chpt 4
phage typing.
A bacteriophage is a virus that inserts its DNA into a bacterium. Commonly called a phage, it adheres only to a select bacterial strain for which each phage type has a specific adhesion factor. Many phages are so specialized for their particular bacterial strain that scientists have used phages to identify and classify bacteria. Such identification is called
A recent advance in microscopy utilizes minuscule, pointed electronic probes to magnify more than 100,000,000×. There are two variations of probe microscopes: scanning tunneling microscopes and atomic force microscopes.
A scanning tunneling microscope (STM) passes a metallic probe, sharpened to end in a single atom, back and forth across and slightly above the surface of a specimen. Rather than scattering a beam of electrons into a detector, as in scanning electron microscopy, a scanning tunneling microscope measures the flow of electrons to and from the probe and the specimen's surface. The amount of electron flow, called a tunneling current, is directly proportional to the distance from the probe to the specimen's surface. A scanning tunneling microscope can measure distances as small as 0.01 nm and reveal three-dimensional details on the surface of a specimen at the atomic level (FIGURE 4.12a). A requirement for scanning tunneling microscopy is that the specimen be electrically conductive. An atomic force microscope (AFM) also uses a pointed probe, but it traverses the tip of the probe lightly on the surface of the specimen rather than at a distance. This might be likened to the way a person reads Braille. Deflection of a laser beam aimed at the probe's tip measures vertical movements, which when translated by a computer reveals the three-dimensional atomic topography. Unlike tunneling microscopes, atomic force microscopes can magnify specimens that do not conduct electrons. They can also magnify living specimens because neither an electron beam nor a vacuum is required (FIGURE 4.12b). Researchers have used atomic force microscopes to magnify the surfaces of bacteria, viruses, proteins, and amino acids. Recent studies using them have examined single living bacteria in three dimensions as they grow and divide.
There are two general types of electron microscopes: transmission electron microscopes and scanning electron microscopes.
A transmission electron microscope (TEM) generates a beam of electrons that ultimately produces an image on a fluorescent screen (FIGURE 4.10a). The path of electrons is similar to the path of light in a light microscope. From their source, the electrons pass through the specimen, through magnetic fields (instead of glass lenses) that manipulate and focus the beam, and then onto a fluorescent screen that changes some of their energy into visible light (FIGURE 4.10b). Dense areas of the specimen block electrons, resulting in a dark area on the screen. In regions where the specimen is less dense, the screen fluoresces more brightly. As with light microscopy, contrast and resolution can be enhanced through the use of electron-dense stains, which are discussed later. The brightness of each region of the screen corresponds to the number of electrons striking it. Therefore, the image on the screen is composed of light and dark areas. The screen can be folded out of the way to enable the electrons to strike a photographic film, located in the base of the microscope. Prints made from the film are called transmission electron micrographs or TEM images (FIGURE 4.10c). Photographers often colorize such images to emphasize certain features. A scanning tunneling microscope (SEM) also uses magnetic fields within a vacuum tube to manipulate a beam of electrons; however, rather than passing electrons through a specimen, a SEM rapidly focuses the electrons back and forth across a specimen's surface, which has previously been coated with a metal such as platinum or gold. The electrons knock other electrons off the surface of the coated specimen, and these scattered electrons pass through a detector and a photomultiplier, producing an amplified signal that is displayed on a monitor. Typically, scanning microscopes are used to magnify up to 10,000× with a resolution of about 20 nm. One advantage of scanning microscopy over transmission microscopy is that whole specimens can be observed because sectioning is not required. Scanning electron micrographs can be beautifully realistic and appear three-dimensional (FIGURE 4.11). Two disadvantages of a scanning electron microscope are that it magnifies only the external surface of a specimen and that, like TEM, it requires a vacuum and thus can examine only dead organisms.
