2 A Brief Journey to the Microbial World Green sulfur bacteria are I Seeing the Very Small 25 phototrophic microorganisms 2.1 Some Principles of Light Microscopy 25 that form their own phyloge- 2.2 Improving Contrast in Light Microscopy 26 netic lineage and were some 2.3 Imaging Cells in Three Dimensions 29 of the first phototrophs to 2.4 Electron Microscopy 30 evolve on Earth. II Cell Structure and Evolutionary History 31 2.5 Elements of Microbial Structure 31 2.6 Arrangement of DNA in Microbial Cells 33 2.7 The Evolutionary Tree of Life 34 III Microbial Diversity 36 2.8 Metabolic Diversity 36 2.9 Bacteria 38 2.10 Archaea 41 2.11 Phylogenetic Analyses of Natural Microbial Communities 43 2.12 Microbial Eukarya 43 CHAPTER 2 • A Brief Journey to the Microbial World 25 for which resolution is considerably greater than that of the light I Seeing the Very Small microscope. UNIT 1 istorically, the science of microbiology blossomed as the The Compound Light Microscope ability to see microorganisms improved; thus, microbiology H The light microscope uses visible light to illuminate cell struc- and microscopy advanced hand-in-hand. The microscope is the tures. Several types of light microscopes are used in microbiol- microbiologist’s most basic tool, and every student of microbiol- ogy: bright-field, phase-contrast, differential interference contrast, ogy needs some background on how microscopes work and how dark-field, and fluorescence. microscopy is done. We therefore begin our brief journey to the With the bright-field microscope, specimens are visualized microbial world by considering different types of microscopes because of the slight differences in contrast that exist between and the applications of microscopy to imaging microorganisms. them and their surrounding medium. Contrast differences arise because cells absorb or scatter light to varying degrees. The com- 2.1 Some Principles of Light Microscopy pound bright-field microscope is commonly used in laboratory Visualization of microorganisms requires a microscope, either a courses in biology and microbiology; the microscopes are called light microscope or an electron microscope. In general, light compound because they contain two lenses, objective and ocular, microscopes are used to examine cells at relatively low magnifi- that function in combination to form the image. The light source cations, and electron microscopes are used to look at cells and is focused on the specimen by the condenser (Figure 2.1). Bacte- cell structures at very high magnification. rial cells are typically difficult to see well with the bright-field All microscopes employ lenses that magnify (enlarge) the microscope because the cells themselves lack significant contrast image. Magnification, however, is not the limiting factor in our with their surrounding medium. Pigmented microorganisms are ability to see small objects. It is instead resolution—the ability to an exception because the color of the organism itself adds con- distinguish two adjacent objects as distinct and separate—that trast, thus improving visualization (Figure 2.2). For cells lacking governs our ability to see the very small. Although magnification pigments there are ways to boost contrast, and we consider these can be increased virtually without limit, resolution cannot, methods in the next section. because resolution is a function of the physical properties of light. We begin with the light microscope, for which the limits of Magnification and Resolution resolution are about 0.2 ␮m (␮m is the abbreviation for microm- The total magnification of a compound light microscope is the 2 eter, 10 6 m). We then proceed to the electron microscope, product of the magnification of its objective and ocular lenses Magnification Light path ,100؋, 400؋ ؋ Visualized 1000 image Eye Ocular lenses Specimen on glass slide 10؋ Ocular lens Intermediate image (inverted from that Objective lens of the specimen) Stage Condenser 10؋, 40؋, or Objective lens (100؋ (oil Focusing knobs Specimen None Light source Condenser lens Light source Carl Zeiss, Inc. (a) (b) Figure 2.1 Microscopy. (a) A compound light microscope. (b) Path of light through a compound light microscope. Besides 10* , ocular lenses are available in 15–30* magnifications. 