Chapter 4: Structure and Function of Bacteria and Archaea PDF

Chapter 4 C’est un grand progrè, monsieur. – LOUIS PASTEUR, 1881* n Chapter 1 we compared and contrasted prokaryotes with eukaryotes and affirmed that prokaryotes have simpler shapes and structures than eukaryotic organisms. Nonetheless, prokaryotes have all the necessary structural features for pe...

Chapter 4 C’est un grand progrè, monsieur. – LOUIS PASTEUR, 1881* n Chapter 1 we compared and contrasted prokaryotes with eukaryotes and affirmed that prokaryotes have simpler shapes and structures than eukaryotic organisms. Nonetheless, prokaryotes have all the necessary structural features for performing essential life functions, including metabolism, growth, and reproduction. And more complex prokaryotic organisms have additional structures and life cycles not found in the simpler species. This chapter introduces the various unicellular and multicellular shapes of prokaryotes and the types of cell division found among them. It also discusses the structure, chemical composition, and function of common morphological features found in prokaryotic organisms. However, before discussing the properties of these microorganisms in greater detail, we need to recognize the special instruments, techniques, and procedures used to study them. Microbiology did not become a scientific discipline until appropriate instruments and techniques were developed to enable scientists to observe and study these small organisms (see Chapter 2). Of foremost importance was the development of quality microscopes. Indeed, it is not surprising that microorganisms were discovered by a lens maker, Antony van Leeuwenhoek, rather than by a biologist— an early illustration of the importance of instrumentation in microbiology. In this chapter, then, we focus first on microscopes, the instruments routinely used by microbiologists, then discuss the structure and function of prokaryotic organisms. MICROSCOPY The human eye has some ability to magnify objects. Natural magnification is simply achieved by bringing the object closer to the eye. The closer the object, the larger it appears, because the image on the retina is larger (Figure 4.l). Maximum enlargement depends on how close the object can be brought to the eye and still remain in focus. This near point is typically 250 mm for an adult human, and the distance increases as people age. Objects smaller than about 0.1 mm (the “eye” of a needle is about 1.0 mm wide) cannot be seen distinctly because their image does not occupy a sufficiently large area on the retinal surface. Therefore, the unaided human eye cannot see small organisms such as prokaryotes, which typically have a cell diameter of only 0.001 mm (1.0 µm, or l0–6 m). Microscopes have been developed *“That is important progress, sir.” Remark made by Louis Pasteur to Robert Koch at the International Medical Congress in London, 1881, after Koch demonstrated his pure culture methods. 62 CHAPTER 4 Im1 Ob1 Ob2 Im2 Figure 4.1 How the human eye magnifies Diagram illustrating the magnifying capacity of the human eye. The object (Ob) is shown at two locations, Ob1 and Ob2. The image formed on the retina is smaller when the object is farther away from the eye (Im1) than when it is closer (Im2). to aid the eye by increasing its ability to magnify. We discuss here several different types of microscopes commonly used by microbiologists. Simple Microscope The simplest optical device that can be used to assist the eye in enlarging objects is appropriately called the simple microscope, which in principle is a magnifying glass. Antoni van Leeuwenhoek constructed his own simple microscopes specifically to observe microorganisms (see Figure 2.2). Magnifying glasses are not particularly powerful. They usually magnify objects about fivefold. It is a tribute to Leeuwenhoek that his simple microscopes not only could magnify 200- to 300-fold but did so with great clarity. Indeed, the images Leeuwenhoek produced were comparable to those obtained with light microscopes of similar magnification that are used today. Compound Microscope Another Dutchman, Zacharias Jansen (late sixteenth and early seventeenth century), is generally given credit for the development of the compound light microscope, although the origins of this instrument are somewhat obscure. It is interesting to note that compound microscopes were available at the time Leeuwenhoek made his momentous discoveries. However, the early compound microscopes were inferior to Leeuwenhoek’s simple microscope—Robert Hooke, the English scientist who coined the term “cell,” could not confirm Leeuwenhoek’s reports of microorganisms with the early compound microscopes available to him. The compound light microscope is named for the two lenses that separate the object from the eye. The objective lens is placed next to the object or specimen to be viewed; the eyepiece or ocular lens is located next to the eye. The object to be viewed is normally placed on a glass slide and illuminated with a light source. The viewer uses the microscope to form