Microbiology chapter 2

who is Antonie van Leeuwenhoek

"the Father of Microbiology," is typically credited as the first person to have created microscopes powerful enough to view microbes

Note

Electron microscopy can be used to observe biofilms, but only after dehydrating the specimen, which produces undesirable artifacts and distorts the specimen. In addition to these approaches, it is possible to follow water currents through the shapes (such as cones and mushrooms) of biofilms, using video of the movement of fluorescently coated beads

. The steps of the Gram stain procedure are listed below and illustrated in Figure 2.33.

First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color. Next, Gram's iodine, a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; in this case, Gram's iodine acts like a trapping agent that complexes with the crystal violet, making the crystal violet-iodine complex clump and stay contained in thick layers of peptidoglycan in the cell walls. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. 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.

Some uses of biofilm

In nature, biofilms are abundant and frequently occupy complex niches within ecosystems (Figure 2.25). In medicine, biofilms can coat medical devices and exist within the body. Because they possess unique characteristics, such as increased resistance against the immune system and to antimicrobial drugs, biofilms are of particular interest to microbiologists and clinicians alike.

note

Though flagella staining is uncommon in clinical settings, the technique is commonly used by microbiologists, since the location and number of flagella can be useful in classifying and identifying bacteria in a sample. When using this technique, it is important to handle the specimen with great care; flagella are delicate structures that can easily be damaged or pulled off, compromising attempts to accurately locate and count the number of flagella.

smear)

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 2.31). 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

Does a microscope has high resolution?

True, it can be difficult to distinguish small structures in many specimens because microorganisms are relatively transparent.

True or false with an example The 20th century saw the development of microscopes that leveraged nonvisible light,

Trure, such as fluorescence microscopy,

Gram stain procedure

procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 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 Figure 2.33.

There are two types of scanning probe microscope

scanning tunneling microscope (STM) and the atomic force microscope (AFM).

Girolamo Fracastoro

the first person to formally postulate that disease was spread by tiny invisible seminaria, or "seeds of the contagion." In

the result of biofilm of being thick?

they cannot be observed very well using light microscopy; slicing a biofilm to create a thinner specimen might kill or disturb the microbial community. Confocal microscopy provides clearer images of biofilms because it can focus on one z-plane at a time and produce a three-dimensional image of a thick specimen. Fluorescent dyes can be helpful in identifying cells within the matrix. Additionally, techniques such as immunofluorescence and fluorescence in situ hybridization (FISH), in which fluorescent probes are used to bind to DNA, can be used.

Acid-Fast Stains

Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. An acid-fast stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not.

Various types of microscopes use different features of light or electrons to increase contrast—visible differences between the parts of a specimen (see

Additionally, dyes that bind to some structures but not others can be used to improve the contrast between images of relatively transparent objects

scanning tunneling microscope (STM)

An STM uses a probe that is passed just above the specimen as a constant voltage bias creates the potential for an electric current between the probe and the specimen. This current occurs via quantum tunneling of electrons between the probe and the specimen, and the intensity of the current is dependent upon the distance between the probe and the specimen. The probe is moved horizontally above the surface and the intensity of the current is measured. Scanning tunneling microscopy can effectively map the structure of surfaces at a resolution at which individual atoms can be detected

atomic force microscope (AFM

, AFMs have a thin probe that is passed just above the specimen. However, rather than measuring variations in the current at a constant height above the specimen, an AFM establishes a constant current and measures variations in the height of the probe tip as it passes over the specimen. As the probe tip is passed over the specimen, forces between the atoms (van der Waals forces, capillary forces, chemical bonding, electrostatic forces, and others) cause it to move up and down. Deflection of the probe tip is determined and measured using Hooke's law of elasticity, and this information is used to construct images of the surface of the specimen with resolution at the atomic level

Differential Interference Contrast Microscopes

1. (also known as Nomarski optics) 2. are similar to phase-contrast microscopes in that they use interference patterns to enhance contrast between different features of a specimen. 3. In a DIC microscope, two beams of light are created in which the direction of wave movement (polarization) differs. 4. Once the beams pass through either the specimen or specimen-free space, they are recombined and effects of the specimens cause differences in the interference patterns generated by the combining of the beams. 5. This results in high-contrast images of living organisms with a three-dimensional appearance. These microscopes are especially useful in distinguishing structures within live, unstained specimens.

