Why is staining important

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. All other cell types will be decolorized. Methylene blue is then used as a counterstain. In the end, acid-fast bacteria AFB will be stained a bright pink color, and all other cell types will appear blue.

Capsule : The polysaccharide goo that surrounds some species of bacteria and a few types of eukaryotic microbes is best visualized when the cells are negative stained. In this method, the bacteria are first mixed with the stain, and then a drop of the mixture is spread across the surface of a slide in the thin film.

With this method, capsules appear as a clear layer around the bacterial cells, with the background stained dark. Metachromatic granules or other intracytoplasmic bodies : Some bacteria may contain storage bodies that can be stained.

Various staining methods are used to visualize intracytoplasmic bodies in bacteria, which often provide an identification clue when observed in cells. Endospores are dormant forms of living bacteria and should not be confused with reproductive spores produced by fungi.

These structures are produced by a few genera of Gram-positive bacteria, almost all bacilli, in response to adverse environmental conditions. Two common bacteria that produce endospores are Bacillus or Clostridum.

Both live primarily in soil and as symbionts of plants and animals, and produce endospores to survive in an environment that change rapidly and often. The process of endosporulation the formation of endospores involves several stages. After the bacterial cell replicates its DNA, layers of peptidoglycan and protein are produced to surround the genetic material.

Once fully formed, the endospore is released from the cell and may sit dormant for days, weeks, or years. When more favorable environmental conditions prevail, endospores germinate and return to active duty as vegetative cells. Mature endospores are highly resistant to environmental conditions such as heat and chemicals and this permits survival of the bacterial species for very long periods. Endospores formed millions of years ago have been successfully brought back to life, simply by providing them with water and food.

Because the endospore coat is highly resistant to staining, a special method was developed to make them easier to see with a brightfield microscope. For example, hematoxylin is a stain that turns cell nuclei blue.

When used in conjunction with eosin, which turns the other parts of the cell red or pink, it provides a stronger contrast and makes the nuclei easier to differentiate. PAP smears and blood marrow samples are easier to examine when these two stains are used together.

Gram's Stain: Hospital workers use Gram's stain to identify harmful bacteria. This is actually a series of colorants that have different effects on different types of bacteria and give doctors an important diagnostic tool. Gram's stain is a three-part process.

In the first, Hucker's crystal violet is added, which stains all bacteria a uniform violet color. In the next stage, iodine stain is added, which causes the color to adhere to Gram-positive cells, which are primarily Staphylococcus and Streptococcus. The stain is washed away, leaving the Gram-positive cells with a distinct violet color; then a third stain, Safranine O, is introduced to enhance the contrast between the Gram-negative bacteria and the rest of the material in the slide.

When preparing a specimen on a slide, you can dry-mount or wet-mount it, you can slice it into a thin section or you can smear it. When using a stain, the usual procedure is to wet-mount the specimen, which means to place a drop of water on the slide, set the specimen in the water and cover it with a cover slip.

You then apply the stain to a corner of the slide with a dropper and allow it to be drawn toward the specimen by capillary action. It helps to put a paper towel on the opposite side of the slide to attract the water. Once the stain has spread over the entire slide, the specimen is ready for examination. If you were to observe a cell or microorganism under a microscope in its natural state it would be difficult to understand what you were looking at. This is because most microbes and cells lack colour and contrast.

Laboratory staff and scientists use different mounting techniques, combined with dyes and microbiology stains to add contrast to specimens and make them easier to observe at a microscopic level and aid with identification. In microbiology laboratories, especially clinical settings, light microscopes are most commonly to view specimens.

Before a sample can be viewed it needs to be mounted onto a microscope slide. There are two methods commonly used to mount specimens on to slides making them ready to be viewed under a microscope:. Wet mounting — live samples and specimens are fixed to a slide using water, a stain or other liquid. A cover slide is placed over the top and the sample is then ready to view. Fixing the specimen in this way also helps to keep the sample within the field of view once it is under the microscope.

Fixation — the aim is to preserve the shape and structure of a specimen before viewing it under a microscope.