chapter 3 LearnSmart
Electron Microscopes - ***Electron microscopy is in some ways comparable to light microscopy, but it can clearly magnify an object 100,000×. Rather than using glass lenses, visible light, and the eye to observe the specimen, an electron microscope uses electromagnetic lenses, electrons, and a fluorescent screen to produce the magnified image (figure 3.10). That image can be photographed, creating a picture called an electron photomicrograph. The image is black-and-white, but may be artificially colored to make certain components stand out or to add visual interest. electrons^^ ***Some electron micrographs are "color enhanced." Why would this be done? Electrons have a wavelength about 1,000 times shorter than visible light, so the resolving power of electron microscopes is about 1,000-fold more than light microscopes—about 0.3 nanometers (nm) or 0.3 × 10-3 μm (see figure 1.7). Consequently, considerably more detail can be observed with electron microscopy.^^ ***Electron microscopes are complex instruments because the lenses and specimen must typically be in a vacuum to avoid air molecules that would otherwise interfere with the path of electrons. The need for a vacuum means that specimen preparation is complicated and makes it impossible to observe living cells. Techniques are being developed that do not require a vacuum, making it possible to view live cells, but images obtained are not as clear as with conventional methods.^^
***FIGURE 3.10 Principles of Light and Electron Microscopy For the sake of comparison, the light source for the light microscope has been inverted (the light is shown at the top and the ocular lens at the bottom).^^
Transmission Electron Microscopes (TEMs) - ***A transmission electron microscope (TEM) is used to observe fine details of cell structure (figure 3.11). It works by directing a beam of electrons that either pass through the specimen or scatter (change direction), depending on the density of the region. The darker areas of the resulting image correspond to the denser portions of the specimen.^^
***FIGURE 3.11 Transmission Electron Microscopy (TEM) (a) Bacillus licheniformis, prepared by thin-sectioning; (b) Pseudomonas aeruginosa, prepared by freeze-etching.a: ©Lee D. Simon/Science Source; b: ©Dr. Tony Brain/Science Source^^
Fluorescent Dyes and Tags - ***Fluorescence can be used to observe total cells, a subset of cells, or cells with certain proteins on their surface, depending on the procedure (figure 3.20). One example is a fluorescent dye that binds to structures in all cells; it can be used for determining the total number of microbial cells in a sample. Another fluorescent dye binds to all cells but is changed by cellular processes, so it can be used to distinguish between live and dead cells (see figure 3.8). Some fluorescent dyes bind to the mycolic acids in the cell walls of Mycobacterium species, making the dyes useful in a staining procedure similar to the acid-fast stain. mycolic acids^^ ***How can fluorescent dyes and tags be used to identify bacteria? A special technique called immunofluorescence is used to tag specific cell components with a fluorescent dye attached to an antibody (see figure 18.6). By tagging a protein unique to a given microbe, immunofluorescence can be used to detect and identify that organism. Antibodies, and how they are obtained, will be described in chapters 15 and 18. antibody^^
***FIGURE 3.20 Fluorescent Dyes and Tags (a) To detect a Mycobacterium species in a sputum sample, a dye that binds mycolic acids and fluoresces yellow is used in a modification of the acid-fast technique. In this example, acridine orange was used to stain all other organisms. (b) Fluorescent antibodies tag specific molecules—in this case, the antibody binds to a molecule unique to Streptococcus pyogenes.a: Source: CDC; b: ©Evans Roberts^^
Peptidoglycan - The strength of the Gram-positive and Gram-negative bacterial cell walls is due to a layer of peptidoglycan, a material found only in bacteria (figure 3.32). ***The basic structure of peptidoglycan is an alternating series of two major subunits related to glucose: N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). These subunits are covalently joined to one another to form a linear polymer called a glycan chain (glyco means "sugar"), which serves as the backbone of the peptidoglycan molecule.^^
***FIGURE 3.32 Components and Structure of Peptidoglycan Why might it be medically significant that peptidoglycan is found only in bacteria? Peptidoglycan Biosynthesis^^
Fluorescence Microscopes - ***A fluorescence microscope is used to observe cells or other materials that are either naturally fluorescent or stained with fluorescent dyes (figure 3.7). Fluorescent molecules absorb light at one wavelength (usually ultraviolet light) and then emit light of a longer wavelength. The microscope then captures only the light emitted by the fluorescent molecules, allowing fluorescent cells to stand out as bright objects against a dark background. fluorescent dyes and tags^^
***FIGURE 3.7 Fluorescence Microscopy Rod-shaped bacterial cells stained with a fluorescent molecule.©Evans Roberts^^
Endospore Stain - ***Members of certain genera, including Bacillus and Clostridium, form a special type of resistant, dormant cell called an endospore. These structures do not stain with the Gram stain, but they can often be seen as clear, smooth objects within stained cells^^ To make endospores easier to see, an endospore stain is used. This multistep procedure often uses malachite green as a primary stain, with gentle heating to help the dye enter endospores. The smear is then rinsed with water, which removes the dye from everything but the endospores. After that, the smear is counterstained, most often with the red dye safranin. Using this method, the endospores will be green while all other cells will be pink (figure 3.18).