Fluorescence Microscopes

1. A fluorescence microscope uses fluorescent chromophores called fluorochromes, which are capable of absorbing energy from a light source and then emitting this energy as visible light. 2. Fluorochromes include naturally fluorescent substances (such as chlorophylls) as well as fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4',6'-diamidino-2-phenylindole (DAPI) and acridine orange. The microscope transmits an excitation light, generally a form of EMR with a short wavelength, such as ultraviolet or blue light, toward the specimen; the chromophores absorb the excitation light and emit visible light with longer wavelengths. The excitation light is then filtered out (in part because ultraviolet light is harmful to the eyes) so that only visible light passes through the ocular lens. This produces an image of the specimen in bright colors against a dark background. Fluorescence microscopes are especially useful in clinical microbiology. They can be used to identify pathogens, to find particular species within an environment, or to find the locations of particular molecules and structures within a cell. Approaches have also been developed to distinguish living from dead cells using fluorescence microscopy based upon whether they take up particular fluorochromes. Sometimes, multiple fluorochromes are used on the same specimen to show different structures or features. One of the most important applications of fluorescence microscopy is a technique called immunofluorescence, which is used to identify certain disease-causing microbes by observing whether antibodies bind to them. (Antibodies are protein molecules produced by the immune system that attach to specific pathogens to kill or inhibit them.) There are two approaches to this technique: direct immunofluorescence assay (DFA) and indirect immunofluorescence assay (IFA). In DFA, specific antibodies (e.g., those that the target the rabies virus) are stained with a fluorochrome. If the specimen contains the targeted pathogen, one can observe the antibodies binding to the pathogen under the fluorescent microscope. This is called a primary antibody stain because the stained antibodies attach directly to the pathogen. In IFA, secondary antibodies are stained with a fluorochrome rather than primary antibodies. Secondary antibodies do not attach directly to the pathogen, but they do bind to primary antibodies. When the unstained primary antibodies bind to the pathogen, the fluorescent secondary antibodies can be observed binding to the primary antibodies. Thus, the secondary antibodies are attached indirectly to the pathogen. Since multiple secondary antibodies can often attach to a primary antibody, IFA increases the number of fluorescent antibodies attached to the specimen, making it easier visualize features in the specimen

Microscopy and Antibiotic Resistance

1. As the use of antibiotics has proliferated in medicine, as well as agriculture, microbes have evolved to become more resistant. 2. Strains of bacteria such as methicillin-resistant S. aureus (MRSA), which has developed a high level of resistance to many antibiotics, are an increasingly worrying problem, so much so that research is underway to develop new and more diversified antibiotics. 3. Fluorescence microscopy can be useful in testing the effectiveness of new antibiotics against resistant strains like MRSA. 4.In a test of one new antibiotic derived from a marine bacterium, MC21-A (bromophene), researchers used the fluorescent dye SYTOX Green to stain samples of MRSA. 5. SYTOX Green is often used to distinguish dead cells from living cells, with fluorescence microscopy. 6. 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. 7. In this particular case, MRSA bacteria that had been exposed to MC21-A did, indeed, appear green under the fluorescence microscope, leading researchers to conclude that it is an effective antibiotic against MRSA. 8. 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. 9. 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?

Capsule Staining

1. Certain bacteria and yeasts have a protective outer structure called a capsule. 2. Since the presence of a capsule is directly related to a microbe's virulence (its ability to cause disease). 3. the ability to determine whether cells in a sample have capsules is an important diagnostic tool. 4. Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is typically used for capsule staining. 5. The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell. 6. The specimen does not need to be heat-fixed prior to negative staining. 7. One common negative staining technique for identifying encapsulated yeast and bacteria is to add a few drops of India ink or nigrosin to a specimen. 8. Other capsular stains can also be used to negatively stain encapsulated cells (Figure 2.37). 9. Alternatively, positive and negative staining techniques can be combined to visualize capsules: 10. The positive stain colors the body of the cell, and the negative stain colors the background but not the capsule, leaving halo around each cell.

note

1. Endospore-staining techniques are important for identifying Bacillus and Clostridium, two genera of endospore-producing bacteria that contain clinically significant species. 2. Among others, B. anthracis (which causes anthrax) has been of particular interest because of concern that its spores could be used as a bioterrorism agent. C. difficile is a particularly important species responsible for the typically hospital-acquired infection known as "C. diff."

