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Chromophores have a positive or negative charge depending on whether the dye is basic (pH>7) or acidic (pH<7). The charge of the chromophores controls how the dye interacts with bacterial cells during the staining process.A basic stain contains positively charged (+) chromophores. Because the cell membrane is slightly negative (-) in charge and opposite charges tend to attract, basic dyes (+) have a high affinity for the cell’s surface. Basic dyes (+) are used for direct stains which only color the cell’s cytoplasm and leave the background outside of the bacterial cell colorless. Examples of basic dyes are crystal violet, methylene blue, safranin, basic fuchsin, and malachite green.An acidic stain contains negatively charged (-) chromophores. Acidic dyes (-) are used for negative stains in whichthe dye is repelled from the cell’s negatively charged (-) surface. Negative stains color only the background or environment outside the cell. Congo red, india ink, and nigrosin are examples of acidic dyes. See Figure 6.
Figure 6. Direct and negative staining.During staining, a thin film of cells called a smear is applied to a blank microscope slide. Often the cells are preserved on the slide through a fixation process, whereby a flame is used to kill the bacteria and firmly affix the cells to the slide. It is important to note that heating causes the cell to shrink in size and some cell features may become distorted. Overheating can cause the cells to deteriorate, removing them from the surface. In negative staining, the bacteria are not affixed by heating. In fact, if the bacteria is still alive it can act as a potential contaminant or pathogen.All bacteria cells can be classified into two groups based on differences in the structure and composition of the cell wall. A widely used form of differential staining, the Gram stain, was developed in 1884 by the physician Hans Christian Gram. The Gram stain uses two dyes to distinguish Gram-positive and Gram-negative bacteria. Like simple stains, differential stains can provide information on cell morphology such as shape, size, and arrangement.Gram-positive cells have a thick outer cell wall composed of a mesh-like polymer called peptidoglycan, adjacent to an interior plasma membrane. See Figure 7. Teichoic acids are only found in the cell wall of Gram-positive bacteria. The teichoic acids are a group of molecules that run perpendicular to the peptidoglycan sheets and provide structural support. The size of the cell wall of Gram-positive cells ranges from 20 – 80 nm in thickness and is cross-linked in two dimensions making a strong, more rigid framework.
Image Copyright Alila Medical Images, 2013. Used under license from Shutterstock.com.Figure 7. Cell wall structure of Gram-positive bacteria. Gram-negative cells