an image by “focusing” the specimen, moving the objective lens and ocular (eyepiece) lens together relative to the specimen until the image is clear. When the specimen has been properly focused, the objective lens produces a real image (one that can be displayed on a screen) within the ocular diaphragm of the microscope. The viewer looking through the ocular lens sees a virtual image, which cannot be displayed on a screen (Figure 4.2A). The magnification of a compound light microscope is determined by multiplying the magnification of the objective lens by that of the eyepiece lens. Usually the eyepiece magnification is 10×. Most microscopes of this type have several separate objective lenses, mounted on a rotating nosepiece, that typically give magnifications of 10× (low power), 60× (high, dry power), and 100× (oil immersion). The resulting magnifications attainable from this microscope would be 100×, 600×, and 1,000×, respectively. The typical compound microscope (Figure 4.2B) also has a third lens system. Light from a lamp or other source is focused on the specimen by a condenser lens. The condenser lens, which is not directly involved in image formation, is necessary to provide high-intensity light, because the brightness of the object becomes a limiting factor as the specimen is increasingly magnified. The light intensity is adjusted by opening or closing a diaphragm called the iris diaphragm, located in the condenser. It might seem reasonable to assume that one could increase the magnification indefinitely using the compound light microscope simply by constructing increasingly powerful objective and eyepiece lenses. In reality, the light microscope has a useful magnification maximum of only 1,000× to 2,000×. Although magnification beyond that level is attainable, it is referred to as empty magnification because it does not provide any greater detail of the specimen. Improving Image Formation in Light Microscopy The ability of an optical system to produce a detailed image is termed its resolving power. The resolving power, d, is defined as the distance between two closely spaced points in an object that can be separated by the lens in the formation of an image (Figure 4.3). The equation for resolving power is: d= 0.61 λ η sin θ where λ is the wavelength of light, η is refractive index, and sin θ is the angular aperture. The lower the value of d, the greater is the resolving power. Therefore, to attain the best resolving power, or resolution, the following conditions are required: STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA (A) The viewer focuses on the object by adjusting the distance between the eyepiece and objective lenses until a sharp image forms. The eye perceives an enlarged virtual image of the real image. Virtual image 63 Objective lens A real image is formed in the ocular diaphragm of the microscope. Object Retina Eyepiece lens Real image Retinal image Lens of eye Eye (B) Figure 4.2 Compound light microscope (A) Image formation by a compound microscope. (B) A typical compound light microscope with binoculars, a rotating nosepiece Perryobjective Staley Lory Microbiology 2/e with lenses, and a condenser lens system with a built-in Sinauer Associates light source. Special knobs are used for focusing and orienting the Figure 04.02a 12/10/01 specimen in the X–Y directions on the stage. Photo courtesy of Nikon Corporation. • Short-wavelength light (λ is low) • A suspending medium (for the specimen) of high refractive index (η is high) • A high angular aperture, the angle at which light enters the objective (θ, and thus sin θ, is high) The light microscope cannot be operated with wavelengths of less than 400 to 500 nm (violet light), as the eye cannot see shorter wavelengths. For maximum resolution, then, the shortest wavelength that can be used is about 500 nm (0.5 µm). By increasing the refractive index, a measure of the ability of a material to bend light, more light from the specimen enters the objective lens, resulting in greater resolution. The crown glass used in lenses has a high refractive index: air has a refractive index of 1.0 (essen- Eyepieces Binocular head Rotating nosepiece Objective Specimen stage X and Y stage travel controls Condenser Fine and coarse focusing knobs tially identical to that of a vacuum), water 1.33, and crown glass 1.5. To permit a continuous, high refractive index between the condenser and the objective, immersion oils of high refractive index are placed on the specimen and on the condenser lens system. Such homogeneous immersion systems allow the objective to be brought closer to the specimen, further increasing resolution (Figure 4.4). The final factor affecting resolution is Two points within the object Two points appear Two points are the aperture or opening of the objective cannot be distinguished as overlapping disks. visible as separate itself. Because the objective lens has a finite from each other. The points They are only partially entities. They are are unresolved. resolved. fully resolved. aperture, a point on the specimen is not imaged as a point on the image but as a disk (called an “Airy disk”) with alternate dark and light rings. The size of this disk can be decreased by increasing the aperFigure 4.3 Resolving power or resolution ture of the lens, but there is a limit to this. Resolution is the degree to which two separate points in an object can be As a result, two closely spaced points on distinguished. Improved lenses allow increased resolution. 