Why staining bacteria

Trichomonas , Strongyloides , some fungi, and some protozoa cysts also have a Gram reaction. Very small bacteria or bacteria without a cell wall, such as Treponema , Mycoplasma , Chlamydia , or Rickettsia do not have a gram reaction.

The characterization of any new bacteria must include their gram reaction. Typically a differential stain has four components; the primary stain, a mordant that sets the stain, a decolorizing agent to remove the primary stain, and a counter stain. In the Gram stain, the primary stain is crystal violet.

This gives the cell an intense purple color. The mordant, iodine, forms a complex with the crystal violet inside the cell wall. Gram positive cells will retain the dye complex and remain purple. The dye rinses out in gram negative cells. The large iodine-crystal violet complex is retained within the cell walls of gram positive cells because of the molecular structure of the many layers of peptidoglycan in the cell wall.

There are lots of cross-linked teichoic acids and the iodine-dye complex cannot physically get out. There is also less lipid in the membrane and the decolorizing agent cannot get to it as well. Gram negative cells have an outer membrane and only one layer of peptidoglycan, with more lipid.

The crystal violet dye is easily washed out. The accuracy of the Gram stain is dependent on the integrity of the bacterial cell wall. There are a variety of things that can influence the cell wall integrity; old cells i.

Under these conditions, gram positive cells will come out as gram-negative. If you de-colorize too long, Gram-positive cells will look like Gram-negative cells. Conversely, if you do not decolorize enough, Gram-negatives will look like Gram-positives. The only way you can trust your results it to always run a known Gram-positive and a known Gram-negative on the same slide. If they stain as predicted you can be pretty sure the result of your unknown sample is reliable.

The Gram staining takes practice to get right. Do not expect to get a good Gram stain on your first try. It is a good idea to hold your slide with a clothespin; your gloves will get pretty psychedelic as will everything you touch! The Congo Red Capsule stain is a modification of the nigrosin negative stain you may have done previously.

The bacteria take up the congo red dye and the background is stained then with acid fuchsin dye. The capsule or slime layers, highly hydrated polymers, exclude both dyes. The background will appear blue, the bacterial cells will appear pink, and the clear halos are the capsules. Clinically, the capsules of some highly pathogenic bacteria i.

The bacteria are suspended in the antisera and then mixed with methlyene blue. In the antisera staining procedure, the bacteria will appear blue surrounded by a clear halo and then surrounded by a thin blue line where the antisera have attached to the capsule.

Endospore formation is characteristic of Clostridum and Bacillus spp. The ability to concentrate and coat their protoplasm allows them to survive the adverse environmental conditions they experience in their soil habitat. This also allows the spores to resist staining. Endospores are typically highly refractile, light striking them is deflected. Many Bacillus species have inclusion bodies that are highly refractile. These inclusion bodies may look like endospores with regular staining.

The presence of endospores must be confirmed with endospore specific stains. The presence, and characteristic shape and position of endospores require special procedures to permeate the endospore coat. Most endospore stains involve heating the slides while keeping them continually moist with the dye.

While quicker, it produces volatile chemicals and is just a big mess. The same results can be obtained by letting the dye sit on the slide for 30 minutes. It is a good idea to start this slide first and work on another stain while you are waiting for the dye to permeate the endospore. You will be using a Bacillus species for the endospore stain. The shape and position of B.

Bacillus does not start forming spores until it runs out of food. If the cultures are too young, you will mostly see just the pink rods of the bacteria.

If the cultures are too old, you will mostly see just the small green ovals of the endospores. Ideally, you should see the green oval bodies of the endospore surrounded by the pink vegetative bacterial cell.

Select a sample from the middle of a colony with the straight inoculating needle for the best results. The edge of the colony is still actively growing and will have few endospores. Microbiology Resource Center. There are two important things to consider when preparing a slide for staining: The bacteria must be evenly and lightly dispersed. Contrast, however, can be improved by either using a different type of optical system, such as phase contrast or a differential interference contrast microscope, or by staining the cells or the background with a chromogenic dye that not only adds contrast, but gives them a color as well.

