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Staining- Pharmaceutical MIcrobiology
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- 2. Simple staining Smearpreparation: A drop of water is placed in the centre of a slide One loopfuls of organisms is transferred to the centre of slide Spread the organisms over the slide The smear is allowed to dry Slide is passed through flame several times to heat-kill and fix organisms A bacterial stain is stained with crystal violet (fuchsin, methylene blue) 1 min Observe under microscope 2
- 3. Basic Dyes Methylene Blue Crystal Violet Carbol Fuchsin Safranin Malachite Green Acidic Dyes Picric Acid Nigrosin India Ink Eosin 3
- 4. DIFFERENCIAL STAINING Twoor more reagents Distinguish Bacterial groups Specific Structures Example Gram stain Acid Fast Stain 4
- 5. GRAM STAINING 1884Christian Gram Staining technique that separates bacteria into two groups: Gram-positive bacteria Gram-negative bacteria It is based on the composition of their cell wall. Gram staining uses crystal violet to stain cell walls ,iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria. Gram-negative organisms appear red or pink because they are counterstained. Because of presence of higher lipid content, after alcohol-treatment, the porosity of the cell wall increases, hence the CVI complex (crystal violet – iodine) can pass through. Thus, the primary stain is not retained. Also, in contrast to most Gram- positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide 5
- 6. Time Frame 1) 1minute 2) 1 minute 3) 15 seconds 4) 1 minute Rinse with water between each step 6
- 7. Ziehl-Neelsen Acid-Fast Stain - Acid fast retain red color in cell walls It is a special bacteriological stain used to identify acid- fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of this group because it is responsible for tuberculosis (TB) 7
- 8. The slideis flooded with Carbol Fuchsin, heated over steam bath at 90°C for 4min. The slide is then flooded with a mild solution of hydrochloric acid in isopropyl alcohol to destain the Carbol Fuchsin, thus removing the stain from cells that are unprotected by a waxy lipid layer. Thereafter, the cells are stained in methylene blue and viewed on a microscope. 8
- 9. ENDOSPORE STAINING Generaof bacteria that produce endospores Bacillus & Clostridium Other genera: Desulfotomaculum, Sporosarcina, Sporolactobacillus, Oscillospira, Thermoactinomyces Schaeffer-Fulton Method for Endospore Staining Preparation of a Bacterial Smear-Air dry Application of Primary Dye (Malachite Green) w/ concurrent application of heat Allow slide to cool Rinse with water Application of Counterstain (Safranin) 9
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- 12. NEGATIVE STAINING Acid Dye (-) chromogen Repelled by (-) cell wall Cells Colorless Seen against dark background 12
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