An oil immersion lens
An oil immersion lens increases not only magnification but also resolution. As we have seen, light refracts as it travels from air into glass and also from glass into air; therefore, some of the light passing out of a glass slide is bent so much that it bypasses the lens (FIGURE 4.5a). Placing immersion oil (historically, cedarwood oil; today, more commonly a synthetic oil) between the slide and an oil immersion objective lens enables the lens to capture this light because light travels through immersion oil at the same speed as through glass. Because light is traveling at a uniform speed through the slide, the immersion oil, and the glass lens, it does not refract (FIGURE 4.5b). Immersion oil increases the numerical aperture, which increases resolution because more light rays are gathered into the lens to produce the image. Obviously, the space between the slide and the lens can be filled with oil only if the distance between the lens and the specimen, called the working distance, is small.
Dyes used as microbiological stains for light microscopy are usually salts. A salt is composed of a positively charged cation and a negatively charged anion. At least one of the two ions in the molecular makeup of dyes is colored; this colored portion of a dye is known as the chromophore. Chromophores bind to chemicals via covalent, ionic, or hydrogen bonds. For example, methylene blue chloride is composed of a cationic chromophore, methylene blue, and a chloride anion. Because methylene blue is positively charged, it ionically bonds to negatively charged molecules in cells, including DNA and many proteins. In contrast, anionic dyes, for example, eosin, bind to positively charged molecules, such as some amino acids.
Anionic chromophores are also called acidic dyes because they stain alkaline structures and work best in acidic (low pH) environments. Positively charged, cationic chromophores are called basic dyes because they combine with and stain acidic structures; further, they work best under basic (higher pH) conditions. In microbiology, basic dyes are used more commonly than acidic dyes because most cells are negatively charged. Acidic dyes are used in negative staining, which is discussed shortly.
Analysis of Nucleic Acids
As we have discussed, the sequence of nucleotides in nucleic acid molecules (either DNA or RNA) provides a powerful tool for classifying and identifying microbes. In many cases, nucleic acid analysis has confirmed classical taxonomic hierarchies. In other cases, as in Woese and Fox's discovery of archaea, curious new organisms and relationships not obvious from classical methodologies have come to light. Techniques of nucleotide sequencing and comparison, such as polymerase chain reaction (PCR) (Chapter 8), are best understood after we have discussed microbial genetics (Chapter 7). For our present discussion, suffice it to say that scientists most commonly use an approach called 16S sequencing to describe bacteria in clinical and research samples. This method does not require culturing the bacterium; therefore, 16S sequencing is faster than identification methods that are based on staining, growth, or biochemical tests. Further, researchers can describe the 16S sequences of species that scientists have not cultured in the laboratory. Scientists compare the 16S sequence of a bacterium to all known 16S sequences in a database. If there is a match, they have identified the bacterium; if not, then they can find similar 16S sequences in the database and thus find related microbes. 16S sequencing is now the "gold standard" for bacterial identification. Determining the percentage of a cell's DNA that is guanine and cytosine, a quantity referred to as the cell's G+C content (or G+C percentage), has also become a part of prokaryotic taxonomy. Scientists express the content as follows: G+CA+T+G+C×100 G+C content varies from 20% to 80% among prokaryotes. Often (but not always), organisms that share characteristics have similar G+C content. Organisms that were once thought to be closely related but have widely different G+C percentages are, in almost all cases, not as closely related as had been thought.
Phage Typing
Bacteriophages (or simply phages) are viruses that infect and usually destroy bacterial cells. Just as antibodies are specific for their target antigens, phages are specific for the hosts they can infect. Phage typing, like serological testing, works because of such specificity. One bacterial strain may be susceptible to a particular phage while a related strain is not. In phage typing, a technician spreads a solution containing the bacterium to be identified across a solid surface of growth medium and then adds small drops of solutions containing different types of bacteriophage. Wherever a specific phage is able to infect and kill bacteria, the resulting lack of bacterial growth produces within the bacterial lawn a clear area called a plaque (FIGURE 4.24). A microbiologist can identify an unknown bacterium by comparing the phages that form plaques with known phage-bacteria interactions.
Microorganisms are spread in liquid across the surface of a slide using a circular motion. After drying in the air, the smear is passed through the flame of a Bunsen burner to fix the cells to the glass. Alternatively, chemical fixation can be used. Why must a smear be fixed to the slide?
