Only the decolorized cells take up the pink dye safranin, which explains the difference in color between the two types of cells. This permits the crystal violet dye to escape. Gram negative cell walls have an outer membrane (also called the envelope) that dissolves during the alcohol wash. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink.Īlthough Gram didn’t know it at the time, the main difference between these two types of bacterial cells is their cell walls. Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. First cells are stained with crystal violet, followed by the addition of a setting agent for the stain (iodine). 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. Today we use Gram’s staining techniques to aid in the identification of bacteria, beginning with a preliminary classification into one of two groups: Gram positive or Gram negative. Although it did little in the way of treatment for the disease, the Gram stain method made it much easier to diagnose the cause of a person’s death at autopsy. 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. In 1884, physician Hans Christian Gram was studying the etiology (cause) of respiratory diseases such as pneumonia. The most commonly encountered bacterial shapes (cocci and bacilli) and their possible arrangements are shown in Figures 3 and 4.įigure 5. Both the shape and arrangement of cells are characteristics that can be used to distinguish among bacteria. Arrangements are particularly obvious with non-motile bacteria, because the cells tend to stay together after the fission process is complete. Often bacteria create specific arrangements of cells, which form as a result of binary fission by the bacteria as they reproduce. There is greater diversity of shapes among Archaea and other bacteria found in ecosystems other than the human body. Most heterotrophic and culturable bacteria come in a few basic shapes: spherical cells (coccus/cocci), rod-shaped cells (bacillus/bacilli), or rod-shaped cells with bends or twists (vibrios and spirilla, respectively). 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). See Figures 1 and 2 for examples of both.įigure 2. Anionic dyes are repelled by the cells, and therefore the cells are bright against the stained background. Cationic dyes bind the bacterial cells which can be easily observed against the bright background. 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. Heat fixed smears are ready for staining. 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. To prevent the bacteria from washing away during the staining steps, the smear may be chemically or physically “fixed” to the surface of the slide. Before you can begin the staining procedure, the cells have to be mounted (smeared) and fixed onto a glass slide.Ī bacterial smear is simply that-a small amount of culture spread in a very thin film on the surface of the slide. Some involve a single stain and just a few steps, while others use multiple stains and a more complicated procedure. There are many different stains and staining procedures used in microbiology. Contrast, however, can be improved by either using a different type of optical system, such as phase contrast or a differential interference contrast microscope, or by staining the cells (or the background) with a chromogenic dye that not only adds contrast, but gives them a color as well. Resolution is a limitation that we can’t do much about, since most bacterial cells are already near the resolution limit of most light microscopes. Two of the most important concerns are resolution and contrast. The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular.
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