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Bacterial stains Most bacteria are difficult to see under the bright field microscope. Bacteria are almost colorless and therefore show little contrast with the broth in which they are suspended. To visualize bacteria, either dyes or stains, are used. Since staining of bacterial cells is relatively fast, inexpensive, and simple. Staining microorganisms enables one to: 1. see greater contrast between the organism and the background. 2. differentiate various morphological types (by shape, arrangement, gram reaction, etc). 3. observe certain structures (flagella, capsules, endospores, etc.). Stains (dyes): are chemicals containing chromophores, groups that impart color. There are several staining methods that are used routinely with bacteria. These methods may be classified as: 1. Simple stain (nonspecific): a) Positive (basic stain). b) Negative (acidic stain) 2. Differential (specific). a) Gram stain. b) Acid-fast stain. 3. Special stain a) Capsular stain b) Endospore stain c) Flagellar stain 1 Simple Stain: The simple stain consists of one dye. The dye adheres to the cell wall and colors the cell making it easier to see. a) Positive (basic stain): are cationic; exhibits a positive charge. its bind to negatively charged cell structures like nucleic acids. Methylene blue, crystal violet and Carbolfuchsin are common basic stains. Dye binds to the specimen. b) Negative (acidic stain): are anionic; exhibits a negative charge. its bind to positively charged cell structures like proteins. Eosin , India ink and nigrosin are common acidic stains. Dye does not bind to the specimen, but rather around the specimen. Positive (direct)stain procedure: 1. Prepare a heat fixed smear of the culture you wish to examine 2. Cover the smear with methylene blue (1 minute) 3. Wash the excess stain off the slide by distilled water 4. Blot off excess stain using bibulous paper. DO NOT rub the slide, rather place the slide between two sheets of bibulous paper and press down gently. Paper will absorb excess dye. 5. Examine the slide under the bright field microscope. 2 Negative (Indirect) Stains: Because the cell wall is also negatively charged only the background around the cells will become stained, leaving the cells unstained. 3 Negative stain procedure: 1. Place a small drop (one-two loopfuls) of the negative stain (India ink) near the end of the slide. 2. Transfer one loop=full of the bacterial sample to the india ink and mix the two together. 3. Hold a clean slide at about a 20o angle to the first slide. Touch the edge of the clean slide to the bacteria/stain mixture so that the mixture spreads across the edge. 4. Spread the suspension across the surface of the slide by drawing the clean slide away from the mixture. 5. Air dry the slide. DO NOT HEAT FIX. Differential Stains : uses two or more dyes to differentiate between cells or structures; A.Gram stain Principle: Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the crystal violet-iodine complex that occurs during staining, while Gram-negative cells have only a thin layer of peptidoglycan. Thus Gram-positive cells do not decolorize with ethanol, and Gram-negative cells do decolorize. This allows the Gram-negative cells to accept the counter stain safranin. Gram-positive cells will appear blue to purple, while Gram-negative cells will appear pink to red. 4 Procedure: • Prepare a Gram stain 1) Using an inoculating loop prepare smears of the bacteria by spreading a thin suspension of each bacteria on a glass slide 2) Heat-fix the smears by passing them through the Bunsen burner flame a 3 times. 3) Place the slides on the staining rack. 4) Cover the smears with the primary dye, crystal violet, and let it stand for 30 seconds. 5) Rinse the slides with water. 6) Apply the mordant, Gram’s Iodine, by covering the smears and let it stand for 1 minute. 7) Rinse the slides with water. 8) Decolorize with ethanol for 15-30 seconds. Add the ethanol slowly until the crystal violet no longer floats up from the slide. Be careful not to decolorize for too long. 9) Rinse the slides with water. 10) Cover the smears with the counter stain, safranin, and let stand for 1 minute. 11) Rinse the slides with water. 12) Blot dry using the bibulous paper. 