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. In Figure 5, there are two distinct types of bacteria, distinguishable by Gram stain reaction, and also by their shape and arrangement.
Below, describe these characteristics for both bacteria:. Some bacteria produce the waxy substance mycolic acid when they construct their cell walls.
Mycolic acid acts as a barrier, protecting the cells from dehydrating, as well as from phagocytosis by immune system cells in a host.
This waxy barrier also prevents stains from penetrating the cell, which is why the Gram stain does not work with mycobacteria such as Mycobacterium , which are pathogens of humans and animals. For these bacteria, the acid — fast staining technique is used.
To perform the acid-fast stain, a heat-fixed smear is flooded with the primary stain carbol fuchsin, while the slide is heated over a steaming water bath. Then the slide is allowed to cool and a solution of acid and alcohol is added as a decolorizer. All other cell types will be decolorized. Methylene blue is then used as a counterstain. However, this method is not suitable for the samples with very low concentrations of cells and there is no way to grow these cells.
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Article of the Year Award: Outstanding research contributions of , as selected by our Chief Editors. Read the winning articles. Journal overview. Special Issues. Academic Editor: Rony Seger. Received 14 Sep Accepted 04 Nov Published 17 Nov Abstract Cell staining is a necessary and useful technique for visualizing cell morphology and structure under a microscope.
Materials and Methods 2. Figure 1. Spreading cells on a slide with a glass spreader. The glass spreader was made from a glass transfer pipette by heating it for a few seconds to minutes over an alcohol burner. Figure 2. PMA induces macrophage-like differentiation. TF-1a cells were treated with PMA for 72 hours, after which the cells were collected and slides were prepared and then stained following the protocol described in Section 2.
Figure 3. TF-1a cells were treated with PMA for 48 hours, after which the cells were collected and slides were prepared following the method described in Section 2. Figure 4. Bay induces apoptosis in TF-1 but not in MV cells. Log-phase TF-1 a and MV b cells were treated with Bay for 24 hours, after which the cells were collected and slides were prepared and stained following the protocol described in Section 2. References C.
Shanholtzer, P. Schaper, and L. View at: Google Scholar J. Hanenberg and S. Fitzgerald and C. Maschmann, R. Goelz, S. Witzel et al. Olson, C. Shanholtzer, K.
Willard, and L. This should be mixed to dilute and stored in a dark flask. This can be used directly. This is then washed in tap water,. This is also referred to as pap-staining or pap smear. It is used for the purposes of examining cell samples that have been obtained from body fluids.
The technique involves the combination of chemicals that include:. A substitute of Scott's Tap water is prepared through mixing a part of Scott's Tap water substitute concentrate with 9 parts of deionized water. This is then followed by filtering papanicolaou staining system reagents before use.
A 50 for two minutes,. Pusiol et al. CytoJournal 5 Acid fuchsin is a magenta red acid dye that is largely used for plasma staining whereas basic fuchsin is a magenta basic dye largely used to stain the nucleus. The technique is also referred to as acid fast staining. The acid fast bacteria have a waxy substance mycolic acid on their cell wall that makes them impermeable to staining procedures.
The term acid fast is used since they resist decolourization with acid alcohol. Carbol fuchsin , the primary stain contains phenol, which helps solubilize the cell wall whereas heat is used to increase the penetration of the stain. On using alcohol to decolorize, cells will be decolorized except for acid fast ones. Methylene blue is used as the counterstain to any cell that was decolorized. Preparation of carbol fuchsin by mixing two solutions:.
Solution 1- 0. Solution 2- 5 grams phenol and 90 ml of distilled water,. A photomicrograph of Mycobacterium smegmatis pink and Micrococcus luteus blue x magnification. Mycobacterium smegmatis is acid-fast, retaining the carbol fuchsin dye, thus appearing pink.
Micrococcus luteus is not acid-fast, loses the carbol fuchsin during decolorization, and is counter-stained with methylene blue. This is a Romanowsky type of metachromatic stain that is prepared by mixing specially treated methylene blue dye with eosin. The acidic portion of the stain unites with the basic components of the cells such as hemoglobin, and thus they are referred to as eosinophilic and are stained pink or red.
The acidic components of the cell, such as the nucleic acids on the other hand take the basic dye and stain blue or purple. PH has to be controlled using a buffer of 6. These steps should be repeated two times for marrow smears.
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