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Enhancing Contrast with Indirect Negative Staining

Learn how to use electron-dense stains to highlight bacteria against the background, preserving fine details and morphology. Negative staining with nigrosine reveals capsule structures without distortion. Follow specific steps for precise results.

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Enhancing Contrast with Indirect Negative Staining

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  1. Indirect (negative) staining • Most biological materials show little contrast with their surroundings unless they are stained. • In the case of light microscopy, contrast can be enhanced by using colored stains which selectively absorb certain wavelengths.

  2. Indirect (negative) staining • To heighten the contrast between bacteria and the background, use is made of electron-dense "stains". • These are usually compounds of heavy metals of high atomic number, that serve to scatter the electrons from regions covered with the stain. • If bacterial particles are coated with stain (positive staining), fine detail may be obscured.

  3. Indirect (negative) staining • Negative staining overcomes this problem by staining the background and leaving the bacteria relatively untouched. • The negative stain is molded round the bacterial particle, outlining its structure, and is also able to penetrate between small surface projections and to delineate them.

  4. Indirect (negative) staining • The acidic dye nigrosine will be used to visualize the capsule or sheath that surrounds some bacteria in a process called negative staining. • Capsules are composed primarily of polysaccharides or glycoproteins and are gelatinous in texture.

  5. Indirect (negative) staining • They are readily destroyed by heating and hence direct staining methods cannot be utilized. • In general, the size and shape of microorganisms is often less distorted with indirect staining procedures, especially when sampled from a broth culture

  6. Indirect (negative) staining • Therefore negative staining is useful whenever the morphology of individual bacteria is in question. • Morphology can often be determined with confidence with only the high dry lens. Consider that this procedure does not necessarily kill the organism, so be careful.

  7. Indirect (negative) staining • After preparing a clean, greaseless slide, a small drop of nigrosine is mixed with a small drop from a broth culture or with a quantity of dry material. • The drop is spread across the slide using the edge of another slide as a spreader. This same procedure is used for blood smears.

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