![]() ![]() (b) Another method for heat-fixing a specimen is to hold a slide with a smear over a microincinerator. (a) A specimen can be heat-fixed by using a slide warmer like this one. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples (Figure 1c).įigure 1. Chemical fixatives are often preferable to heat for tissue specimens. To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear), and the slide is then briefly heated over a heat source (Figure 1b). In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation. Fixation is often achieved either by heating ( heat fixing) or chemically treating the specimen. The “fixing” of a sample refers to the process of attaching cells to a slide. The second method of preparing specimens for light microscopy is fixation. Once the liquid has been added to the slide, a coverslip is placed on top and the specimen is ready for examination under the microscope. Sometimes the liquid used is simply water, but often stains are added to enhance contrast. Solid specimens, such as a skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Some specimens, such as a drop of urine, are already in a liquid form and can be deposited on the slide using a dropper. The simplest type of preparation is the wet mount, in which the specimen is placed on the slide in a drop of liquid. There are two basic types of preparation used to view specimens with a light microscope: wet mounts and fixed specimens. In clinical settings, light microscopes are the most commonly used microscopes. Here, we will focus on the most clinically relevant techniques. Indeed, numerous methods have been developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the microscope. ![]() We have already alluded to certain techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for sample preparation in greater detail. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens. In their natural state, most of the cells and microorganisms that we observe under the microscope lack color and contrast. Explain the procedures and name clinical applications for Gram, endospore, acid-fast, negative capsule, and flagella staining.Describe the unique features of commonly used stains.Differentiate between simple and differential stains.Air dry, Blot dry and Observe under Microscope.Add safranin for about 1 minute and wash with water.Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.Flood the gram’s iodine for 1 minute and wash with water.Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.Prepare the smear of suspension on the clean slide with a loopful of sample.A decolorizer made of acetone and alcohol (95%).Then when again stained with safranin, they take the stain and appears red in color. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in color. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms. Gram Staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. ![]()
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