Endospore Staining

1. Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions. 2. Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. 3. Endospore staining uses two stains to differentiate endospores from the rest of the cell. 4. The Schaeffer-Fulton method (the most commonly used endospore-staining technique) uses heat to push the primary stain (malachite green) into the endospore. 5. Washing with water decolorizes the cell, but the endospore retains the green stain. 6. The cell is then counterstained pink with safranin. 7. The resulting image reveals the shape and location of endospores, if they are present. 8. The green endospores will appear either within the pink vegetative cells or as separate from the pink cells altogether. 9. If no endospores are present, then only the pink vegetative cells will be visible

Flagella Staining

1. Flagella (singular: flagellum) are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes. 2. Because they are so thin, flagella typically cannot be seen under a light microscope without a specialized flagella staining technique. 3.Flagella staining thickens the flagella by first applying mordant (generally tannic acid, but sometimes potassium alum), 4.which coats the flagella; then the specimen is stained with pararosaniline (most commonly) or basic fuchsin

Ligh Microscopy

1. Many types of microscopes fall under the category of light microscopes 2. which use light to visualize images. 3. Examples of light microscopes include brightfield microscopes, darkfield microscopes, phase-contrast microscopes, differential interference contrast microscopes, fluorescence microscopes, confocal scanning laser microscopes, and two-photon microscopes. 4. These various types of light microscopes can be used to complement each other in diagnostics and research.

Phase-Contrast Microscopes

1. Phase-contrast microscopes use refraction and interference caused by structures in a specimen to create high-contrast, high-resolution images without staining. 2. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. 3. To create altered wavelength paths, an annular stop is used in the condenser. 4.The annular stop produces a hollow cone of light that is focused on the specimen before reaching the objective lens. 5. The objective contains a phase plate containing a phase ring. 6. As a result, light traveling directly from the illuminator passes through the phase ring while light refracted or reflected by the specimen passes through the plate. 7. This causes waves traveling through the ring to be about one-half of a wavelength out of phase with those passing through the plate. 8. Because waves have peaks and troughs, they can add together (if in phase together) or cancel each other out (if out of phase). 9. When the wavelengths are out of phase, wave troughs will cancel out wave peaks, which is called destructive interference. 10. Structures that refract light then appear dark against a bright background of only unrefracted light. More generally, structures that differ in features such as refractive index will differ in levels of darkness 11.Because it increases contrast without requiring stains, phase-contrast microscopy is often used to observe live specimens. 12.Certain structures, such as organelles in eukaryotic cells and endospores in prokaryotic cells, are especially well visualized with phase-contrast microscopy

Preparing Specimens for Electron Microscopy

1. Samples to be analyzed using a TEM must have very thin sections. 2. But cells are too soft to cut thinly, even with diamond knives. To cut cells without damage, the cells must be embedded in plastic resin and then dehydrated through a series of soaks in ethanol solutions (50%, 60%, 70%, and so on). The ethanol replaces the water in the cells, and the resin dissolves in ethanol and enters the cell, where it solidifies. Next, thin sections are cut using a specialized device called an ultramicrotome (Figure 2.42). Finally, samples are fixed to fine copper wire or carbon-fiber grids and stained—not with colored dyes, but with substances like uranyl acetate or osmium tetroxide, which contain electron-dense heavy metal atoms.