Fixation causes the specimen to adhere to the glass so that it does not easily wash off during staining.
Gram Stain
In 1884, the Danish scientist Hans Christian Gram (1853-1938) developed the most frequently used differential stain, which now bears his name. The Gram stain differentiates between two large groups of microorganisms: purple-staining Gram-positive cells and pink-staining Gram-negative cells. These cells differ significantly in the chemical and physical structures of their cell walls (see Figure 3.16). Typically, a Gram stain is the first step a medical laboratory scientist performs to identify bacterial pathogens; knowing the Gram reaction of a bacterium can help a provider select an appropriate antimicrobial drug.
genera
In Linnaeus's system, which forms the basis of modern taxonomy, similar species are grouped into Genera 79 and similar genera into still larger taxonomic categories. That is, genera sharing common features are grouped together to form families; similar families are grouped into orders; orders are grouped into classes; classes into phyla;80 and phyla into kingdoms (FIGURE 4.20).
dark-field microscopes
In dark-field microscopes, the specimen is made to appear light against a dark background. These microscopes prevent light from directly entering the objective lens. Instead, light rays are reflected inside the condenser, so that they pass into the slide at such an oblique angle that they completely miss the objective lens. Only light rays scattered by a specimen can enter the objective lens and be seen, so the specimen appears light against a dark background—the field. Dark-field microscopy increases contrast and enables observation of more details than are visible in bright-field microscopy. Dark-field microscopes are especially useful for examining small or colorless cells.
MALDI-TOF Mass Spectrometry
Medical laboratory scientists can use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, more simply known as MALDI-TOF (mal′de-toff) to identify microbial species based upon unique proteins that the microbes synthesize. The technique uses a matrix to stabilize ions of large organic molecules, such as proteins, which have been ionized with laser energy. The organic molecules remain stable and retain their structure so that scientists can sort them by mass spectrometry—a process that sorts chemicals based on their mass-to-charge ratios. Though the details of MALDI-TOF and mass spectrometry are beyond the scope of this text, scientists use MALDI-TOF to sort and identify proteins unique to specific microbes and thereby show that particular microbes are present in a sample. Scientists and physicians use MALDI-TOF to identify species, to predict resistance to particular antibiotics, and to rapidly diagnose diseases.
Biochemical Tests
Microbiologists distinguish many prokaryotes on the basis of differences in their ability to utilize or produce certain chemicals
confocal microscopes.
Microscopes that use lasers to illuminate fluorescent chemicals in a thin plane of a specimen are called confocal microscopes. Confocal microscopes 75 also use fluorescent dyes or fluorescent antibodies, but these microscopes use ultraviolet lasers to illuminate the fluorescent chemicals in only a single plane that is no thicker than 1.0μm; the rest of the specimen remains dark and out of focus. Visible light emitted by the dyes passes through a pinhole aperture that helps eliminate blurring that can occur with other types of microscopes and increases resolution by up to 40%. Each image from a confocal microscope is thus an "optical slice" through the specimen, as if it had been thinly cut. Once individual images are digitized, a computer is used to construct a three-dimensional representation, which can be rotated and viewed from any direction (FIGURE 4.9 on the next page). Confocal microscopes have been particularly useful for examining the relationships among various organisms within complex microbial communities called biofilms. Regular light microscopy cannot produce clear images of structures within a living biofilm, and removing surface layers from a biofilm would change the dynamics of a biofilm community.
staining
Most microorganisms are colorless and difficult to view with bright-field microscopes. Microscopists use stains to make microorganisms and their parts more visible because stains increase contrast between structures and between a specimen and its background. Staining helps medical personnel identify pathogens, which in turn helps them prescribe optimal treatments. Electron microscopy also requires that specimens be treated with stains or coatings to enhance contrast.
Differential Stains
Most stains used in microbiology are differential stains, which use more than one dye so that different cells, chemicals, or structures can be distinguished when microscopically examined. Common differential stains are the Gram stain, the acid-fast stain, the endospore stain, Gomori methenamine silver stain, and hematoxylin and eosin stain.