5 Procedure Reagent Cell color Gram Positive Gram Negative Primary stain Crystal Violet PURPLE PURPLE Mordant Iodine PURPLE PURPLE Decolorizer Alcohol PURPLE COLORLESS Counter stain Safranin PURPLE RED B. Acid-fast Stain (Ziehl Neelsen): Mycobacterium and many Nocardia species are called acid-fast because during an acid-fast staining procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent acid-alcohol (95% ethanol with 3% HCl). Nearly all other genera of bacteria are nonacid-fast. The acid-fast genera have the waxy hydroxy-lipid called mycolic acid in their cell walls. It is assumed that mycolic acid prevents acid-alcohol from decolorizing protoplasm. pink to red acid-fast cells and blue non-acid-fast cells; distinguishes the genera Mycobacterium and Nocardia from other bacteria; differential stain. A : acid fast bacteria B : non acid fast bacteria Ziehl Neelsen Acid-fast stain 6 Procedure Reagent Cell color Primary dye Carbolfuchsin Acid-fast Bacteria Acid-fast Bacteria RED RED Decolorizer Acid-alcohol RED COLORLESS Counter stain Methylene blue RED BLUE Procedure: 1. Add one loopful of sterile water to a microscope slide. 2. Make a heavy smear of tested bacteria. 3. Air dry and heat fix well. 4. Cover the smear with carbolfuchsin dye. Place a piece of paper towel on top of the dye. Be sure the paper towel is saturated with the dye. 5. Place the slide on the rack over dry heat for 2 minutes. 6. Cool and rinse with water. 7. Decolorize by placing a drop of acid alcohol on the slide and allowing it to sit for 15 seconds. 8. Wash the top and bottom of slide with water and clean the slide bottom well. 9. Counterstain with Methylene Blue for 30 seconds to 1 minute. 10.Wash and blot the slide with bibulous paper. 7 Special stains: A. Endospore stain: Principle: Spores have a durable outer coating that is composed of the protein keratin. This keratin coat resists staining so in order to stain a spore the primary stain, malchite green, must be heated to drive the stain into the spores. Vegatative cells are then decolorized with water and 0.5% safranin is used to counterstain. Thus endospores are stained green, while vegetative cells are stained red. (e.g. Bacillus spp., Clostridium spp.) Procedure: 1. Malachite green is the primary stain .which is placed on blotting paper over the smear gently heating over a warm water bath to penetrate the spore coat. 2. The bacteria are decolorized with water. leaves the endospores green as the stain is driven into the endospore . The malachite green is washed out of the vegetative cells with the water. 3. It is then counterstained with safranin. 8 B. Capsular stain: Capsules are structures composed of carbohydrate or glycoprotein that lay outside of an organism's cell wall and thus are in direct contact with the environment. Many bacteria produce capsules under the right conditions. Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. In this stain we use acidic and basic dyes: Acidic dye as India Ink and Nigrosen use to stain the background of the slide but basic dye as methylene blue and crystal violet use to stain the cell results in background being dark; cells are unstained or stained with simple stain; this reveals bacterial capsules- Klebsiella pneumonia. Procedures 1. Use an inoculating needle to suspend the organism in a drop of India Ink at one end of the slide. 2. Place the short end of a clean microscope slide into the suspension and spread the mixture across the slide to form a thin layer. 3. Allow to air dry. Do not heat fix. 4. Cover the smear with methylene blue for 2-3 minutes. 5. Rinse gently with water and allow to air dry. 9 Capsules appear as clear zones (halos) around the organism. Examples: Bacteria with capsules: Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas spp. . C. Flagellar stain: Purpose: To determine the presence/absence and location of flagella on various microorganisms Principle: Because bacterial flagella are very thin and fragile a special stain (flagella stain) is prepared that contains a mordant. This mordant allows piling of the stain on the flagella, increasing the thickness until they become visible. Various arrangements of flagella are seen on different cells. Preparation of smear : 1. Handling the suspension carefully remove a large loopful of the suspension and place it at one end of the rectangular area. 2. Tilt the slide to permit the suspension to run down to the other end of the slide. If the drop fails to run, add another drop. 3. Air dry the film. Do not heat. 4. Place the slide on a horizontal staining rack. Staining procedure : 10 1. Add about 1 ml. of the *Flagella Stain solution (one dropper full) to the smear and allow to stain for 10-15 minutes. 2. Flood off the stain by adding tap water to the slide while it remains on the rack. 3. Drain and flood the slide with Methylene blue for one minute. Rinse by flooding. Drain and air dry. Do not blot. *Flagella Stain solution: Nacl 1% Tannic acid 3% Basic fuchsine 1.25% Solvent in ethyl alcohol 95% 11