26 UNIT 1 • Principles of Microbiology angles (that would otherwise be lost to the objective lens) to be collected and viewed. MiniQuiz • Define and compare the terms magnification and resolution. • What is the useful upper limit of magnification for a bright-field microscope? Why is this so? T. D. Brock (a) 2.2 Improving Contrast in Light Microscopy In microscopy, improving contrast typically improves the final image. Staining is an easy way to improve contrast, but there are many other approaches. Staining: Increasing Contrast for Bright-Field Microscopy Dyes can be used to stain cells and increase their contrast so that Norbert Pfennig they can be more easily seen in the bright-field microscope. Dyes (b) are organic compounds, and each class of dye has an affinity for Figure 2.2 Bright-field photomicrographs of pigmented microor- specific cellular materials. Many dyes used in microbiology are ganisms. (a) A green alga (eukaryote). The green structures are chloro- positively charged, and for this reason they are called basic dyes. plasts. (b) Purple phototrophic bacteria (prokaryote). The algal cell is Examples of basic dyes include methylene blue, crystal violet, and ␮ ␮ about 15 m wide, and the bacterial cells are about 5 m wide. We safranin. Basic dyes bind strongly to negatively charged cell com- contrast prokaryotic and eukaryotic cells in Section 2.5. ponents, such as nucleic acids and acidic polysaccharides. Because cell surfaces tend to be negatively charged, these dyes * (Figure 2.1b). Magnifications of about 2000 are the upper limit also combine with high affinity to the surfaces of cells, and hence for light microscopes. At magnifications above this, resolution are very useful general-purpose stains. does not improve. Resolution is a function of the wavelength of To perform a simple stain one begins with dried preparations light used and a characteristic of the objective lens known as its of cells (Figure 2.3). A clean glass slide containing a dried sus- numerical aperture, a measure of light-gathering ability. There is pension of cells is flooded for a minute or two with a dilute a correlation between the magnification of a lens and its numeri- solution of a basic dye, rinsed several times in water, and blot- cal aperture: Lenses with higher magnification typically have ted dry. Because their cells are so small, it is common to higher numerical apertures (the numerical aperture of a lens is observe dried, stained preparations of bacteria with a high- stamped on the lens alongside the magnification). The diameter power (oil-immersion) lens. of the smallest object resolvable by any lens is equal to 0.5␭/numerical aperture, where ␭ is the wavelength of light used. Differential Stains: The Gram Stain Based on this formula, resolution is highest when blue light is Stains that render different kinds of cells different colors are used to illuminate a specimen (because blue light is of a shorter called differential stains. An important differential-staining pro- wavelength than white or red light) and the objective has a very cedure used in microbiology is the Gram stain (Figure 2.4). On high numerical aperture. For this reason, many light microscopes the basis of their reaction to the Gram stain, bacteria can be come fitted with a blue filter over the condenser lens to improve divided into two major groups: gram-positive and gram-negative. resolution. After Gram staining, gram-positive bacteria appear purple-violet As mentioned, the highest resolution possible in a compound and gram-negative bacteria appear pink (Figure 2.4b). The color ␮ light microscope is about 0.2 m. What this means is that two difference in the Gram stain arises because of differences in the ␮ objects that are closer together than 0.2 m cannot be resolved cell wall structure of gram-positive and gram-negative cells, a as distinct and separate. Microscopes used in microbiology have topic we will consider in Chapter 3. After staining with a basic 9 * ocular lenses that magnify 10 20 and objective lenses of dye, typically crystal violet, treatment with ethanol decolorizes 9 * * ␮ 10 100 (Figure 2.1b). At 1000 , objects 0.2 m in diameter gram-negative but not gram-positive cells. Following counter- * can just be resolved. With the 100 objective, and with certain staining with a different-colored stain, typically safranin, the two other objectives of very high numerical aperture, an optical- cell types can be distinguished microscopically by their different grade oil is placed between the specimen and the objective. colors (Figure 2.4b). Lenses on which oil is used are called oil-immersion lenses. The Gram stain is one of the most useful staining procedures Immersion oil increases the light-gathering ability of a lens by in microbiology. Typically, one begins the characterization of a allowing some of the light rays emerging from the specimen at new bacterium by determining whether it is gram-positive or CHAPTER 2 • A Brief Journey to the Microbial World 27 I. Preparing a smear Step 1 Flood the heat-fixed smear with crystal violet for 1 min UNIT 1 Result: All cells purple Spread culture in thin Dry in air film over slide Step 2 Add iodine solution II. Heat fixing and staining for 1 min Result: All cells remain purple Step 3 Decolorize with Pass slide through Flood slide with stain; alcohol briefly flame to heat fix rinse and dry — about 20 sec Result: Gram-positive III. Microscopy cells are purple; gram-negative cells are colorless –100؋ Step 4 G Slide Counterstain with Oil safranin for 1–2 min Result: Place drop of oil on slide; Gram-positive examine with 100؋ objective (G+) cells are purple; + lens G gram-negative (G–) cells are pink to red Figure 2.3 Staining cells for microscopic observation.