So, the decolorizing solution dehydrates the peptidoglycan layer trapping all the CVI complexes inside the cell wall and bacteria retain the purple or violet color of crystal violet. Whereas in Gram-Positive bacteria, there is no outer membrane, and the peptidoglycan layer is also thick with higher cross-linkage. This causes cells to lose most of the CVI complexes. The peptidoglycan layer is thin with less cross-linking in the Gram-Negative cell wall, hence becoming leaky. The outer membrane of the Gram-Negative bacterial cell wall is dissolved exposing the peptidoglycan layer. When decolorizing solution (ethanol or a mixture of ethanol and acetone) is added it interacts with lipids in the cell wall. When Gram’s Iodine is added as mordant, the iodine (I – or I -3 ion) interacts with CV + ion and forms CV-I complex within cytoplasm and cell membrane and cell wall layers. The CV + ion interacts with negatively charged components of the cell wall. These ions easily penetrate the cell wall components of both positive and negative bacteria. In an aqueous solution of crystal violet dye, their molecules dissociate into CV + and Cl – ions. Bacteria having a thin peptidoglycan layer with lesser cross-linkage lose primary stain during decolorizing and gain counter stain appearing pink or red. Bacteria having cell walls with a thick layer of peptidoglycan will resist decolorization of primary stain and appear violet or purple. Gram staining and differentiation are based on the differences in cell wall structure and composition of bacteria. Using this staining technique, bacteria can be differentiated into two groups hence it is called the differential staining technique. These bacteria are called Gram-Negative bacteria. The other group of bacteria with Gram-Negative cell wall will lose primary stain and take up the counterstain and appears pink or red under the microscope. These bacteria are termed Gram-Positive bacteria. Those bacteria with Gram-positive cell walls will retain primary stain and appear violet or purple. This staining technique uses two stains crystal violet as primary stain and safranine as a counterstain. It is generally the first test performed on bacteria during their identification and observation process. It is the most widely used and the most important staining technique in bacteriology, especially in medical bacteriology. The method is reproducible and has consistently resulted in precipitate-free sections.Gram staining is a differential bacterial staining technique used to differentiate bacteria into Gram Positive and Gram Negative types according to their cell wall composition. The staining procedure described here is a simple means of preventing any extensive stain-air contact since this will cause the formation of precipitate in the staining solution and consequently on the sections. Lever (8) suggested cleaning the stained sections by dipping them in 1 per cent potassium hydroxide. Highly alkaline lead stains were found by Karnovsky (7) to be more stable than those of a lower pH. Millonig (6) added tartrate to the lead hydroxide solution to stabilize the lead salt so that protection from air was not necessary. Dalton and Zeigel (5), after staining sections with lead acetate, exposed them to ammonium hydroxide vapors thus, the formation of lead hydroxide occurred in the sections themselves. Other investigators proposed variations in stain preparations and staining procedures designed to discourage contamination. A more complicated mechanism which serves as a barrier between the lead hydroxide stain and air was built by Huxley and Zubay (4). Parsons and Darden (3) designed a 592 B R I E F N O T E Sstaining apparatus with which six grids could be stained at once without the danger of precipitate formation. The cover of the syringe was filled with a carbon dioxide trap consisting of sodium hydroxide, anhydrous calcium chloride, and cotton. In order to prevent the stain from coming in contact with air, Peachey (2) used a plastic, air-free syringe as a receptacle for the stain and as a mechanism for staining sections. However, when lead hydroxide is exposed to the carbon dioxide of air, lead carbonate forms and is deposited on the sections as fine, needle-like crystals, large amor-phous or polygonal precipitates, or small granules. Lead hydroxide, as suggested by Watson (1), has become increasingly popular as a stain for electron microscopy. Staining of thin sections results in greater contrast which facilitates focusing and the observation of fine structure.
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