Special Stains 101 Special stains are simple stains designed to reveal specific microbial structures. Three types of special stains are negative stains, flagellar stains, and fluorescent stains (which we already discussed in the section on fluorescence microscopy).
Negative (Capsule) Stain Most dyes used to stain bacterial cells, such as crystal violet, methylene blue, malachite green, and safranin, are basic dyes. These dyes stain cells by attaching to negatively charged molecules within them. Acidic dyes, by contrast, are repulsed by the negative charges on the surface of cells and, therefore, do not stain them. Such stains are called negative stainsbecause they stain the background and leave cells colorless. Eosin and nigrosin are examples of acidic dyes used for negative staining. Negative stains are used primarily to reveal the presence of negatively charged bacterial capsules. Therefore, they are also called capsule stains. Encapsulated cells appear to have a halo surrounding them (FIGURE 4.18). Flagellar Stain Bacterial flagella are extremely thin and thus normally invisible with light microscopy, but their presence, number, and arrangement are important in identifying some species, including some pathogens. Flagellar stains, such as pararosaniline and carbolfuchsin, in combination with mordants—chemicals that combine with a dye and make it less soluble and therefore affix it in a material—are applied in a series of steps. Flagellar stains bind to flagella, increasing their diameter and colorizing them, which increases contrast and makes the flagella visible (FIGURE 4.19).
A principle of microscopy is that resolution is dependent on (1) the wavelength of the electromagnetic radiation and (2) the numerical aperture of the lens, which refers to the ability of a lens to gather light.
Resolution may be calculated using the following formula: resolution=0.61×wavelength. divided by numerical aperture
Physical Characteristics
Scientists can usually identify protozoa, fungi, algae, and parasitic worms based solely on their morphology (shape). Linnaeus categorized prokaryotic cells into two genera based on two prevalent shapes. He classified spherical prokaryotes in the genus "Coccus,"86 and he placed rod-shaped cells in the genus Bacillus.87
compound microscopes.
Simple microscopes have been replaced in modern laboratories by compound microscopes. A compound microscope uses a series of lenses for magnification (FIGURE 4.4a). Many scientists, including Galileo Galilei (1564-1642), made compound microscopes as early as 1590, but it was not until about 1830 that scientists developed compound microscopes that exceeded the clarity and magnification of van Leeuwenhoek's simple microscope. magnification is achieved as light rays pass through a specimen and into an objective lens, which is the lens immediately above the object being magnified An objective lens is really a series of lenses that not only create a magnified image but also are engineered to reduce aberrations in the shape and color of the image. Most light microscopes used in biology have three or four objective lenses mounted on a revolving nosepiece. The objective lenses on a typical microscope are scanning objective lens (4×), low-power objective lens (10×), high-power lens or high dry objective lens (40×), and oil immersion objective lens (100×).
Endospore Stain
Some bacteria—notably those of the genera Bacillus and Clostridium (klos-trid′ē-ŭm), which contain species that cause such diseases as anthrax, gangrene, and tetanus—produce endospores. These dormant, highly resistant cells form inside the cytoplasm of the bacteria and can survive environmental extremes such as desiccation, heat, and harmful chemicals. Endospores cannot be stained by normal staining procedures because their walls are practically impermeable at room temperature. The Schaeffer-Fulton endospore stain uses heat to drive the primary stain, malachite green, into the endospore. After cooling, the slide is decolorized with water and counterstained with safranin. This staining procedure results in green-stained endospores and red-colored vegetative cells (FIGURE 4.17). Heat from steam is used to drive the green primary stain into the endospores. Counterstaining is performed at room temperature, and the thick, impermeable walls of the endospores resist the counterstain.
The smear is air-dried completely and then attached or fixed to the surface of the slide. In heat fixation, developed more than a hundred years ago by Robert Koch, the slide is gently heated by passing the slide, smear up, through a flame. Alternatively, chemical fixation involves applying a chemical such as methyl alcohol to the smear for one minute. Desiccation (drying) and fixation kill the microorganisms, attach them firmly to the slide, and generally preserve their shape and size. It is important to smear and fix specimens properly so that they are not lost during staining.
