Basic staining: The bacterial cell wall is a complex structure that has an overall negative charge due to the

proteins and other components in and on it. Basic staining takes advantage of this by using a stain that has a positive charge, which allows the stain to adhere to the negatively charged bacterial cell wall and stain it. This leaves the background of the slide unstained, so the bacteria are clearly visible. Basic stains belong to a class of stains known as simple stains. These allow us to visualize size, shape and arrangement of an organism under the microscope. Examples of basic stains include: carbon fuchsin, 1% crystal violet, methylene blue, safranin. Before an organism can be stained it must be placed on a slide. This is called a bacterial smear. When a bacterial smear is made, a heat fixing step is required to allow the organism to adhere to the slide better, but this step causes cell damage and shrinking so one has to compromise the observance of the organisms distorted shape in order to make it visible via staining.

Negative Staining: The process of negative staining is used when we want to cause minimal damage to a cell. This is because negative staining does not include a heat fixing step, which is damaging to cells. A negative stain is also known as an acidic stain. It possesses a negative charge, which will be repelled by the bacterial cell wall since it has a negative charge (same charges repel each other). This will stain the background of the slide leaving the organism unstained, but visible. By using this technique we are able to visualize size shape and arrangement. Examples of negative stains include: Nigrosin, picric acid, and India pink

Gram Staining: Gram staining became one of the most important tools to microbiologists because it allowed for the organization of most organisms (except for mycobacterium, mycoplasma and a few others) into two groups, the Gram-positive organisms and the Gram-negative organisms. This type of staining is known as differential staining since it not only allows for the visualization of size, shape and arrangement of an organism, but it also allows for the placement of it into a group Gram staining takes advantage of the differences in the cell wall structures of organisms and how each retains a certain stain. Mainly the difference in peptidoglycan (composed of amino acids and sugars), which provides the thickness of the cell wall Gram-positive organisms stain purple while Gram-negative stain pink. This type of staining is more complex and it also involves a de-colorization step using ethanol. Several factors can influence the quality of the Gram stain. The most important is the age of the cell. As cells get older their cell walls will become more loose and they will not retain the dye. This is known as Gram variability.

Things to remember: 1) Any time you are staining you have to still practice your aseptic technique because you dont want to contaminate the sample you are working with. 2) Although the images show you almost covering the slide completely with the stain, you should not do that because it becomes messy and it is a waste of stain. You should only add enough drops to cover your smear (3-4 drops are ok for a medium size smear). 3) The Negative stain procedure is completely different from the basic and Gram stain. Pay attention to the steps. 4) We are not doing the Gram stain in week 2. Bring this handout back for week 3 when we do the Gram stain.