FIGURE 3.18 Endospore Stain Endospores retain the primary stain, malachite green. Counterstaining with safranin colors other cells pink.
Arrangements - Most prokaryotes divide by binary fission, a process in which one cell divides into two. Cells often stick to each other following division, forming characteristic arrangements (figure 3.23). Cells that divide in one plane can form chains of varying length. Cocci that typically occur in pairs are routinely called diplococci. An important clue in the identification of Neisseria gonorrhoeae is its characteristic diplococcus arrangement. Other cells form long chains, a characteristic typical of some members of the genus Streptococcus (strepto means "twisted chain"). Cocci often divide in more than one plane. Those that divide in perpendicular planes form cubical packets. Members of the genus Sarcina form such packets. Cocci that divide in Page 58several planes at random may form clusters. Staphylococcus species, which typically form grape-like clusters, are an example (staphylo means "bunch of grapes").
FIGURE 3.23 Common Cell Arrangements (a) Chains; (b) packets; (c) clusters. (SEM).a (top): ©Dennis Kunkel Microscopy/SPL/Science Source; a (bottom): ©BSIP SA/Alamy Stock Photo; b: Source: Betsy Crane/CDC; c: ©Eye of Science/Science Source
The Role of the Cytoplasmic Membrane in Energy Transformation - The cytoplasmic membrane of prokaryotic cells plays a crucial role in transforming energy—converting the energy of food Page 62or sunlight into ATP, the energy currency of a cell. This is an important difference between prokaryotic and eukaryotic cells; in eukaryotic cells, this process occurs in membrane-bound organelles, which will be discussed later in this chapter. ATP As part of their energy-transforming processes, most prokaryotes have a series of protein complexes—collectively referred to as the electron transport chain (ETC)—embedded in their cytoplasmic membrane. Details of how the ETC works will be explained in chapter 6, but the net result is that protons are moved out of the cell. This creates an electrochemical gradient across the membrane—positively charged protons are concentrated immediately outside the membrane, whereas negatively charged hydroxide ions remain inside the cell (figure 3.28). The charged ions attract each other, so they stay close to the membrane. Inherent in the gradient is a form of energy called proton motive force, which is analogous to the energy stored in a battery. electron transport chain The energy of the proton motive force is harvested by mechanisms that allow protons to move back into the cell. That energy is used to drive certain cellular processes, including ATP synthesis. It is also used to power one of the transport systems discussed next and some forms of motility.
FIGURE 3.28 Proton Motive Force The electron transport chain, a series of protein complexes within the membrane, moves protons out of the cell.
Structure and Arrangement of Flagella - A flagellum has three basic parts: a basal body, a hook, and a filament (figure 3.39). The basal body anchors the structure to the cell wall and cytoplasmic membrane. The hook is a flexible curved segment that extends out from the basal body, connecting it to the filament. The filament, which extends out into the external environment, is made up of identical subunits of a protein called flagellin. These subunits form a chain that twists into a helical structure with a hollow core.
FIGURE 3.39 The Structure of a Flagellum in a Gram-Negative Bacterium The flagellum is composed of three basic parts—a filament, a hook, and a basal body.
Pili - Pili (singular: pilus) are considerably shorter and thinner than flagella, and their function is quite different (figure 3.42). However, one part of their structure has a theme similar to the filament of a flagellum: a string of protein subunits arranged helically to form a long molecule with a hollow core. Many types of pili allow cells to attach to specific surfaces; these pili are also called fimbriae. Strains of E. coli that cause watery diarrhea have pili that allow them to attach to cells that line the small intestine. Without the ability to attach, these cells would simply move Another type of pilus, called a sex pilus, is used to join one bacterium to another for a specific type of DNA transfer. This and other mechanisms of DNA transfer will be described in chapter 8. DNA transfer
FIGURE 3.42 Pili (a) Pili on an Escherichia coli cell. The short pili (fimbriae) allow adherence, whereas the sex pilus is involved in DNA transfer (SEM). (b) Escherichia coli attaching to epithelial cells in the small intestine of a pig (TEM).
