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Staining of microorganisms (focus on bacteria) Staining: coloring a microbe with a dye that binds to and emphasizes certain structures Preparation: Fixing • Before staining, a sample of bacteria must be fixed to a microscope slide. • A smear of bacteria (thin film containing cells) is spread over the slide, and the cells are killed and fixed in place by exposure to dry heat. • Now we are ready to STAIN! What are dyes? • Dyes are salts composed of a positive and negative ion, one of which is colorful - this is called the chromophore. • Basic dye: chromophore is positive ion • Acidic dye: chromophore is negative ion Bacteria are somewhat negatively charged, so… Basic dyes bind to bacterial structures Examples: crystal violet, methylene blue Acidic dyes cause negative staining, since they may bind to background and avoid binding to the bacterium (charge repulsion) Staining techniques • Simple stain - solution of single basic dye Generally highlights entire microorganism. Mordant improves binding of dye to sample • Differential staining - divides bacteria into groups according to their reaction to the staining procedure - most popular = • Gram stain! The Gram Stain • Divides bacteria into two large groups, the gram-positive and the gram-negative. • Developed by Hans Christian Gram in 1884 to aid in bacterial identification. • So, how do you do it?... Gram stain procedure • 1. Apply a basic dye (usually crystal violet). This stains all the cells purple or blue and is called the primary dye. • 2. After a short time, wash off dye and apply iodine, the mordant: after it is washed off all cells appear purple. Gram staining cont’d • 3. Wash preparation with alcohol, a decolorizing agent which removes the stain from SOME bacteria. • 4. Apply another basic stain, safranin, which colors the unstained bacteria red or pink. Safranin is the counterstain. • When it is all over, – Gram-positive cells look purple – Gram-negative cells look red Why do bacteria react differently? • Structural differences in their cell walls account for the different reactions to the Gram stain. More about this soon! • Also popular, especially clinically, is the ACID-FAST STAIN. Why? Because it preferentially distinguishes bacteria of the genus Mycobacterium, which cause tuberculosis. Acid-fast Stain • Procedure is generally similar to the Gram stain. • 1. Apply a basic dye (carbolfuchsin). This stains all the cells red and is called the primary dye. • 2. Carbolfuchsin preferentially binds to cell walls rich in a certain type of wax. Acid-fast cont'd • 3. Wash preparation with acid-alcohol, a decolorizing agent which removes the stain from SOME bacteria (those without the waxy substance) • 4. Apply another basic stain, methylene blue, which colors the unstained bacteria blue. Methylene blue is the counterstain. • When it is all over, – Acid-fast cells look red – Non acid-fast cells look blue Special stains exist to... • Visualize microbial capsules (negative capsule staining); use an acid stain (colors background), then safranin (colors entire bacterium EXCEPT for capsule; capsule appears as a halo). (Capsules are related to virulence of pathogens) • Highlight endospores • Highlight flagella