When the background is a very dark gray color I b equals 0. By lightening the background to a somewhat lighter gray color I b equals 0. Although the optical systems found in modern microscopes may be capable of producing high resolution images at high magnifications, such a capability is worthless without sufficient contrast in the image.
Contrast is not an inherent property of the specimen, but is dependent upon interaction of the specimen with light and the efficiency of the optical system coupled to its ability to reliably record this image information with the detector.
Control of image contrast in a microscope optical system is dependent upon several factors, primarily the setting of aperture diaphragms, degree of aberration in the optical system, the optical contrast system employed, the type of specimen, and the optical detector. There are several sites in the microscope that allow adjustment of contrast.
These consist of the field aperture, condenser aperture, additional magnification for video detectors, electronic camera gamma, film gamma, printing paper gamma, image processing in real time, as well as specimen staining.
Because the human eye perceives an object by the contrast generated in its image, one can be easily led astray unless there is knowledge of the optical events that occur to produce contrast in the image.
Figure 2 illustrates three photomicrographs of the same viewfield containing transparent, colorless human cheek cells imaged with an optical microscope under differing contrast modes: brightfield, phase contrast, and Hoffman modulation contrast.
It is quite apparent that the three images appear different, and because of these variations, a microscopist might arrive at a different conclusion from each viewfield.
The cells illustrated in Figure 2 a were imaged with a microscope operating in brightfield mode with the condenser aperture closed down enough to render the specimen edges visible. An identical viewfield of the cheek cells using phase contrast optics is shown in Figure 2 b.
Note the dark inner areas and the bright outer areas surrounding the edges of objects such as the cell membranes and nuclei called halos, and are artifacts. In this view, it is difficult to specify the edges of the cells and to interpret the cause of the dark and light areas in the image.
The cells also appear quite flat. Finally, the cells illustrated in Figure 2 c were imaged using Hoffman modulation contrast, where one side of the image is dark while its opposite side is bright, leading to the perception of a pseudo three-dimensional object. Each viewfield in Figure 2 provides a different specimen image and leads to different interpretations, which can only be deciphered with knowledge about how the microscope created these images. For many specimens in optical microscopy, especially unstained or living material, contrast is so poor that the specimen remains essentially invisible regardless of the ability of the objective to resolve or clearly separate details.
Often, for just such specimens, it is important not to alter them by killing or treatment with chemical dyes or fixatives. This necessity has led microscopists to experiment with contrast enhancing techniques for over a hundred years in an attempt to improve specimen visibility and to bring more detail to the image without altering the specimen itself. It is a common practice to reduce the condenser aperture diaphragm below the recommended size or to lower the substage condenser to increase specimen contrast.
Unfortunately, while these maneuvers will indeed increase contrast, they also seriously reduce resolution and sharpness. It is useful to consider the characteristics of how light and matter interact before discussing methods of controlling contrast in the light microscope. Light responsible for illuminating the specimen may be either spatially coherent, incoherent, or partially coherent.
For example, the light beam can be shaped in the form of a slit or annulus, and can be composed of selected wavelengths that vibrate in all directions perpendicular to the direction of propagation or in a single direction due to polarization.
Some specimens are considered amplitude objects because they absorb light partially or completely, and can thus be readily observed using conventional brightfield microscopy. Others that are naturally colored or artificially stained with chemical color dyes can also be clearly imaged with the microscope. These stains or natural colors absorb some part of the white light passing through and transmit or reflect other colors.
Often, stains are combined to yield contrasting colors, e. It is a common practice to utilize stains on specimens that do not readily absorb light, thus rendering such images visible to the eye. Other specimens do not absorb light and are referred to as phase objects.
Because the human eye can only detect intensity and color differences, the phase changes due to objects must be converted to intensity differences. Phase specimens are characterized by several criteria including their shape typically round or flat , the density of internal light scattering elements, thickness, and unique chemical or electrical structural properties collectively grouped as refractive index.
Thick specimens may be relatively clear and contain only a few light scattering elements, or they may contain many scattering elements that do not pass light and make the specimen effectively opaque to transmitted illumination. These specimens are often termed reflected light specimens. When considering optical methods to enhance specimen contrast, it is useful to consider various characteristics of a specimen that can be manipulated to create intensity variations of those characteristics to render them visible.
A primary question is which characteristic of the object will be transformed into a different intensity under a unique set of circumstances. Specimen details and edges that have a size approximating the wavelength of imaging light will diffract or scatter light, provided there is a difference in refractive index between the specimen and its surrounding medium the surround.
Refractive index is defined as the ratio of the speed of light through air or a vacuum divided by the speed of light through the object. Because the speed of light through any material is less than the speed of light in a vacuum, the refractive index always exceeds a value of 1.
In order to resolve small distances between objects and to reproduce their shape with reasonable fidelity, a large angle of diffracted light must be captured by the microscope objective. Diffracted or deviated light gathered by the objective must be brought into a sharp focus at the image plane in order to generate specimen detail, as illustrated in Figure 3. At the image plane, light waves comprising the diffracted light undergo interference with undiffracted light. The visibility of light after interference is very much dependent upon the coherency of light illuminating the specimen, with visibility increasing proportionally by increasing coherence.
In the optical microscope, the condenser aperture diaphragm opening size controls the spatial coherence of light impinging on the specimen. Decreasing the diaphragm opening size yields a greater spatial coherence. Microscopists have long relied on decreasing the condenser aperture diaphragm opening size to increase visibility of particles and edges in phase specimens.
