Show Caption Hide Anthrax gram stain. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Decolorizer: Acetone 1 volume Ethyl alcohol 95 percent 1 volume Mix 1 volume of acetone with 1 volume of ethyl alcohol and store in a tightly sealed bottle.
Safranine 0 counterstain: Safranine 0 0. Filter and store away from direct sunlight. Procedure for Gram's Staining After the smear has been dried, heat-fixed, and cooled off, proceed as follows: Place slide on staining rack and cover specimen with crystal violet.
Principle of Gram's Stain The crystal violet stain is the primary stain, which stains everything in the smear blue. Reading and Reporting Smears Place a drop of oil in the slide and, using the oil immersion objective of the microscope, read the smear. The hospital corpsman reports only what he or she sees. Accept and continue. Solution A: 2 gm Crystal violet certified 20 ml Ethyl alcohol 95 percent. Solution B: 0. Mix solutions A and B, store for 24 hours, filter, and store at room temperature, in a dark bottle, in a dark place, away from direct sunlight.
Grind iodine and potassium iodide in mortar. Acetone 1 volume Ethyl alcohol 95 percent 1 volume. Mix 1 volume of acetone with 1 volume of ethyl alcohol and store in a tightly sealed bottle. Safranine 0 0. Some involve a single stain and just a few steps, while others use multiple stains and a more complicated procedure.
Before you can begin the staining procedure, the cells have to be mounted smeared and fixed onto a glass slide. A bacterial smear is simply that—a small amount of culture spread in a very thin film on the surface of the slide.
Heat fixing is an easy and efficient method, and is accomplished by passing the slide briefly through the flame of a Bunsen burner, which causes the biological material to become more or less permanently affixed to the glass surface. Heat fixed smears are ready for staining. In a simple stain, dyes that are either attracted by charge a cationic dye such as methylene blue or crystal violet or repelled by charge an anionic dye such as eosin or India ink are added to the smear.
Cationic dyes bind the bacterial cells which can be easily observed against the bright background. Anionic dyes are repelled by the cells, and therefore the cells are bright against the stained background. See Figures 1 and 2 for examples of both. Probably the most important feature made obvious when you stain bacterial cells is their cellular morphology not to be confused with colonial morphology, which is the appearance of bacterial colonies on an agar plate.
There is greater diversity of shapes among Archaea and other bacteria found in ecosystems other than the human body. Often bacteria create specific arrangements of cells, which form as a result of binary fission by the bacteria as they reproduce. Arrangements are particularly obvious with non-motile bacteria, because the cells tend to stay together after the fission process is complete. Both the shape and arrangement of cells are characteristics that can be used to distinguish among bacteria.
The most commonly encountered bacterial shapes cocci and bacilli and their possible arrangements are shown in Figures 3 and 4. In microbiology, differential staining techniques are used more often than simple stains as a means of gathering information about bacteria. Differential staining methods, which typically require more than one stain and several steps, are referred to as such because they permit the differentiation of cell types or cell structures.
The most important of these is the Gram stain. Other differential staining methods include the endospore stain to identify endospore-forming bacteria , the acid-fast stain to discriminate Mycobacterium species from other bacteria , a metachromatic stain to identify phosphate storage granules, and the capsule stain to identify encapsulated bacteria.
We will be performing the Gram stain and endospore staining procedures in lab, and view prepared slides that highlight some of the other cellular structures present in some bacteria.
In , physician Hans Christian Gram was studying the etiology cause of respiratory diseases such as pneumonia. He developed a staining procedure that allowed him to identify a bacterium in lung tissue taken from deceased patients as the etiologic agent of a fatal type of pneumonia.
The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain crystal violet by resisting a decolorization process. Gram staining involves four steps. First cells are stained with crystal violet, followed by the addition of a setting agent for the stain iodine.
Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink. Gram negative cell walls have an outer membrane also called the envelope that dissolves during the alcohol wash.
This permits the crystal violet dye to escape. Only the decolorized cells take up the pink dye safranin, which explains the difference in color between the two types of cells. At the conclusion of the Gram stain procedure, Gram positive cells appear purple, and Gram negative cells appear pink.
When you interpret a Gram stained smear, you should also describe the morphology shape of the cells, and their arrangement. The primary limitation of the technique is that it yields erroneous results if mistakes are made in the technique.
Practice and skill are needed to produce a reliable result. Also, an infectious agent may not be bacterial. Eukaryotic pathogens stain gram-negative. However, most eukaryotic cells except fungi including yeast fail to stick to the slide during the process. Not all bacteria identified by the Gram stain are associated with diseases, but a few important examples include:.
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