Brightfield Microscopes

1. The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more lenses that produce a dark image on a bright background. 2. Some brightfield microscopes are monocular (having a single eyepiece), though most newer brightfield microscopes are binocular (having two eyepieces) 3. each eyepiece contains a lens called an ocular lens 4. The ocular lenses typically magnify images 10 times (10⨯). 5.At the other end of the body tube are a set of objective lenses on a rotating nosepiece. 6. The magnification of these objective lenses typically ranges from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses work together to create a magnified image. 7.The total magnification is the product of the ocular magnification times the objective magnification

note

1. When samples are prepared for viewing using an SEM, they must also be dehydrated using an ethanol series. However, they must be even drier than is necessary for a TEM. 2. Critical point drying with inert liquid carbon dioxide under pressure is used to displace the water from the specimen. 3. After drying, the specimens are sputter-coated with metal by knocking atoms off of a palladium target, with energetic particles. Sputter-coating prevents specimens from becoming charged by the SEM's electron beam

Robert Hooke (1635-1703),

1. also made important contributions to microscopy, publishing in his book Micrographia 2. many observations using compound microscopes. 3. Viewing a thin sample of cork through his microscope, he was the first to observe the structures that we now know as cells 4. Hooke described these structures as resembling "Honey-comb," and as "small Boxes or Bladders of Air," noting that each "Cavern, Bubble, or Cell" is distinct from the others (in Latin, "cell" literally means "small room"). They likely appeared to Hooke to be filled with air because the cork cells were dead, with only the rigid cell walls providing the structure.

Darkfield Microscopy

1.is a brightfield microscope that has a small but significant modification to the condenser. 2. A small, opaque disk (about 1 cm in diameter) is placed between the illuminator and the condenser lens. 3.This opaque light stop, as the disk is called, blocks most of the light from the illuminator as it passes through the condenser on its way to the objective lens, producing a hollow cone of light that is focused on the specimen. 4. The only light that reaches the objective is light that has been refracted or reflected by structures in the specimen. 5. The resulting image typically shows bright objects on a dark background

why the microscope is very important?

It is often necessary to increase contrast to detect different structures in a specimen.

Using Microscopy to Diagnose Tuberculosis

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, can be detected in specimens based on the presence of acid-fast bacilli. Often, a smear is prepared from a sample of the patient's sputum and then stained using the Ziehl-Neelsen technique (Figure 2.36). If acid-fast bacteria are confirmed, they are generally cultured to make a positive identification. Variations of this approach can be used as a first step in determining whether M. tuberculosis or other acid-fast bacteria are present, though samples from elsewhere in the body (such as urine) may contain other Mycobacterium species. An alternative approach for determining the presence of M. tuberculosis is immunofluorescence. In this technique, fluorochrome-labeled antibodies bind to M. tuberculosis, if present. Antibody-specific fluorescent dyes can be used to view the mycobacteria with a fluorescence microscope.

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. Figure 2.40 provides more detail.

Note

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

Note

Preparation and Staining for Other Microscopes

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(111). Toluene vapor is a common fixative.

Who invented the microscope? (1)

Some argue that this designation should belong to Hans and Zaccharias Janssen, Dutch spectacle-makers who may have invented the telescope,

fixation

The "fixing" of a sample refers to the process of attaching cells to a slide. 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.

Using Microscopy to Diagnose Syphilis

The causative agent of syphilis is Treponema pallidum, a flexible, spiral cell (spirochete) that can be very thin (<0.15 μm) and match the refractive index of the medium, making it difficult to view using brightfield microscopy. Additionally, this species has not been successfully cultured in the laboratory on an artificial medium; therefore, diagnosis depends upon successful identification using microscopic techniques and serology (analysis of body fluids, often looking for antibodies to a pathogen). 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. It is also possible to use fluorescence or electron microscopy to view Treponema

