Reasons why bacteria need to be fixed before staining in microscopy experiments:
Killing cells is easier for dyes to stain.
Make bacteria adhere to glass slides and not easily washed away by water.
When fixing, it is necessary to air dry slowly. If it is necessary to speed up the process, you can dry it a few times from a distance above the flame of the alcohol lamp. Do not dry the glass slide until it heats up, otherwise it may damage the bacterial life form
Gram staining and microscopic observation of bacteria
Experimental principle
The principle of Gram staining: Different reactions of bacteria to Gram staining are caused by differences in the composition and structure of their cell walls. The cell wall of Gram positive bacteria is mainly composed of a network of peptidoglycans. When treated with ethanol, dehydration causes a decrease in the pore size and permeability of the network structure, making it difficult for the crystal violet iodine complex to be washed out and retained inside the cell. After decolorization and counterstaining, the blue purple color of the initial dye is still retained. The peptidoglycan layer on the cell wall of Gram negative bacteria is thin and has a high lipid content. Therefore, when decolorized, the lipid is dissolved in ethanol (or acetone), increasing the permeability of the cell wall and making it easier to wash out the crystal violet iodine complex. After counterstaining with a counterstain, the cells are stained with the red color of the counterstain. Microscope imaging principle: Modern ordinary optical microscopes use two sets of lens systems, an eyepiece and an objective lens, to magnify and image, so they are often referred to as compound microscopes. In the optical system of a microscope, the performance of the objective lens is the most critical, and the magnification of the oil lens is the highest, which is the most important for microbiological research. Adding lens oil between the glass slide and the lens is mainly to increase illumination brightness and enhance the resolution of the microscope. Using oil instead of air as the medium for light propagation reduces the refraction or total reflection of light, making the observed image clearer.
