- Learn the differences between simple staining and differential staining techniques.
- Learn how to prepare a bacterial smear from cultured organisms.
- Learn the differences between gram positive and gram negative bacteria.
- Learn how to perform the gram stain procedure.
- Use microscopy to examine gram stained cells.
- Learn about some special staining procedures, and view examples of these under oil immersion.
Most types of cells do not have much natural pigment and are therefore difficult to see under the light microscope unless they are stained. Several types of stains are used to make bacterial cells more visible. In addition, specific staining techniques can be used to determine the cells’ biochemical or structural properties, such as cell wall type and presence or absence of endospores. This type of information can help scientists identify and classify microorganisms, and can be used by health care providers to diagnose the cause of a bacterial infection.
One type of staining procedure that can be used is the simple stain, in which only one stain is used, and all types of bacteria appear as the color of that stain when viewed under the microscope. Some stains commonly used for simple staining include crystal violet, safranin, and methylene blue. Simple stains can be used to determine a bacterial species’ morphology and arrangement, but they do not give any additional information.
Scientists will often choose to perform a differential stain, as this allows them to gather additional information about the bacteria they are working with. Differential stains use more than one stain, and cells will have a different appearance based on their chemical or structural properties. Some examples of differential stains are the Gram stain, acid-fast stain, and endospore stain. In this lab you will learn how to prepare bacterial cells for staining, and learn about the gram staining technique.
The Gram Stain
This very commonly used staining procedure was first developed by the Danish bacteriologist Hans Christian Gram in 1882 (published in 1884) while working with tissue samples from the lungs of patients who had died from pneumonia. Since then, the Gram stain procedure has been widely used by microbiologists everywhere to obtain important information about the bacterial species they are working with. Knowing the Gram reaction of a clinical isolate can help the health care professional make a diagnosis and choose the appropriate antibiotic for treatment.
Gram stain results reflect differences in cell wall composition. Gram positive cells have thick layers of a peptidoglycan (a carbohydrate) in their cell walls; Gram negative bacteria have very little. Gram positive bacteria also have teichoic acids, whereas Gram negatives do not. Gram negative cells have an outer membrane that resembles the phospholipid bilayer of the cell membrane. The outer membrane contains lipopolysaccharides (LPS), which are released as endotoxins when Gram negative cells die. This can be of concern to a person with an infection caused by a gram negative organism.
Figure 3.1.1 shows the major differences between the Gram positive and Gram negative cell walls (also refer to your textbook for additional information). The differences in the cell wall composition are reflected in the way the cells react with the stains used in the Gram stain procedure.
Gram stains are best performed on fresh cultures—older cells may have damaged cell walls and not give the proper Gram reaction. Also, some species are known as Gram-variable, and so both Gram positive and Gram negative reactions may be visible on your slide.
Although the vast majority of bacteria are either Gram positive or Gram negative, it is important to remember that not all bacteria can be stained with this procedure (for example, Mycoplasmas, which have no cell wall, stain poorly with the Gram stain).
There are a variety of staining procedures used to identify specific external or internal structures that are not found in all bacterial species (see table at the end of this exercise for a comparison of staining procedures). You will do some of these staining procedures in the next lab (acid-fast staining and endospore staining). In today’s lab, you will observe prepared slides of special stains: a capsule stain (Klebsiella pneumoniae), flagella stain (Proteus vulgaris) and spirochete stain (Treponema pallidum).
Some bacteria secrete a polysaccharide-rich structure external to the cell wall called a glycocalyx. If the glycocalyx is thin and loosely attached, it is called a slime layer; if it is thick and tightly bound to the cell, it is called a capsule. The glycocalyx can protect the cell from desiccation and can allow the cell to stick to surfaces like tissues in the body. They may also provide cells with protection against detection and phagocytosis by immune cells and contribute to the formation of a biofilm: in this way a glycocalyx can act as a virulence factor; (contributes to the ability of an organism to cause disease).
Capsules can be detected using a negative staining procedure in which the background (the slide) and the bacteria are stained, but the capsule is not stained. The capsule appears as a clear unstained zone around the bacterial cell. Since capsules are destroyed by heat, the capsule staining procedure is done without heat-fixing the bacteria.
Flagella (long whip-like structures used for bacterial motility) and some bacteria (e.g. spirochetes) are too thin to be observed with regular staining procedures. In these cases, a silver stain is used. Silver nitrate is applied to the bacteria along with a special mordant; the silver nitrate precipitates around the flagella or the thin bacteria, thus thickening them so they can be observed under the light microscope.
Gram stain, bacterial smear, simple stain, differential stain, Gram positive, Gram negative, Gram variable, capsule, spirochete, flagella, negative staining, silver stain