64 CHAPTER 4 1 In nonhomogeneous immersion, air, with a refractive index of 1.0, is between the lens and specimen. 2 In homogeneous immersion, the specimen is immersed in oil and the refractive index between the lens and specimen is 1.5, with a corresponding increase in resolution. Objective lens Objective lens 60° Air η = 1.0 Light source Location of specimen 90° Oil η = 1.5 Figure 4.4 Nonhomogeneous and homogeneous immersion Immersion in oil increases resolving power, not only by increasing refractive index but also by allowing the specimen to be brought closer to the lens, thus increasing the angular aperture, θ. the object are seen as fuzzy disks on the image, and if they are too close may actually overlap and not be separate points (Figure 4.3). The theoretical maximum value for the sine of the angular aperture is 1.0. When these ideal values of the shortest possible wavelength (η = 0.5 µm), homogeneous oil immersion (λ = 1.5), and the angular aperture (sin θ = 1.0) are substituted into the equation for resolving power, then d = 0.61(0.5 µm)/1.5, which is about 0.2 µm. Since typical prokaryotic cells are about 1.0 µm in diameter, the compound light microscope has a satisfactory resolution for observation of these cells. It is not well suited, however, for observing internal cell structures, which are much smaller. Early versions of the compound microscope were plagued by lens aberrations, which are of two types: Correction of these aberrations was made possible by the use of multicompo- • Staining the cells with dyes to produce higher contrast • Using a modified compound microscope, such as the phase contrast microscope or the darkfield microscope Dyes and Stains Microorganisms to be observed in the Light source • Chromatic aberration, in which light of different wavelengths entering the objective from the specimen is focused at different planes in the formation of the image • Spherical aberration, in which light rays from the specimen that enter the periphery of the objective lens are focused at a different place from those that enter the center of the lens nent lens systems (Figure 4.5) in the objective lens. These developments, and the introduction of the condenser, transpired over a period of about two centuries. The compound light microscope was finally perfected in the late nineteenth century by opticians including Ernst Abbe in Germany (1840–1905). Because the refractive index of prokaryotic cells is similar to that of water, prokaryotes are almost invisible when viewed with an ordinary compound light microscope. Two approaches have been taken to overcome this problem: light microscope are often stained with dyes, because dyes increase the contrast between the cells and their environment. Most dyes used to stain microorganisms are aniline dyes, intensely pigmented organic salts derived from coal tar. They are called basic dyes if the chromophore (pigmented portion) of the molecule is positively charged. For example, crystal violet and methylene blue are basic dyes (Figure 4.6). Other basic dyes commonly used to stain microorganisms are basic fuchsin, malachite green, and safranin. Under normal growth conditions, most prokaryotes have an internal pH near neutrality (pH 7.0) and a negatively charged cell surface, so basic dyes are generally the most effective staining agents. Acid dyes such as nigrosin, Congo red, eosin, and acid fuchsin have a negatively charged chromophore and are useful in staining positively charged cell components such as protein. Simple stains of microorganisms are made by spreading a suspension of the organism on a glass slide, allowing it to dry, and then gently heating it to fix it to the slide (a fixative, in this case heat, allows the specimen to adhere—like frying an egg in a pan without oil). Such a preparation is called a smear. The stain is added, and after a brief period of exposure the excess dye is removed by gentle rinsing. The slide is then viewed with the microscope. Differential stains, such as the Gram stain (see Box 4.4), distinguish one microbial group from another, in this instance, grampositive bacteria from gram-negative bacteria. Likewise, only acid-fast bacteria such as those in the genus Mycobacterium retain the Figure 4.5 Objective lens Cross-sectional view of a microscope objective lens showing its multicomponent lens system. Courtesy of Carl Zeiss, Inc. STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA + [(CH3)2NC6H4]2C color of a dye when the stained preparation is rinsed in a solution of ethanol containing hydrochloric acid at a final concentration of 3%. Some staining procedures allow the identification