Specimens prepared for electron microscopy must be dry because water vapor from a wet specimen would stop an electron beam. As we have seen, transmission electron microscopy requires that the desiccated sample also be sliced very thin, generally before staining. Specimens for scanning electron microscopy are coated, not stained.
condenser lens (or lenses),
directs light through the specimen, as well as one or more mirrors or prisms that deflect the path of the light rays from an objective lens to the ocular lens. Some microscopes have mirrors or prisms that direct light to a camera through a special tube. A photograph of such a microscopic image is called a light micrograph (LM); micrograph refers to any microscopic image.
magnifies- other than light
electrons
acid fast stain
The acid-fast stain is another important differential stain because it stains cells of the genera Mycobacterium and Nocardia (nō-kar′dē-ă), which cause many human diseases, including tuberculosis, leprosy, and other lung and skin infections. Cells of these bacteria have large amounts of waxy lipid in their cell walls, so they do not readily stain with the water-soluble dyes used in Gram staining. Microbiological laboratories can use a variation of the acid-fast stain developed by Franz Ziehl (1857-1926) and Friedrich Neelsen (1854-1894) in 1883. Their procedure is as follows: Cover the smear with a small piece of tissue paper to retain the dye during the procedure. Flood the slide with the red primary stain, carbolfuchsin, for several minutes while warming it over steaming water. In this procedure, heat is used to drive the stain through the waxy wall and into the cell, where it remains trapped. Remove the tissue paper, cool the slide, and then decolorize the smear by rinsing it with a solution of hydrochloric acid (pH < 1.0) and alcohol. The bleaching action of acid-alcohol removes color from both non-acid-fast cells and the background. Acid-fast cells retain their red color because the acid cannot penetrate the waxy wall. The name of the procedure is derived from this step; that is, the cells are colorfast in acid. Counterstain with methylene blue, which stains only bleached, non-acid-fast cells. The Ziehl-Neelsen acid-fast staining procedure results in pink acid-fast cells, which can be differentiated from blue non-acid-fast cells, including human cells (FIGURE 4.16). The presence of acid-fast bacilli (AFBs) in sputum is indicative of mycobacterial infection.
species
The definition of species as "a group of organisms that interbreed to produce viable offspring" works relatively well for sexually reproducing organisms, but it is not satisfactory for asexual organisms such as most microorganisms. As a result, some scientists define a microbial species as a collection of strains or serotypes—populations of cells that arose from a single cell—that share many stable properties, differ from other strains, and evolve as a group. Alternatively, biologists define a microbial species as cells that share at least 97% common genetic sequences. Not surprisingly, these definitions sometimes result in disagreements and inconsistencies in the classification of microbial life. Some researchers question whether unique microbial species exist at all.
bright-field microscopes
The most common microscopes are bright-field microscopes, in which the background (or field) is illuminated. There are two basic types of bright-field microscopes: simple microscopes and compound microscopes.
Wavelength of Radiation
These various forms of radiation differ in wavelength—the distance between two corresponding parts of a wave. For example, the wavelength of electrons at 10,000 volts (V) is 0.01 nm; that of electrons at 1,000,000 V is 0.001 nm. As we will see, using radiation of smaller wavelengths results in enhanced microscopy.
Taxa.
They sort organisms on the basis of mutual similarities into nonoverlapping groups called
Resolution,
also called resolving power, is the ability to distinguish two points that are close together. An optometrist's eye chart is a test of resolution at a distance of 20 feet (6.1 m). Van Leeuwenhoek's microscopes had a resolving power of about 1 μm; that is, he could distinguish objects if they were more than about 1 μm apart, whereas objects closer together than 1 μm appeared as a single object. The better the resolution, the better the ability to distinguish two objects that are close to one another.
Simple stains
are composed of a single basic dye, such as crystal violet, safranin, or methylene blue. They are "simple" because they involve no more than soaking the smear in the dye for 30-60 seconds and then rinsing off the slide with water. (A properly fixed specimen will remain attached to the slide despite this treatment.) After carefully blotting the slide dry, the microbiologist observes the smear under the microscope. Simple stains are used to determine size, shape, and arrangement of cells (FIGURE 4.14). Such characteristics can help a medical laboratory scientist identify microbes.
