In contrast, most gram-negative bacteria...

Gram-Stain is the most common and cost-effective technique used for staining detection of bacterial organisms. This method was originally developed in 1884 by Danish bacteriologist Hans Christian Gram. PRINCIPLE gram staining method is based on the ability of bacterial cell wall to keep the crystal violet dye in the bleaching treatment bleaching agent. Cell wall of gram-positive bacteria have a higher peptidoglycan and lower lipid content than gram-negative bacteria. Bacterial cell wall, originally painted with crystal violet. Iodine is added as a fixative to form a complex way that paint can not be easily removed. This step is usually called fixing dyes. Further treatment decolouriser, a mixture of ethanol and acetone dissolves the lipid layer of gram-negative cells. Removal of lipid layer allows leaching crystal violet stain from the cells. When the counter stain safranina added discolored gram-negative bacteria stain pink. Unlike solvent dehydrates the thick gram-positive cell walls, closing the pores, as compacted cell wall. As a result, violet-iodine complex and kept the cells remain purple. The basic procedure for staining 1) Make a thin layer of cells of bacteria to clean glass slide with fresh (18-24 hours of growth), an isolated colony from nutrient agar plate growth. Allow to air dry. 2) Secure with 95% methanol for 2 minutes, air dry. This is the current recommended method of fixation, because it does not distort the cells. Heating slides (which is the traditional method) can cause artifacts and correct for sample slide well. 3) flood the slide with crystal (or gentian) violet staining reagent for thirty seconds. 4) Rinse the slide with a gentle and indirect stream of tap water for two seconds. Tilt the slide to drain excess water. 5) flood the slide with iodine staining reagent for thirty seconds. 6) Repeat step 4. 7) Add decolouriser to slide from its slope until the color is not washed off, about 10 30 seconds. 8) Repeat step 4. 9) Flood slide with safranina (pink) against staining reagent for thirty seconds. 10) Wash the glass in a gentle, indirect stream of tap water until the color is not washed off. Air dry and pat dry slide filter paper. 11) View the slides using a light microscope under oil immersion. Gram-positive bacteria appear blue or purple and gram-negative bacteria appear pink-red. TROUBLESHOOTING Problem Gram smear preparation: If the smear is too thick, the cells may appear gram-positive in a very large area. You can see the variability of gram-large to the small area. SOLUTION: Try to make a single layer of cells. Run positive and negative control may help (previously prepared slides with controls that are available for purchase). PROBLEM: If the cells prepared in hyper-or hypotonic solutions, the morphology may be affected. SOLUTION: a swab of cells on the slide dry sterile toothpick. PROBLEM: The warming smear (this happens most often when smears returns to being fully dried in the air, or when burning too much to fix the slides) will cause all cells appear Gram-negative. SOLUTION: Dry slide completely to fix the heat, be very careful when using fire. PROBLEM: Old culture smears can cause cells to appear Gram-negative (low wall). SOLUTION: Prepare smears only from fresh, log-phase growth (usually at night culture). PROBLEM: Wednesday, from which the colony is important. Often, if you take the colonies to liquid media or selective media, Gram-negative cells appear more coccoid. SOLUTION: Take the example of fresh colonies on nutrient agar medium. Coloring Problem: Over-bleaching: This is a very common problem, often caused by using or keeping a strong decolouriser decolouriser on the slide for too long. Use decolouriser of the same company as your stains, and follow their protocol. Check your decolouriser solution using different times (5, for example, 10, 15 seconds) with a positive, negative and some positive cultures slightly, until you find the method that works well for you. PROBLEM: Gram variability: This may be due to the organism, not staining. SOLUTION: Most gram-negative microorganisms, partly gram-positive. Typical Gram-variable organisms (eg Corynebacterium variabilis), or those whose membrane change with age and appear Gram-variable (for example, Arthrobacterium srr.) Grouped with gram-positive microorganisms. Thus, they are regarded as Gram-positive microorganisms. GRAM coloring options are normally training gram of iodine unstable and can lose up to 60% available iodine for 30 days when stored at 25C. Gram-positive microorganisms can paint badly, when 40% of the available iodine is lost. A solution of iodine stabilized some manufacturers adding sodium bicarbonate in solution, and a stabilized iodine-polyvinylpyrrolidone iodine. Additional methods to distinguish between gram-positive and gram-negative bacteria, Gram results are not always conclusive tests to indicate the structure of the bacterial cell wall. Some gram-positive bacteria lose some of their cell wall properties with age or exposure to harsh or bacterial static / bactericidal agents that cause them the appearance of Gram-negative and gram-variable. In addition, many sp Bacillus. and Clostridium sp. spot false gram-negative. Some additional tests may be performed to assist in determining the Gram reaction, although none of them correct. A. L-alanine-4-nitroanilida (LANA) can be divided into aerobic and facultative anaerobic. Reagent commercially soaked in cotton swabs, which turn yellow when touched to the colonies of Gram-negative bacteria. 2. Potassium hydroxide (KOH) test also detect gram-negative bacteria. The cycle of growth of colonies on the surface of glass mixed in a suspension of 3% KOH and stirred continuously for 60 seconds. Gram-negative cell wall collapse and when the cycle gently out of the suspension will be thick and stringy. 3. Vancomycin is an antimicrobial agent that acts on the wall of most gram-positive bacteria. Major impart body to the surface of agar plates of sheep blood and put in 5 mcg vancomycin disk grafted area. Any zone of growth inhibition after overnight incubation usually indicates gram-positive bacteria. Many lactic acid bacteria are usually resistant to vancomycin. Some gram-negative microorganisms, particularly Moraxella and Acinetobacter, may be susceptible to vancomycin. In contrast, most Gram-negative bacteria are sensitive to polymyxin at 10ug/ml. 4. Maconkey agar selective for the growth of Gram-negative bacteria. Gram-positive bacteria inhibited crystal violet and bile acid salts. 5. Columbia agar kolistyn-nalidixic strattera 25mg acid (CNA) is selective for the growth of gram-positive cocci. .