MICROBIOLOGY LAB 5 BIDISHA PAUL 261-BH Why Stain? Bacteria is colorless. Staining enhances the contrast between bacteria and the surrounding material and enhance visualization of the cell. Greater details of the cell components can be observed using stain The morphology and arrangement of the cells can be seen. Bacterial morphologies BASIC SHAPES OF BACTERIA ARRANGEMENT Dr Ekta Chourasia Other shapes of bacteria Comma shaped Spirochetes Spirilla Morphology and arrangement can only be determined by 100 X lens Do NOT use staphylococcus as the arrangement. Instead use staphylo as arrangement Morphology and arrangement are interpretation Example: If the result of a bacterial stain is that the cell is round and in grape like cluster; the INTERPRETATION is cocci and staphylo STAINING CLASSIFICATION Chromophore - A chromophore is the color possessing portion of the solution and is therefore responsible for the stain color BASIC- If the chromophore is made up of positive ion, the stain is basic. Eg- Methylene blue, Crystal Violet, Carbol Fuchsin, Safranin, Malachite Green ACIDIC- If the chromophore is made up of negative ion, stain is acidic. Eg Picric Acid, Nigrosin, India Ink, Eosin STAINING TECHNIQUES Simple stain- only one reagent is used. All bacteria are stained similarly. Differential stain- multiple reagents used. Bacteria react to different reagents differently Direct Stain Negative Stain Simple Stain DIRECT STAIN Simple stain: One reagent used Bacteria stained Basic dye (+) chromophore (-) cell wall Shows morphology Size Shape Arrangement Examples Methylene Blue CF Crystal Violet SMEAR Smear is a distribution of bacterial cells on a slide for the purpose of viewing them under the microscope Simple Stain SIMPLE STAIN OF S.epidermidis Why Heat Fix ? Kills the organisms without serious distortion Adhere better to the slide Take up dye more easily Before Starting Wash your hands Disinfect tables Wear your lab coats Take Staining tray, rack, wax pencil, clothes pin, striker, loop, bibulous paper, wash bottles Things to note Use small drop of distilled water for faster drying. Used slides should be placed in amphyl pan. Do not heat fix for too long else it will distort the cells. Use very small amount of bacteria while preparing smears. You can save your slides by storing in slide box kept at the last table. Affix a masking tape label to the back of the slides. If your hands get stained use a stain remover cream located next to each sink and then wash off your hands.