An objective lens
bends the light rays, which then pass up through one or two ocular lenses, which are the lenses closest to the eyes. Microscopes with a single ocular lens are monocular, and those with two are binocular. Ocular lenses magnify the image created by the objective lens, typically another 10×.
gram positive
cell wall, thick layer of peptidoglycan,
Simple Microscopes
contains a single magnifying lens, is more similar to a magnifying glass than to a modern microscope. They were capable of approximately 300× magnification and achieved excellent clarity, far surpassing other microscopes of his time.
cultured
cultured (grown) for 12-24 hours, though this time can be greatly reduced by the use of rapid identification tools.
Carlous Linnaeus (1707-1778)
current system of taxonomy
Serological Tests
erology is the study of serum, the liquid portion of blood after the clotting factors have been removed and an important site of antibodies. In its most practical application, serology is the study of antigen-antibody reactions in laboratory settings. Antibodies are immune system proteins that bind very specifically to target antigens (Chapter 16). In this section, we briefly consider the use of serological testing to identify microorganisms. Many microorganisms are antigenic; that is, within a host organism they trigger an immune response that results in the production of antibodies. Suppose, for example, that a scientist injects a sample of Borrelia burgdorferi (bō-rē′lē-ă burg-dōr′fer-ē), the bacterium that causes Lyme disease, into a rabbit. These antibodies can be isolated from the rabbit's serum and concentrated into a solution known as an antiserum (plural: antisera). Antisera bind to the antigens that triggered their production. In a procedure called an agglutination test, antiserum is mixed with a sample that potentially contains its target cells. If the antigenic cells are present, antibodies in the antiserum will clump (agglutinate) the antigen (FIGURE 4.23). Other antigens, and therefore other organisms, remain unaffected because antibodies are highly specific for their targets. Her work is memorialized as what are now known as Lancefield groupings, a common way to identify streptococci. (Chapter 17 examines other serological tests, such as enzyme-linked immunosorbent assay, or ELISA, and immunoblotting.)
absorb electrons in electron dense stains
heavy metals
Domain Eukarya
includes all eukaryotic cells, all of which contain eukaryotic rRNA sequences. Domains Bacteria and Archaea include all prokaryotic cells. They contain bacterial and archaeal rRNA sequences, respectively, which differ significantly from one another and from those in eukaryotic cells. In addition to differences in rRNA sequences, cells of the three domains differ in many other characteristics, including the lipids in their cell membranes, transfer RNA (tRNA) molecules, and sensitivity to antibiotics. (Chapters 11 and 12, which cover prokaryotes and eukaryotes respectively, discuss the taxonomy of organisms within the three domains.)
Magnification
is an apparent increase in the size of an object. It is indicated by a number and ×, which is read "times." For example, 16,000× is 16,000 times. Magnification results when a beam of radiation refracts (bends) as it passes through a lens.
The total magnification of a compound microscope
is determined by multiplying the magnification of the objective lens by the magnification of the ocular lens. Thus, total magnification using a 10× ocular lens and a 10× low-power objective lens is 100×. Using the same ocular and a 100× oil immersion objective produces 1000× magnification. Some light microscopes, using higher-magnification oil immersion objective lenses and ocular lenses, can achieve 2000× magnification, but this is the limit of useful magnification for light microscopes because their resolution is restricted by the wavelength of visible light.
Taxonomy
is the science of classifying and naming organisms. Taxonomy consists of classification, which is the assigning of organisms to taxa based on similarities; nomenclature, which is concerned with the rules of naming organisms; and identification, which is the practical science of determining that an isolated individual or population belongs to a particular taxon. In this text, we concentrate on classification and identification
stain where you dont add color
mordant
micrometer
one thousandth of a millimeter
group b/w kingdom and class
phylum
Contrast
refers to differences in intensity between two objects or between an object and its background. Contrast is important in determining resolution. For example, although you can easily distinguish two golf balls lying side by side on a putting green 15 m away, at that distance it is much more difficult to distinguish them if they are lying on a white towel.