Chromosome and Plasmids - The prokaryotic chromosome is typically a single, circular double-stranded DNA molecule that contains all the genetic information required by a cell, as well as information that may be helpful but not required. The chromosome folds and twists to form a tightly packed mass within the cytoplasm, creating a gel-like region called the nucleoid (figure 3.43). ***The compact shape is due partially to nucleoid-associated proteins that bind to DNA, creating a structure that bends and folds. In addition, the DNA is twisted, or supercoiled. To understand what is meant by supercoiling, cut a rubber band and then twist one end several times before rejoining the cut ends. The twisting and coiling you see are analogous to supercoiling.^^
FIGURE 3.43 The Chromosome Escherichia coli undergoing cell division, with the DNA shown in red (TEM).
Endospores - An endospore is a unique type of dormant cell produced by certain bacterial species such as members of the genera Bacillus and Clostridium (figure 3.45). The structures may remain dormant for perhaps 100 years, or even longer, and are extraordinarily resistant to damaging conditions including heat, desiccation, toxic chemicals, and ultraviolet (UV) light. Immersion in boiling water for hours may not kill them. An endospore that survives these treatments can germinate, or exit the dormant stage, to become a typical multiplying cell, called a vegetative cell.
FIGURE 3.45 Endospore Clostridium difficile forming an endospore (TEM).©Dr. Kari Lounatmaa/Science Source
Endocytosis and Exocytosis - Endocytosis is the process by which a eukaryotic cell takes up material from the surrounding environment by forming invaginations (inward folds) in its cytoplasmic membrane (figure 3.49). It is significant because it allows cells to take in material too large to fit through transport proteins. The type of endocytosis common to most animal cells is pinocytosis. In this process, the small invaginations bring in liquid along with any dissolved substances. This action ultimately forms a membrane-bound compartment called an endosome, which then fuses with digestive organelles called lysosomes to form an endolysosome. Within this compartment, the enclosed material will be degraded. lysosome
FIGURE 3.49 Endocytosis and Exocytosis These processes allow the cell to take in or remove substances too large to move through a transport protein.
Nucleus - An important distinguishing feature of a eukaryotic cell is the nucleus, which contains the genetic information. The boundary of this structure is the nuclear envelope, composed of two phospholipid bilayer membranes: the inner and outer membranes (figure 3.52). Complex protein structures span the envelope, forming nuclear pores. These allow large molecules such as ribosomal subunits and proteins to be transported into and out of the nucleus. The nucleolus is a region where ribosomal RNAs are synthesized.
FIGURE 3.52 Nucleus Organelle that contains the cell's genetic information (DNA). (a) Diagrammatic representation. (b) Electron micrograph of a pig kidney cell by freeze-fracture technique.©Biophoto Associates/Science Source
Osmosis - Osmosis is the diffusion of water across a selectively permeable membrane. It occurs when the concentrations of solute (dissolved molecules and ions) on two sides of a membrane are unequal. Typical of diffusion, water moves down its concentration gradient from high water concentration (low solute concentration) to low water concentration (high solute concentration). solute When describing osmosis, three terms are used to refer to the solutions on opposing sides of a membrane: hypotonic (hypo means "less"; tonic refers to solute), hypertonic (hyper means "more"), and isotonic (iso means "the same"). Water flows from the hypotonic solution to the hypertonic one (figure 3.27). No net water movement occurs between isotonic solutions.
Osmosis has important biological consequences. The cytoplasm of a cell is a concentrated solution of inorganic salts, sugars, amino acids, and various other molecules. However, the environments in which bacteria and archaea normally grow are typically very dilute (hypotonic). Water moves toward the high solute concentration, so it flows from the surrounding medium into the cell (see figure 3.27a). This inflow of water exerts tremendous osmotic pressure on the cytoplasmic membrane, much more than it typically can resist. However, the strong cell wall surrounding the membrane generally withstands such high pressure. The cytoplasmic membrane is forced against the wall but cannot balloon further. Damage to the cell wall weakens the structure, and consequently, cells may lyse (burst).