Electron Microscopy

The maximum theoretical resolution of images created by light microscopes is ultimately limited by the wavelengths of visible light. Most light microscopes can only magnify 1000⨯, and a few can magnify up to 1500⨯, but this does not begin to approach the magnifying power of an electron microscope (EM), which uses short-wavelength electron beams rather than light to increase magnification and resolution. Electrons, like electromagnetic radiation, can behave as waves, but with wavelengths of 0.005 nm, they can produce much better resolution than visible light. An EM can produce a sharp image that is magnified up to 100,000⨯. Thus, EMs can resolve subcellular structures as well as some molecular structures (e.g., single strands of DNA); however, electron microscopy cannot be used on living material because of the methods needed to prepare the specimens. There are two basic types of EM: the transmission electron microscope (TEM) and the scanning electron microscope (SEM) (Figure 2.21). The TEM is somewhat analogous to the brightfield light microscope in terms of the way it functions. However, it uses an electron beam from above the specimen that is focused using a magnetic lens (rather than a glass lens) and projected through the specimen onto a detector. Electrons pass through the specimen, and then the detector captures the image For electrons to pass through the specimen in a TEM, the specimen must be extremely thin (20-100 nm thick). The image is produced because of varying opacity in various parts of the specimen. This opacity can be enhanced by staining the specimen with materials such as heavy metals, which are electron dense. TEM requires that the beam and specimen be in a vacuum and that the specimen be very thin and dehydrated. The specific steps needed to prepare a specimen for observation under an EM are discussed in detail in the next section. SEMs form images of surfaces of specimens, usually from electrons that are knocked off of specimens by a beam of electrons. This can create highly detailed images with a three-dimensional appearance that are displayed on a monitor (Figure 2.23). Typically, specimens are dried and prepared with fixatives that reduce artifacts, such as shriveling, that can be produced by drying, before being sputter-coated with a thin layer of metal such as gold. Whereas transmission electron microscopy requires very thin sections and allows one to see internal structures such as organelles and the interior of membranes, scanning electron microscopy can be used to view the surfaces of larger objects (such as a pollen grain) as well as the surfaces of very small samples (Figure 2.24). Some EMs can magnify an image up to 2,000,000⨯.1

wet mount

The simplest type of preparation is the wet mount, in which the specimen is placed on the slide in a drop of liquid. Some specimens, such as a drop of urine, are already in a liquid form and can be deposited on the slide using a dropper. Solid specimens, such as a skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid used is simply water, but often stains are added to enhance contrast. Once the liquid has been added to the slide, a coverslip is placed on top and the specimen is ready for examination under the microscope

Confocal Microscopes

Whereas other forms of light microscopy create an image that is maximally focused at a single distance from the observer (the depth, or z-plane), a confocal microscope uses a laser to scan multiple z-planes successively. This produces numerous two-dimensional, high-resolution images at various depths, which can be constructed into a three-dimensional image by a computer. As with fluorescence microscopes, fluorescent stains are generally used to increase contrast and resolution. Image clarity is further enhanced by a narrow aperture that eliminates any light that is not from the z-plane. Confocal microscopes are thus very useful for examining thick specimens such as biofilms, which can be examined alive and unfixed

Two-Photon Microscopes

While the original fluorescent and confocal microscopes allowed better visualization of unique features in specimens, there were still problems that prevented optimum visualization. The effective sensitivity of fluorescence microscopy when viewing thick specimens was generally limited by out-of-focus flare, which resulted in poor resolution. This limitation was greatly reduced in the confocal microscope through the use of a confocal pinhole to reject out-of-focus background fluorescence with thin (<1 μm), unblurred optical sections. However, even the confocal microscopes lacked the resolution needed for viewing thick tissue samples. These problems were resolved with the development of the two-photon microscope, which uses a scanning technique, fluorochromes, and long-wavelength light (such as infrared) to visualize specimens. The low energy associated with the long-wavelength light means that two photons must strike a location at the same time to excite the fluorochrome. The low energy of the excitation light is less damaging to cells, and the long wavelength of the excitation light more easily penetrates deep into thick specimens. This makes the two-photon microscope useful for examining living cells within intact tissues—brain slices, embryos, whole organs, and even entire animals. Currently, use of two-photon microscopes is limited to advanced clinical and research laboratories because of the high costs of the instruments. A single two-photon microscope typically costs between $300,000 and $500,000, and the lasers used to excite the dyes used on specimens are also very expensive. However, as technology improves, two-photon microscopes may become more readily available in clinical settings.