reaction with antibodies
serological testing
microscope that is a magnifying glass
simple
gram neg
thin layer of peptidoglycan and an extra outer membrane
Fluorescence microscopes
use invisible ultraviolet light to cause specimens to radiate visible light, a phenomenon called fluorescence. Molecules that absorb energy from invisible radiation (such as ultraviolet light) and then radiate the energy back as a longer, visible wavelength are said to be fluorescent. Fluorescence microscopes use an ultraviolet (UV) light source to fluoresce objects. UV light increases resolution because it has a shorter wavelength than visible light, and contrast is improved because fluorescing structures are visible against a nonfluorescing, black background. Some cells—for example, the pathogen Pseudomonas aeruginosa (soo-dō-mō′nas ā-roo-ji-nō′să)—and some cellular molecules (such as chlorophyll in photosynthetic organisms) are naturally fluorescent. Other cells and cellular structures can be stained with fluorescent dyes. When these dyes are bombarded with ultraviolet light, they emit visible light and show up as bright orange, green, yellow, or other colors (see Figure 3.34b). Some fluorescent dyes are specific for certain cells. For example, the dye fluorescein isothiocyanate attaches to cells of Bacillus anthracis (ba-sil′ŭs an-thrā′sis), the causative agent of anthrax, and appears apple green when viewed in a fluorescence microscope. Another fluorescent dye, auramine O, stains Mycobacterium tuberculosis (mī′kō-bak-tēr′ē-ŭm too-ber-kyū-lō′sis) (FIGURE 4.7) Fluorescence microscopy is also used in a process called immunofluorescence. First, fluorescent dyes are chemically linked to Y-shaped immune system proteins called antibodies (FIGURE 4.8a). Antibodies will bind specifically to complementary-shaped antigens, which are portions of molecules that are present, for example, on the surface of microbial cells. When viewed under UV light, a microbial specimen that has bound dye-tagged antibodies becomes visible (FIGURE 4.8b). In addition to identifying pathogens, including those that cause syphilis, rabies, and Lyme disease, scientists use immunofluorescence to locate and make visible a variety of proteins of interest..
Phase microscopes
use the alignment or misalignment of light waves to achieve the desired contrast between a living specimen and its background. Scientists use phase microscopes to examine living microorganisms or specimens that would be damaged or altered by attaching them to slides or staining them. Basically, phase microscopes treat one set of light rays differently from another set of light rays. When a phase microscope's lenses bring the two sets of rays together, contrast is created. There are two types of phase microscopes: phase-contrast and differential interference contrast microscopes. The simplest phase microscopes, phase-contrast microscopes, produce sharply defined images in which fine structures can be seen in living cells. These microscopes are particularly useful for observing cilia and flagella. Differential interference contrast microscopes (also called Nomarski74 microscopes) significantly increase contrast and give an image a dramatic three-dimensional and shadowed appearance, almost as though light were striking the specimen from one side. This technique can also produce unnatural colors, which enhance contrast.
gram procedure
❶ Flood the smear with the basic dye crystal violet for 1 minute, and then rinse with water. Crystal violet, which is called the primary stain, colors all cells. ❷ Flood the smear with an iodine solution for 1 minute, and then rinse with water. Iodine is a mordant, a substance that binds to a dye and makes it less soluble. After this step, all cells remain purple. ❸ Rinse the smear with a solution of ethanol and acetone for 10-30 seconds, and then rinse with water. This solution, which acts as a decolorizing agent, breaks down the thin cell wall of Gram-negative cells, allowing the stain and mordant to be washed away; these Gram-negative cells are now colorless. Gram-positive cells, with their thicker cell walls, remain purple. ❹ Flood the smear with safranin for 1 minute, and then rinse with water. This red counterstain provides a contrasting color to the primary stain. Although all types of cells may absorb safranin, the resulting pink color is masked by the darker purple dye already in Gram- positive cells. After this step, Gram-negative cells now appear pink, whereas Gram-positive cells remain purple. After the final step, the slide is blotted dry in preparation for light microscopy.
