why do we need to stain bacteria

Gram Staining Created by What is Gram Staining? 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. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process. How Does Gram Staining Work? Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin. Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. The process involves three steps:
Cells are stained with crystal violet dye. Next, a Gram s iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water. A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.


Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost. A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However, the decolorized Gram negative cells are stained red. How To- Staining Protocol and Concerns: Reagents: Decolorizer (e. g. ethanol) Make a slide of cell sample to be stained. Heat fix the sample to the slide by carefully passing the slide with a drop or small piece of sample on it through a Bunsen burner three times. Add the primary stain (crystal violet) to the sample/slide and incubate for 1 minute. Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet. Add Gram s iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall. Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells. Add the secondary stain, safranin, to the slide and incubate for 1 minute. Wash with a gentle stream of water for a maximum of 5 seconds. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope.


If the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to appear red when viewed under a microscope. Literature Related Links Teaching Activities This teaching activity uses a step-wise process to aid the student in familiarity with the use of the microscope as well as to increase the success rate with which they are able to stain and view microorganisms. Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope. In some cases, staining is unnecessary, for example when microorganisms are very large or when motility is to be studied, and a drop of the microorganisms can be placed directly on the slide and observed. A preparation such as this is called a wet mount. A wet mount can also be prepared by placing a drop of culture on a coverslip (a glass cover for a slide) and then inverting it over a hollowedout slide. This procedure is called the hanging drop. In preparation for staining, a small sample of microorganisms is placed on a slide and permitted to air dry. The smear is heat fixed by quickly passing it over a flame. Heat fixing kills the organisms, makes them adhere to the slide, and permits them to accept the stain. Simple stain techniques. Staining can be performed with basic dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial cytoplasm. Such a procedure is the simple stain procedure. An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged dyes. They are repelled by the negatively charged cytoplasm and gather around the cells, leaving the cells clear and unstained.


This technique is called the negative stain technique. Differential stain techniques. The differential stain technique distinguishes two kinds of organisms. An example is the Gram stain technique. This differential technique separates bacteria into two groups, Grampositive bacteria and Gramnegative bacteria. Crystal violet is first applied, followed by the mordant iodine, which fixes the stain (Figure ). Then the slide is washed with alcohol, and the Grampositive bacteria retain the crystalviolet iodine stain; however, the Gramnegative bacteria lose the stain. The Gramnegative bacteria subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear red under the oilimmersion lens, while Grampositive bacteria appear blue or purple, reflecting the crystal violet retained during the washing step. Another differential stain technique is the acidfast technique. This technique differentiates species of Mycobacterium from other bacteria. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acidalcohol solution. Mycobacterium species resist the effect of the acidalcohol and retain the carbolfuchsin stain (bright red). Other bacteria lose the stain and take on the subsequent methylene blue stain (blue). Thus, the acidfast bacteria appear bright red, while the nonacidfast bacteria appear blue when observed under oilimmersion microscopy. Other stain techniques seek to identify various bacterial structures of importance. For instance, a special stain technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width.


Flagella so stained can then be observed. A special stain technique is used to examine bacterial spores. Malachite green is used with heat to force the stain into the cells and give them color. A counterstain, safranin, is then used to give color to the nonsporeforming bacteria. At the end of the procedure, spores stain green and other cells stain red. The Gram stain procedure used for differentiating bacteria into two groups. Another differential stain technique is the acid-fast technique. This technique differentiates species of Mycobacterium from other bacteria. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acid-alcohol solution. Mycobacterium species resist the effect of the acid-alcohol and retain the carbolfuchsin stain (bright red). Other bacteria lose the stain and take on the subsequent methylene blue stain (blue). Thus, the acid-fast bacteria appear bright red, while the nonacid-fast bacteria appear blue when observed under oil-immersion microscopy. Other stain techniques seek to identify various bacterial structures of importance. For instance, a special stain technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. Flagella so stained can then be observed. A special stain technique is used to examine bacterial spores. Malachite green is used with heat to force the stain into the cells and give them color. A counterstain, safranin, is then used to give color to the nonsporeforming bacteria. At the end of the procedure, spores stain green and other cells stain red.

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