There are many different stains and staining procedures used in microbiology. Some involve a single stain and just a few steps, while others use multiple stains and a more complicated procedure. Before you can begin the staining procedure, the cells have to be mounted smeared and fixed onto a glass slide. A bacterial smear is simply that—a small amount of culture spread in a very thin film on the surface of the slide.

Heat fixing is an easy and efficient method, and is accomplished by passing the slide briefly through the flame of a Bunsen burner, which causes the biological material to become more or less permanently affixed to the glass surface. Heat fixed smears are ready for staining. In a simple stain, dyes that are either attracted by charge a cationic dye such as methylene blue or crystal violet or repelled by charge an anionic dye such as eosin or India ink are added to the smear.

Cationic dyes bind the bacterial cells which can be easily observed against the bright background. Anionic dyes are repelled by the cells, and therefore the cells are bright against the stained background. See Figures 1 and 2 for examples of both. Probably the most important feature made obvious when you stain bacterial cells is their cellular morphology not to be confused with colonial morphology, which is the appearance of bacterial colonies on an agar plate.

There is greater diversity of shapes among Archaea and other bacteria found in ecosystems other than the human body. Often bacteria create specific arrangements of cells, which form as a result of binary fission by the bacteria as they reproduce. Arrangements are particularly obvious with non-motile bacteria, because the cells tend to stay together after the fission process is complete.

Both the shape and arrangement of cells are characteristics that can be used to distinguish among bacteria. The most commonly encountered bacterial shapes cocci and bacilli and their possible arrangements are shown in Figures 3 and 4. In microbiology, differential staining techniques are used more often than simple stains as a means of gathering information about bacteria.

Differential staining methods, which typically require more than one stain and several steps, are referred to as such because they permit the differentiation of cell types or cell structures. The most important of these is the Gram stain.

Other differential staining methods include the endospore stain to identify endospore-forming bacteria , the acid-fast stain to discriminate Mycobacterium species from other bacteria , a metachromatic stain to identify phosphate storage granules, and the capsule stain to identify encapsulated bacteria. We will be performing the Gram stain and endospore staining procedures in lab, and view prepared slides that highlight some of the other cellular structures present in some bacteria.

In , physician Hans Christian Gram was studying the etiology cause of respiratory diseases such as pneumonia. He developed a staining procedure that allowed him to identify a bacterium in lung tissue taken from deceased patients as the etiologic agent of a fatal type of pneumonia. The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain crystal violet by resisting a decolorization process.

Gram staining involves four steps. First cells are stained with crystal violet, followed by the addition of a setting agent for the stain iodine. Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink. Gram negative cell walls have an outer membrane also called the envelope that dissolves during the alcohol wash.

This permits the crystal violet dye to escape. Only the decolorized cells take up the pink dye safranin, which explains the difference in color between the two types of cells. At the conclusion of the Gram stain procedure, Gram positive cells appear purple, and Gram negative cells appear pink.

When you interpret a Gram stained smear, you should also describe the morphology shape of the cells, and their arrangement.

In Figure 5, there are two distinct types of bacteria, distinguishable by Gram stain reaction, and also by their shape and arrangement. Below, describe these characteristics for both bacteria:.

Some bacteria produce the waxy substance mycolic acid when they construct their cell walls. Mycolic acid acts as a barrier, protecting the cells from dehydrating, as well as from phagocytosis by immune system cells in a host. This waxy barrier also prevents stains from penetrating the cell, which is why the Gram stain does not work with mycobacteria such as Mycobacterium , which are pathogens of humans and animals. For these bacteria, the acid — fast staining technique is used.

To perform the acid-fast stain, a heat-fixed smear is flooded with the primary stain carbol fuchsin, while the slide is heated over a steaming water bath. Then the slide is allowed to cool and a solution of acid and alcohol is added as a decolorizer. The process involves three steps:. Your Account. Gram Staining Created by Monica Z. Bruckner What is Gram Staining?