Two different methods for acid-fast staining are

Ziehl-Neelsen technique and the Kinyoun technique . Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after a decolorizing agent (an acid-alcohol solution) is applied. A secondary counterstain, methylene blue, is then applied, which renders non-acid-fast cells blue. The fundamental difference between the two carbolfuchsin-based methods is whether heat is used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria (AFB). If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the surrounding tissue cells

Antonie van Leeuwenhoek (1632-1723)

being the first person to observe microbes, including bacteria, which he called "animalcules" and "wee little beasties." Even though van Leeuwenhoek's microscopes were simple microscopes they were more powerful and provided better resolution than the compound microscopes of his day. (c) Though more famous for developing the telescope, Galileo Galilei was also one of the pioneers of microscopy.

what Darkfield microscopy can often create?

create high-contrast, high-resolution images of specimens without the use of stains, which is particularly useful for viewing live specimens that might be killed or otherwise compromised by the stains. For example, thin spirochetes like Treponema pallidum, the causative agent of syphilis, can be best viewed using a darkfield microscope (

Joseph Jackson Lister

created an essentially modern light microscope.

Hans Lippershey

developed microscopes and telescopes during the same time frame, and he is often credited with inventing the telescope. The historical records from the time are as fuzzy and imprecise as the images viewed through those early lenses, and any archived records have been lost over the centuries.

What are more information about Antonie van Leeuwenhoek?

he began his career selling fabrics. he later became interested in lens making. and his innovative techniques produced microscopes that allowed him to observe microorganisms as no one had before. In 1674, he described his observations of single-celled organisms, whose existence was previously unknown, in a series of letters to the Royal Society of London. His report was initially met with skepticism, but his claims were soon verified and he became something of a celebrity in the scientific community.

Book De Contagione for Girolamo Fracastoro

he proposed that these seeds could attach themselves to certain objects (which he called fomes [cloth]) that supported their transfer from person to person.

What is the definition of numerical aperture?

is a measure of a lens's ability to gather light. The higher the numerical aperture, the better the resolution.

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.

Scanning Probe Microscopy

it does not use light or electrons, but rather very sharp probes that are passed over the surface of the specimen and interact with it directly. This produces information that can be assembled into images with magnifications up to 100,000,000⨯. Such large magnifications can be used to observe individual atoms on surfaces. To date, these techniques have been used primarily for research rather than for diagnostics.

Biofilm

it is a complex community of one or more microorganism species, typically forming as a slimy coating attached to a surface because of the production of an extrapolymeric substance (EPS) that attaches to a surface or at the interface between surfaces (e.g., between air and water).

What is the second factor that affects resolution?

numerical aperture

Galileo Galilei

used a compound microscope to examine insect parts (Figure 2.9). Whereas van Leeuwenhoek used a simple microscope, in which light is passed through just one lens, Galileo's compound microscope was more sophisticated, passing light through two sets of lenses.

Who invented the microscope?(2)

van Leeuwenhoek and Hooke can thank ample documentation of their work for their respective legacies. Like Janssen, van Leeuwenhoek began his work in obscurity, leaving behind few records. However, his friend, the prominent physician Reinier de Graaf, wrote a letter to the editor of the Philosophical Transactions of the Royal Society of London calling attention to van Leeuwenhoek's powerful microscopes. From 1673 onward, van Leeuwenhoek began regularly submitting letters to the Royal Society detailing his observations. In 1674, his report describing single-celled organisms produced controversy in the scientific community, but his observations were soon confirmed when the society sent a delegation to investigate his findings. He subsequently enjoyed considerable celebrity, at one point even entertaining a visit by the czar of Russia. Similarly, Robert Hooke had his observations using microscopes published by the Royal Society in a book called Micrographia in 1665. The book became a bestseller and greatly increased interest in microscopy throughout much of Europe.

In clinical settings, light microscopes are the most commonly used microscopes. There are two basic types of preparation used to view specimens with a light microscope

wet mounts and fixed specimens.

fluorescence microscopy

which uses an ultraviolet light source, and electron microscopy, which uses short-wavelength electron beams. These advances led to major improvements in magnification, resolution, and contrast.