of structures within the cell. Thus, specific stains exist for bacterial endospores, flagella, capsules, and other cell structures. Lipophilic dyes, such as Sudan black, can be used to specifically stain lipid inclusions such as polyβ-hydroxybutyric acid (see below). Cl– N(CH3)2 Crystal violet N (CH3)2N S+ Cl– Methylene blue N(CH3)2 Brightfield and Darkfield Microscopes An ordinary light microscope is called a brightfield microscope, because the entire field of view containing the specimen and the background is illuminated and appears bright. The brightfield microscope can be modified to produce a darkfield microscope, so named because the cells appear bright against a dark background (Box 4.1). One advantage of the darkfield microscope is that it can be used to view living cells. Cell suspensions Figure 4.6 Dyes Chemical structures of crystal violet and methylene blue, two basic (positively charged) dyes, shown here as their chloride salts. Methods & Techniques Box 4.1 The Darkfield Microscope The darkfield effect in microscopy is produced by illuminating only the cells and not the background. For this procedure, wet-mount preparations are made by placing a droplet of cell suspension on a slide and covering it with a coverslip. Unlike smears, the cells in wet-mount preparations remain alive in their normal growth environment while being observed. The darkfield effect is created by using a special condenser that introduces light onto the specimen at such an angle that the only light entering the objective is light that is scattered into the objective lens from the cells (Figure A). As a result, the cells appear bright against a dark background (Figure B). (A) The only light entering the objective lens is light refracted or diffracted by the specimen. As a result, the specimen appears bright on a dark background. 65 Objective The light passes through the specimen but not through the objective lens. Specimen Cover slip Slide Reflected light rays Condenser (B) Diaphragm with ring opening Light source The diaphragm allows only peripheral light to enter the condenser lens. Figure A The principle of darkfield illumination. Figure B Diatoms (Pleurosigma angulatum) viewed by darkfield microscopy. ©James Solliday/Biological Photo Service. 66 CHAPTER 4 are observed in wet mounts, prepared by placing a coverslip over the live suspension. The movement of motile cells, for example, is readily visible in these preparations. Phase Contrast Microscope As mentioned earlier, most microbial cells appear to be colorless, transparent objects when observed by ordinary brightfield microscopy (Figure 4.7A). A slight difference exists, however, between the refractive index of the cell (η is about 1.35) and that of its aqueous environment (η is 1.33). The phase contrast microscope, or phase microscope, amplifies this slight difference in refractive index and converts it to a difference in contrast. The result is that the cells appear very dark against a bright back- (A) Figure 4.8 Fluorescence microscopy Bacteria and diatoms stained with the fluorescent dye acridine orange and illuminated with ultraviolet light appear green and yellow to the eye. ©Paul W. Johnson/Biological Photo Service. ground (Figure 4.7B). As with darkfield microscopy, cells can be observed while alive, in wet mounts. Fluorescence Microscope (B) Figure 4.7 Phase contrast microscopy Photomicrographs of an unstained bacterium, Bacillus megaterium, showing its appearance by (A) brightfield microscopy and (B) phase contrast microscopy. Courtesy of J. T. Staley. Certain dyes used for staining microorganisms are called fluorescent dyes, because when illuminated by short-wavelength light they emit light of a longer wavelength (they fluoresce). One of the most commonly used fluorescent dyes is acridine orange. It specifically stains nucleic acid components of cells. When stained preparations are illuminated with an ultraviolet or halogen light source, the cells fluoresce green to orange in color (Figure 4.8). One special advantage of fluorescence microscopy is that it allows the observation of cells located on an opaque surface such as a soil particle. These would be impossible to see with an ordinary light microscope in which light must be transmitted through the specimen. In fluorescence microscopes, short-wavelength light is provided by either a mercury lamp (ultraviolet) or a halogen lamp (near ultraviolet). The light is passed through the objective lens system onto the specimen to provide incident illumination, that is, illumination from above the specimen (not transmitted through the specimen as in an ordinary light microscope—although some fluorescence microscopes do use transmitted light). The longer-wavelength light emitted by the fluorescent dyes is visible when viewed through the ocular. Special barrier filters prevent any harmful short-wavelength UV light from reaching the eyes. Confocal Scanning Microscope The confocal scanning microscope is especially useful when viewing microorganisms in three-dimensional space. The preparation, which may be a