Last updated on May 30th, 2021Most motile bacteria possess flagella, the shape, number, and also place of which are vital features in the differentiation of genera and species identification. Staining bacterial flagella differs from staining various other bacterial structures because it normally requires extraordinary care for the slides, stain, and also cells. Flagellar stains are painstakingly ready to coat the surchallenge of the flagella via dye or a steel such as silver.The number and also arrangements of flagella are important in identifying species of motile bacteria.Two techniques for staining flagella are in use:A wet-mount procedure (Ryu method)Dried-smear preparation (Leifkid staining technique)A wet-mount approach for staining bacterial flagella is extremely successful once a stable stain and also regular slides and also coverslips are supplied. This technique is straightforward for regime use once the number and also arrangement of flagella are crucial in identifying species of motile bacteria.The preparations are not long-term because the stain precipitates as the wet mount dries.Method:Grow the organism to be stained at room temperature on blood agar for 16-24 hrs.Add a small drop of water to a microscopic lense slide.Dip a sterile inoculating loop right into the sterile waterTouch the loopful of water to the nest margin briefly (this enables motile cells to swim right into the droplet of water)Touch the loopful of motile cells to the drop of water on the slide. NOTE: Agitating the loop in the droplet of water on the slide causes the flagella to shear off the cell.Cover the faintly turbid drop of water on the slide via a coverslip. A correct wet mount has badepend sufficient liquid to fill the space under a coverslip. Small air spaces around the edge are preferable.Examine the slide immediately under 40× to 50× for motile cells. If motile cells are not checked out, carry out not proceed through the stain.If motile cells are viewed, leave the slide at room temperature for 5 to10 minutes. This enables time for the bacterial cells to adbelow to either the glass slide or the cover slip.Apply 2 drops of RYU flagella stain gently to the edge of the cover slip. The stain will certainly circulation by capillary action and also mix through the cell suspension. Small air pockets about the edge of the wet mount are beneficial in aiding the capillary activity.(Note: The Ryu stain has 2 components. Equipment I, the mordant, has 10 ml of 5% aqueous solution of phenol, 2 g of tannic acid, and also 10 ml of saturated aqueous solution of aluminum potassium sulfate-12 hydprice. Systems II, the stain, is a saturated ethanolic solution of crystal violet (12 g in 100 ml of 95% ethanol).


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The final stain was prepared by mixing 1 component solution Il with 10 parts solution I and also then filtering the mixture through filter paper to remove coarse precipitate)After 5 to 10 minutes at room temperature, research the cells for flagella.Cells with flagella might be observed at 100× (oil) in the zone of optimum stain concentration, about fifty percent means from the edge of the coverslip to the center of the mount.Focmaking use of the microscopic lense on the cells attached to the coverslip quite than the cells attached to the slide facilitates visualization of the flagella. The precipitate from the stain is primarily on the slide fairly than the cover slip. EXPECTED RESULTSObserve the slide and note the following:Presence or absence of flagellaNumber of flagella per cellLocation of flagella per cell
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Quality control:Peritrichous: Escherichia coliPolar: Pseudomonas aeruginosaNegative: Klebsiella pneumoniae