Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample. Cells may also be enumerated by staining cells to determine biomass in an environment of interest.
Cell staining techniques and preparation depend on the type of stain and analysis used. One or more of the following procedures may be required to prepare a sample:.
There are several types of staining media, each can be used for a different purpose. Cells are sometimes also stained to highlight metabolic processes or to differentiate between live and dead cells.
Below is a list of commonly used stains, often for different types of cells. All those listed can be used on fixed non-living cells and any that can be used on living cells are noted at the end of the description with the word "LIVE".
The image above shows how to draw a stain into a prepared slide. With the cover slip in place on top of the specimen, place a drop of stain on the edge of the cover slip. On the opposide side of the cover slip place a paper towel or cloth to draw the liquid out from the cover slip. As the liquid is drawn out, the stain will be pulled in under the cover slip.
Microscope Slide Staining Information Microscope cell staining is a technique used to enable better visualization of cells and cell parts under the microscope. By using different stains, a nucleus or a cell wall are easier to view.
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. The second method of preparing specimens for light microscopy is fixation. Fixation is often achieved either by heating heat fixing or chemically treating the specimen.
In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.
To heat-fix a sample, a thin layer of the specimen is spread on the slide called a smear , and the slide is then briefly heated over a heat source Figure 1b. Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde formalin , and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples Figure 1c.
Figure 1. Chemical fixation kills microorganisms in the specimen, stopping degradation of the tissues and preserving their structure so that they can be examined later under the microscope. In addition to fixation, staining is almost always applied to color certain features of a specimen before examining it under a light microscope.
Stains, or dyes, contain salts made up of a positive ion and a negative ion. Depending on the type of dye, the positive or the negative ion may be the chromophore the colored ion ; the other, uncolored ion is called the counterion.
If the chromophore is the positively charged ion, the stain is classified as a basic dye ; if the negative ion is the chromophore, the stain is considered an acidic dye. Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how the dye will interact with the specimen. In most cases, it is preferable to use a positive stain , a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background.
However, there are scenarios in which it is advantageous to use a negative stain , which is absorbed by the background but not by the cells or organisms in the specimen.
Negative staining produces an outline or silhouette of the organisms against a colorful background Figure 2. Figure 2. Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly used basic dyes such as basic fuchsin , crystal violet , malachite green , methylene blue , and safranin typically serve as positive stains.
On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin , eosin , and rose bengal. Table 2 provides more detail. Some staining techniques involve the application of only one dye to the sample; others require more than one dye. In simple staining , a single dye is used to emphasize particular structures in the specimen.
A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism. In contrast, differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear to be different colors.
Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining. Table 3 provides more detail on these differential staining techniques.
The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques.
The steps of the Gram stain procedure are listed below and illustrated in Table 1. Gram-staining is a differential staining technique that uses a primary stain and a secondary counterstain to distinguish between gram-positive and gram-negative bacteria.
Step 2: Iodine. Cells remain purple or blue. Step 3: Alcohol. Step 4: Safranin. Gram-negative cells appear pink or red. Figure 3. In this specimen, the gram-positive bacterium Staphylococcus aureus retains crystal violet dye even after the decolorizing agent is added. Gram-negative Escherichia coli, the most common Gram stain quality-control bacterium, is decolorized, and is only visible after the addition of the pink counterstain safranin.
The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative Figure 3. However, there are several important considerations in interpreting the results of a Gram stain. First, older bacterial cells may have damage to their cell walls that causes them to appear gram-negative even if the species is gram-positive.
Thus, it is best to use fresh bacterial cultures for Gram staining. Second, errors such as leaving on decolorizer too long can affect the results. In some cases, most cells will appear gram-positive while a few appear gram-negative as in Figure 3. This suggests damage to the individual cells or that decolorizer was left on for too long; the cells should still be classified as gram-positive if they are all the same species rather than a mixed culture.
Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-negative cells have other implications with clinical relevance. For example, Gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. Gram-negative bacteria tend to be more resistant to certain antibiotics than gram-positive bacteria.
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