Contrast for amplitude objects can also be improved by proper adjustment of the condenser aperture. Small objects, edges, and particles will diffract light regardless of whether they belong to an amplitude or phase specimen. Only a portion of this diffracted light is captured by the objective see Figure 3 due to numerical aperture limitations of the objective. Remaining diffracted light that is not collected represents image information that is lost.
The correct setting for the condenser aperture diaphragm opening size is a tradeoff between enhancement of specimen image contrast and the introduction of diffraction artifacts. These are manifested in a loss of resolution, superimposition of diffraction rings, and other undesirable optical effects originating from regions in the specimen that are not in common focus. As the diffraction limit is approached, image contrast becomes lower as object details become smaller and spatial frequencies become larger.
In cases where the distribution of light from the specimen becomes sinusoidal, the modulus of the optical transfer function becomes the modulation transfer function MTF. As the spatial frequency increases, the modulation transfer function decreases and specimen contrast is reduced.
For each objective, the specific modulation transfer function is dependent upon the objective design and numerical aperture, the mode of contrast generation, wavelength of illuminating light, and the numerical aperture of the substage condenser. The edge of the objective rear focal plane acts as a low pass filter for the diffracted light, which must be focused at the image plane for interference to occur and form the image of the particle or edge.
Focusing a microscope brings these diffracted light waves together at the intermediate image plane. The angle of the light wavefront with respect to the specimen determines the degree of difficulty in focusing on the top or bottom of smooth rounded surfaces, which usually contain no diffraction sites. However, the edges of spherical specimens or fibers are easily focused because the edge interface is at a sufficient angle to the wavefront and diffracts light.
When discussing phase specimens, we will define an object as any resolvable portion of the specimen. 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. 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? What is one difference between specimen preparation for a transmission electron microscope TEM and preparation for a scanning electron microscope SEM?
Skip to main content. How We See the Invisible World. Search for:. Staining Microscopic Specimens Learning Objectives 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.
Think about It Explain why it is important to fix a specimen before viewing it under a light microscope. 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. 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? Clinical Focus: Nathan, Part 3 Figure 4.
Using Microscopy to Diagnose Tuberculosis Figure 5. Think about It Why are acid-fast stains useful? Think about It How does negative staining help us visualize capsules? Think about It Is endospore staining an example of positive, negative, or differential staining?
Think about It Why is it important to dehydrate cells before examining them under an electron microscope? Name the device that is used to create thin sections of specimens for electron microscopy. Think about It 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. Microscopy and Antibiotic Resistance As the use of antibiotics has proliferated in medicine, as well as agriculture, microbes have evolved to become more resistant.
Key Concepts and Summary Samples must be properly prepared for microscopy. A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining , capsule staining , endospore staining, and flagella staining.
Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used. Multiple Choice What mordant is used in Gram staining? Iodine is used in Gram staining. Show Answer Answer b. Only the SEM specimen requires sputter-coating.
Show Answer Ziehl-Neelsen staining, a type of acid-fast staining, is diagnostic for Mycobacterium tuberculosis. Show Answer The Gram stain is used to differentiate bacterial cells based on the components of their cell walls. Think about It How could you identify whether a particular bacterial sample contained specimens with mycolic acid-rich cell walls?
You use the Gram staining procedure to stain an L-form bacterium a bacterium that lacks a cell wall. What color will the bacterium be after the staining procedure is finished? Licenses and Attributions. CC licensed content, Shared previously. Step 1: Crystal Violet primary stain added to the specimen smear.
Step 2: Iodine mordant, makes the dye less soluble so it adheres to cell walls. Step 3: Alcohol the decolorizer, washes away stain from gram-negative cell walls.
Step 4: Safranin counterstain allows dye adherence to gram-negative cells. After staining with basic fuchsin, acid-fast bacteria resist decolonization by acid-alcohol. Non-acid-fast bacteria are counterstained with methylene blue.
Used to distinguish acid-fast bacteria such as M. Uses heat to stain endospores with malachite green Schaeffer-Fulton procedure , then cell is washed and counterstained with safranin. Flagella are coated with a tannic acid or potassium alum mordant, then stained using either pararosaline or basic fuchsin.
Negative staining with India ink or nigrosine is used to stain the background, leaving a clear area of the cell and the capsule Counterstaining can be used to stain the cell while leaving the capsule clear.
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.
This method, called the endospore stain , uses either heat or long exposure time to entice the endospores to take up the primary stain, usually a water soluble dye such as malachite green since endospores are permeable to water. Following a decolorization step which removes the dye from the vegetative cells in the smear, the counterstain safranin is applied to provide color and contrast.
When stained by this method, the endospores are green, and the vegetative cells stain pink, as shown in Figure 7. Figure 7. Bacterial cells with endospores, stained with the endospore stain. Although endospores themselves are resistant to the Gram stain technique, bacterial cells captured in the process of creating these structures can be stained.
In this case, the endospores are seen as clear oval or spherical areas within the stained cell. Endospores can also be directly observed in cells by using phase contrast microscopy, as shown in Figure 8. Because many differential staining methods require several steps and take a long time to complete, we will not be performing all of the differential staining methods discussed above. Pre-stained slides will be used to visualize bacterial capsules, metachromatic granules, and acid-fast bacilli.
Obtain one slide of each of the three bacteria listed in the table below. Your environmental isolate may have one or more of these cellular features, and learning to recognize them will aid in identification.
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