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Staining the Purpose of Staining : We Stain Bacteria to Study There : A) Morphology and Arrangement

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Staining the Purpose of Staining : We Stain Bacteria to Study There : A) Morphology and Arrangement Staining The purpose of Staining : We stain bacteria to study there : A) Morphology and Arrangement . B)Differentiated bacteria to groups according to their biochemical composition of cell wall . C)Study structures of bacteria (capsule,flagella) . Stains are classified according to their functions into : 1)Simple stain (methylene blue , safranin) that help to stain the outlines of bacterial cells, giving one the characteristic shape ,size, and arrangement of the cells stained with the simple stain . 2)Differential stain (Gram stain ,acid fast stain) Differential stain will Differentiate between the two cells . 3)Special stains (capsule stain,flagella stain ) stained some structures of bacteria . Preparation of smear : 1. Clean the slide . 2. Place a loop full of water in the center of the slide . 3. Mix a small amount of bacteria using a loop with the water and spread it out . 4. Allow the slide to air dry. 5-Heat-fix the smear by passing the slide through the Benzen burner . 1)Simple stain : methylene blue stain 1. Prepare the smear . 2. Flood the slide with methylene blue stain for 3 mints . 3. Wash the slide with tap water gently,drain off excess water then let the slide dry in air or by using filter paper . 4. Exam it microscopically . 5. The bacteria will appear blue cells . 2) Differential stains : A)Gram staining . B)Ziehl Neelsen (Acid Fast ) staining : Is a differential stain used to identify acid-fast organisms as members of the genus Mycobacterium . 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 . Acid-fast organisms are highly resistant to disinfectants and dry conditions . Procedure : 1. Prepare the smear . 2. Flood the slide with carbol fuchsin . 3. Heat the slide gently over the Bunsen burner for 5 minutes .do not boil it ;if it stops steaming ,add more stain . 4. Rinse the slide gently with water . 5. Decolorize the slide with acid-alcohol . 6. Rinse the slide gently with water . 7. Counterstain with methylene blue for 2 minutes . 8. Rinse the slide gently with water . 9. Carefully blot the slide dry with filter paper . 10. Observe the slide under the microscope ,using proper microscope technique . Acid –fast cells will stain fuchsia ;Non acid-fast cells will stain blue. 3)Special stains: A)Capsule stain : Most bacteria have some kind of CAPSULE. This viscous surface layer is also known as the SLIME LAYER, the GLYCOCALYX and EXTRACELLULAR POLYMERIC SUBSTANCE (EPS). Most bacterial capsules are composed of polysaccharide however some genera produce polypeptide capsules. Capsular material is very moist (slimy) and any heating will cause it to shrink - it is for this reason that we will not heat fix the slide before staining. The polymers which make up the capsule tend to be uncharged and as such they are not easily stained. That is, we use a stain which stains the background against which the uncolored capsule can be seen. Our procedure, the Gin's Method, uses india ink to color the background and crystal violet to stain the bacterial cell "body". This structure helps the bacterial cell to ATTACH TO SURFACES and to AVOID BEING PHAGOCYTOSED. For instance, the oral streptococci produce a glucan based EPS which helps them to attach to the teeth. When this material begins to accumulate on the teeth it is referred to as dental plaque. organisms with capsules tend to be more virulent because of their resistance to phagocytosis and killing. Streptococcus pneumoniae exists in a smooth form (encapsulated) and a rough form (non- encapsulated). Only the smooth form is lethal for mice. Procedure : 1-Use a loop to mix a drop of water, a drop of india ink and a small amount of Klebsiella pneumoniae together at the end of a slide. 2-Use another slide to spread the smear like a blood smear. (As the instructor will demonstrate.) Allow the smear to air dry. DON'T HEAT FIX . 3- Flood the smear with crystal violet, 1 minute. Wash with water . 4- Observe the slide under the microscope. (The background will be dark. The bacterial cells will be stained purple .The capsule (if present) will appear clear against the dark background. (Capsule stain) Endospore Stain These are very resistant structures made by only a few genera of bacteria. The two genera which we will study are: Clostridium is an anaerobic organism that forms spores. Tetanus, botulism, gas gangrene are diseases caused by different species in this genus. Bacillus is a common aerobic genus whose species can form endospores. Anthrax and Bacillus cereus food poisoning are two diseases caused by members of this genus. Spores are extremely resistant structures, difficult to destroy with heat or other physical and chemical disinfecting agents. Endospore destroy with autoclave. Procedure : 1. Perform a bacterial smear of Bacillus or the organism you want to stain. 2. Place a small piece of bibulous paper over the smear. Saturate the paper with malachite green. 3. Heat the slide gently over the Bunsen burner for 5 minutes. Be sure to keep the bibulous paper saturated with malachite green during heating. 4. Remove the bibulous paper from the slide, and rinse the slide gently with water. 5. Counterstain with safranin for 2 minutes. 6. Rinse the slide gently with water. 7. Observe the slide under the microscope Endospores will stain green. Parent cells will stain red. (Endospore Stain) .
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