Why do samples need to be stained in light microscopy

There are several types of staining media, each can be used for a different purpose. Commonly used stains and how they work are listed below. All these stains may be used on fixed, or non-living, cells and those that can be used on living cells are noted.

After staining cells and preparing slides, they may be stored in the dark and possibly refrigerated to preserve the stained slide, and then observed with a microscope.

Next Page ». Your Account. Microscopy Created by Monica Z. This question is unanswerable without proper controls imaged with exactly the same settings as the experimental sample.

Here are some of the most common controls we recommend:. Tissues and cells can have considerable autofluorescence, particularly but not exclusively in the green, cyan and blue region of the spectrum.

Including this control can be very useful when determining whether any signals that are visualized in the experimental samples can be attributed to autofluorescence. Secondary antibodies can bind non-specifically to tissue or, if aggregated, lead to punctate staining throughout a sample.

Including this control helps determine whether a signal visualized in the experimental sample can be explained by a problem with the secondary antibody. Even if there is no non-specific binding of secondary antibodies, and autofluorescence is comparatively low, a strong signal in your experimental sample does not guarantee you are looking at your protein of interest.

Primary antibodies are not necessarily as specific as advertised; the best control for this is to remove the putative target protein from the sample and test staining there. If there is still significant staining in this no target control assuming autofluorescence and non-specific binding of secondary antibodies are not a problem , then the primary antibody is detecting something other than what it should.

No target controls are frequently extremely expensive as they can involve making or purchasing knockdown or knockout cell lines or animals. In fact, they are sometimes impossible to procure because removing the target protein kills the animal or cells in which it is removed.

Nevertheless, they are the only way to prove an antibody is staining what it is advertised to stain, and nothing else. If you are interested in determining whether two or more fluorophores are present in the same location it is useful to have controls with every fluorophore except one, excluding each of those that are candidates for colocalization in turn.

Even if the fluorophores you are using are well separated spectrally, the fluorescence from one channel can always bleed into another, particularly in cases where the amount of protein is much higher in one channel example: overexpression via viral constructs or one of the fluorophores is much stronger than the other.

Following the example above, if you had stained a sample for proteins A and B, looking at the B channel in the condition without staining for B would give us an idea of how much of the signal in channel B is due to bleedthrough from channel A. This type of information will be essential if you want to make any statements about the degree of spatial overlap between proteins A and B.

Make sure to use 1. This is what almost all of the objectives on our microscopes are designed for; other thicknesses will lead to noticeably worse images, particularly using high-resolution objectives.

If you are not using 1. Therefore, if applicable, try to grow your cells on coverslips not slides. Use a coverslip that does not extend all the way to the edge of the slide. Since fixation and staining would kill the cells, darkfield microscopy is typically used for observing live specimens and viewing their movements.

However, other approaches can also be used. For example, the cells can be thickened with silver particles in tissue sections and observed using a light microscope.

Though the stain kills the cells, it increases the contrast to make them more visible. In clinical settings, indirect immunofluorescence is often used to identify Treponema. Samples for fluorescence and confocal microscopy are prepared similarly to samples for light microscopy, except that the dyes are fluorochromes. Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific proteins on specific types of cells immunofluorescence ; others may attach to DNA molecules in a process called fluorescence in situ hybridization FISH , causing cells to be stained based on whether they have a specific DNA sequence.

Sample preparation for two-photon microscopy is similar to fluorescence microscopy, except for the use of infrared dyes. Specimens for STM need to be on a very clean and atomically smooth surface.

They are often mica coated with Au Toluene vapor is a common fixative. What is the main difference between preparing a sample for fluorescence microscopy versus light microscopy? Each case study walks you through a clinical problem using appropriate techniques in microscopy at each step.

After some additional testing, the technician determines that these bacteria are the medically important species known as Staphylococcus aureus , a common culprit in wound infections. Because some strains of S. After testing several antibiotics, the lab is able to identify one that is effective against this particular strain of S. This reduces the risk that any especially resistant bacteria could survive, causing a second infection or spreading to another person.

As the use of antibiotics has proliferated in medicine, as well as agriculture, microbes have evolved to become more resistant. Strains of bacteria such as methicillin-resistant S. Fluorescence microscopy can be useful in testing the effectiveness of new antibiotics against resistant strains like MRSA. Live cells will not absorb the dye, but cells killed by an antibiotic will absorb the dye, since the antibiotic has damaged the bacterial cell membrane. In this particular case, MRSA bacteria that had been exposed to MCA did, indeed, appear green under the fluorescence microscope, leading researchers to conclude that it is an effective antibiotic against MRSA.

Of course, some argue that developing new antibiotics will only lead to even more antibiotic-resistant microbes, so-called superbugs that could spawn epidemics before new treatments can be developed.

For this reason, many health professionals are beginning to exercise more discretion in prescribing antibiotics. Whereas antibiotics were once routinely prescribed for common illnesses without a definite diagnosis, doctors and hospitals are much more likely to conduct additional testing to determine whether an antibiotic is necessary and appropriate before prescribing.

A sick patient might reasonably object to this stingy approach to prescribing antibiotics. To the patient who simply wants to feel better as quickly as possible, the potential benefits of taking an antibiotic may seem to outweigh any immediate health risks that might occur if the antibiotic is ineffective.

But at what point do the risks of widespread antibiotic use supersede the desire to use them in individual cases? Skills to Develop Differentiate between simple and differential stains Describe the unique features of commonly used stains Explain the procedures and name clinical applications for Gram, endospore, acid-fast, negative capsule, and flagella staining. Preparing Specimens for Light Microscopy In clinical settings, light microscopes are the most commonly used microscopes.

What types of specimens should be chemically fixed as opposed to heat-fixed? Why might an acidic dye react differently with a given specimen than a basic dye? Explain the difference between a positive stain and a negative stain. Explain the difference between simple and differential staining. Gram Staining The Gram stain procedure is a differential staining procedure that involves multiple steps. First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color.

Cells that have thick peptidoglycan layers in their cell walls are much less affected by the decolorizing agent; they generally retain the crystal violet dye and remain purple. However, the decolorizing agent more easily washes the dye out of cells with thinner peptidoglycan layers, making them again colorless.

Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized cells pink and is less noticeable in the cells that still contain the crystal violet dye. Explain the role of alcohol in the Gram stain procedure.

What color are gram-positive and gram-negative cells, respectively, after the Gram stain procedure? What does this reveal about their cell walls? Acid-Fast Stains Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. Capsule Staining. Endospore Staining Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions.

Flagella Staining Flagella singular: flagellum are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes. Name the device that is used to create thin sections of specimens for electron microscopy.