natural commu- STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA 67 nity containing microorganisms, is illuminated with a laser beam, which is focused on one point of the specimen using an objective lens mounted between the condenser lens and the specimen. Mirrors are used to pass (scan) the laser beam across the specimen in the x and y directions. The objective lens used for viewing the specimen magnifies the image, which is free from diffracted light, and the image is reconstructed on a video display screen. Epifluorescence scanning microscopy uses a laser beam to illuminate the specimen, which has been stained with a fluorescent dye. The laser beam is focused at a particular plane of the preparation. The image viewed is that of a cross section of the preparation in which only those cells that are in the plane of illumination appear on the display screen (see Figure 24.2). Transmission Electron Microscope (TEM) The light microscope has a useful magnification of about 1,000× to 2,000×. Although greater magnification can be achieved, as noted above, finer detail will not be visible, in part because of the properties of the illuminating source itself—light (see p. 62). It is not possible to observe objects well if they are smaller than the wavelength of the illuminating source. An analogy may help illustrate this point. Suppose you wish to make an impression of your hand and two materials are available: fine particles of wet clay (analogous to a short wavelength) or gravel (a long wavelength). You can make a much more detailed and accurate impression of your hand with the clay. The transmission electron microscope can produce very short wavelengths by using a beam of electrons as the illuminating source. The wavelength is controlled by the voltage applied to an electron gun, which is the source of electrons. If the accelerating voltage is 60,000 volts, the wavelength of the electron beam is 0.005 nm, or 0.000005 µm. This is 100,000 times shorter than the wavelength of violet light (about 500 nm). As a result, the theoretical resolution of the electron microscope is about 2 Å (Å is an angstrom; 1 Å = 10–10 m), which is twice the diameter of the hydrogen atom. In place of optical lenses, the TEM uses electromagnetic lenses to bend the electron beam for focusing. A vacuum is essential in permitting the flow of electrons through the lens system, so the entire electron microscope must have an enclosed chamber and accompanying vacuum pumps. This makes the TEM a much larger instrument than the ordinary light microscope (Figure 4.9). The real image is formed by electrons bombarding a phosphorescent screen, and the photograph, called an electron micrograph, is taken by a camera mounted below the screen, containing film sensitive to electron radiation. The lenses of the TEM are equivalent to those of the compound light microscope—a condenser lens, an objective lens, and a projector (ocular) lens. The fundamental Figure 4.9 Transmission electron microscope (TEM) The TEM is very large because the entire device is contained in a vacuum and it uses electron magnets for lenses. The illuminating source is an electron gun located at the top of the microscope. The specimen is placed between the electron gun and the phosphorescent screen that is used to view the image. Courtesy of JEOL USA, Inc. differences are that electrons are the illuminating source and magnets replace optical lenses (Figure 4.10). Figure 4.11 compares the appearance of bacterial cells in a light microscope and in a TEM. Note the increased detail in the electron micrograph, even though the magnification is about the same for both preparations. Because the preparation to be observed in the electron microscope is placed in a vacuum chamber, it is not possible to observe living cells with the TEM. In place of a glass slide, cell preparations are placed on a small screen (4.0 mm in diameter, about the size of this O) called a grid. A thin plastic film is placed on the grid to hold the cell preparation. In the simplest preparation, cells are placed on the plastic-coated grid and allowed to dry. The whole cell preparation is then stained with a heavy metal stain such as phosphotungstic acid or uranyl acetate, allowed to dry, and then placed in the electron microscope. The major application of the TEM in biology is to observe internal cell structures. This requires a more elaborate procedure for preparing the specimen for observation, a procedure called thin sectioning. Because the specimen will ultimately be observed in a vacuum, some procedure must be used to preserve the structure of the 68 CHAPTER 4 (A) Light microscope (B) Transmission electron microscope (TEM) Light source Electron gun Condenser lens Magnetic condenser lens Specimen Specimen Objective lens Magnetic objective lens Intermediate image Intermediate image Magnetic projector Eyepiece Projector lens Observation screen or photographic plate Retina of the eye or photographic plate (C) Scanning electron microscope (SEM) Electron gun Magnetic condenser lens Scan coil Magnetic objective