different cell types. Gram staining

Gram Staining Procedure

(CV-I) complex

Gram staining principles -

Results for gram stain G –ve and G +ve

Factors affecting the efficiency of Gram staining process 1- Bacterial smear thickness 2- Smear fixation 3- Concentration and freshness of reagents may affect the quality of the stain 4- The nature and age of the bacterial culture 5- The washing water False results G -ve may appear as G +ve G+ ve may appear as G-ve

Acid-fast stain Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids

ZIEHL-NEELSEN STAIN PRINCIPLE OF * * Carbolfuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall

1 2 3 4 5 6 7

How the Acid fast bacteria appear

SPECIAL STAINS 1- Capsules Stain (Negative stain) India ink Nigrosine

Flagella Stain Leifson’s stain Spores Stain Malachite green