lens Specimen Specimen holder Secondary electrons Cathode-ray tube image Figure 4.10 Illumination in light and electron microscopes A comparison of the illuminating paths in the light microscope, the transmission electron microscope, and the scanning electron microscope. Note that all three have illuminating sources, condenser lenses, and objective lenses. Because a virtual image cannot be viewed, a projector lens is needed in the TEM and when cells are photographed in the light microscope. The image of the SEM specimen is viewed in a cathode ray tube. specimen in the absence of water. This is accomplished through a process called dehydration and embedding, in which the cells are taken from their aqueous environment and transferred into a plastic resin. Although more complex, this process is similar to embedding insects in plastic resins, as practiced by some hobbyists. First, cells are harvestThis bacterium, like many prokaryotes, ed from their growth has fimbriae, short appendages made medium by centrifugaof proteinaceous material. In this species the fimbriae are too small to tion. They are gradually be seen by light microscopy. dehydrated by transferring them step by step from the aqueous medium into ethanol solutions of increasing concentration, until they are placed in 100% ethanol. The next step is to (B) transfer the cells through an acetone-ethanol series until they are suspended in 100% acetone. Unlike ethanol and water, acetone is a plastic solvent and is miscible with plastic resins. At this point, the preparation is completely dehydrated. The next step is to embed the cells in a plastic mixture. Again, a series of transfers is made until the cells are in 100% plastic resin. Sufficient time 2 µm is permitted to ensure that the resin 2 µm completely displaces the acetone in the cells. The preparation is “cured” Figure 4.11 Resolution in light and electron microscopy (polymerized to form a solid) by A large unidentified colonial bacterium, obtained from a lake, is viewed under heating at 60°C in an oven. The (A) the electron microscope and (B) the light microscope. Fimbriae are discussed organism is now embedded. later in the chapter (see also Figure 4.54). Courtesy of J. T. Staley and Joanne Tusov. (A) STRUCTURE AND FUNCTION OF BACTERIA AND ARCHAEA W M S 69 the best procedure available to examine colonial forms of microorganisms, such as the fruiting structures of the myxobacteria (Figure 4.13B). An SEM can be equipped with an X-ray analyzer, enabling the researcher to determine the elemental composition of microorganisms or their components. Elements with atomic weights greater than 20 can be assayed in a semiquantitative manner using this instrument (Figure 4.14). The varieties of microscopes and associated procedures described above were instrumental in the developing field of microbiology. In the next section we discuss the various morphological attributes of prokaryotes (A) Figure 4.12 Thin section of a gram-negative bacterium A thin section of Thiothrix nivea showing (from the outside) its sheath (S), cell wall (W), and cell membrane (M). Courtesy of Judith Bland and J. T. Staley. The embedded cells are sliced into thin sections with an ultramicrotome. The ultramicrotome is analogous to a meat slicer, except that it has a diamond knife and cuts extremely thin sections (about 60 nm thick). The thin sections are stained with heavy metals (lead citrate and uranyl acetate) to increase contrast. They are then placed on TEM grids and examined. A typical thin section is shown in Figure 4.12. (B) 100 µm Scanning Electron Microscope (SEM) As in the TEM, electrons are the illuminating source for the scanning electron microscope (SEM), but the electrons are not transmitted through the specimen as in transmission electron microscopy. In SEM the image is formed by incident electrons scanned across the specimen and back-scattered (reflected) from the specimen, as described for the fluorescence microscope. The reflected radiation is then observed with the microscope, in the same manner that we observe objects illuminated by sunlight. Solid metal stubs are used to hold the preparations. Before the organism is viewed, it is dried by critical point drying, carefully controlled drying in which water is removed as vapor so that structural damage to cells is minimized. The specimen is then coated with an electronconducting noble metal such as gold or palladium. The SEM has a larger depth of field than the TEM so there is a greater depth through which the specimen remains in focus. Thus, all parts of even a relatively large specimen, such as a eukaryotic cell, remain in focus when viewed by the SEM (Figure 4.13A). The result is an image that looks three-dimensional. This is Figure 4.13 Scanning electron microscopy (SEM) (A) A radiolarian, a eukaryotic protist with a siliceous (silicacontaining) shell. (B) The fruiting structure of the myxobacterium Stigmatella aurantiaca. The entire structure is about 1 mm in length. A, courtesy of Barbara Reine; B, ©K. Stephens and D. White/Biological Photo Service.