684 MICROBIOLOGY INTRODUCTION Microbiology is the study of organisms which are small enough to require the aid of a microscope to be seen. In a few cases, organisms are included in this group which can be seen by the unaided eye because these organisms are clearly related to the smaller ones. Microorganisms include viruses, bacteria including rickettsia, mycoplasma, fungi (yeast and molds), most algae, protozoa and, if one inter- prets “micro” broadly, certain tiny multicellular plants and animals. The study of cells and tissues from higher plants and animals ( tissue culture ) uses techniques common to the microbiologist and is frequently considered part of modern microbiology. Cells in general vary greatly in size but have many simi- larities in internal organization. Among the most primitive type of cells, it is impossible to clearly distinguish whether they are distinctly “plants” or “animals” since they may have some of the properties of each type. Viruses, on the other hand, are not cells at all. Instead of arguing endlessly about whether a micro- organisms is more plant-like or more animal-like and worrying how to assign viruses, many scientists have divided organisms in general into those which have (1) only animal characteristics, (2) only plant characteristics and (3) the Protista (Table 1), which have some characteristics of both plants and animals. Some protists, viruses, may have characteristics not shared by either plants or animals, that is, crystallizability and ability to reproduce only by infecting some cell and using the cell’s manufacturing machinery. PHYSICAL CHARACTERISTICS OF MICROORGANISMS Protists vary greatly in size, shape and internal architecture. Protists are subdivided into prokaryotes, and eukaryotes. Prokaryotes do not have their genetic material (chromo- somes) separated from the rest of the cell by a membrane whereas eukaryotes have a true nucleus ( eu —true, karyo — nucleus) separated from the rest of the cell by a nuclear membrane. Viruses (virions) are usually included among the prokaryotes. There are 9 types of prokaryotes. Prokaryotes 1) Viruses are the smallest protists. They range in size from about 30–300 nm. The smallest viruses can only be visualized with an electron micro- scope while the largest can be seen with a light microscope. Viruses are composed of two general molecular types (1) only one nucleic acid, either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and (2) a group of proteins also called pro- tein subunits or capsomeres, which surround the TABLE 1 Characteristics of the Protista Virion (virus) 30–300 nm icosahedron, hollow cylinder icosahedral head ϩ tail RNA, DNA requires participation of host machinery Mycoplasma 100–300 nm pleomorphic prokaryotes DNA fission True bacteria 250–3000 nm spherical, rod, spiral rods, prokaryotes DNA fission Higher bacteria 500–5000 nm spherical, rod, spiral rods, filamentous, prokaryotes DNA fission, budding Prokaryotic algae 500–5000 nm spherical, rods in chains, spiral rods in chains DNA fission, internal septation, gonidia Eukaryotic algae 500 nm to macroscopic unicellular or multicellular, filamentous, leafy DNA in nucleus, chloroplasts, mitochondria asexual or sexual simple fission to complex life cycles Protozoa 500–500,000 nm unicellular or colonial various forms DNA in nucleus, mitochondria asexual or sexual simple fission to complex life cycles C013_004_r03.indd 684C013_004_r03.indd 684 11/18/2005 10:41:56 AM11/18/2005 10:41:56 AM © 2006 by Taylor & Francis Group, LLC MICROBIOLOGY 685 nucleic acid and form a protective coat or capsid. The smallest viruses appear spherical but magnifi- cation in the order of 150,000–700,000 ϫ reveals that they are icosahedrons (20 triangular faces and 12 corners) for example, wart virus. Other viruses, for example, tobacco mosaic virus (TMV), the first virus crystallized in 1935 by Wendell Stanley, is grossly rodlike. Tobacco mosaic virus is composed of a central, spirally-attached RNA to which capso- meres are attached to form the outside of a cylinder. The center of the RNA spiral of TMV is hollow. Structurally, the most complicated viruses are some which attack bacteria and blue-green algae. These complicated viruses are composed of an icosahedral head, containing DNA, a protenaceous tail and sometimes accessory tail structures which are important for the attach- ment of the virus to its host cell. 2) Mycoplasma are prokaryotes which overlap viruses in size. They range from 100–300 nm in size. They are highly pleomorphic: they do not have one typi- cal shape but rather can appear coccoid, filamentous, or highly branched. Unlike most other prokaryotes, they do not have cell walls external to their cell membranes. Their cell membranes usually contain sterols, which are thought to lend strength to these cell-limiting membranes (see also Table 2). 3) The true bacteria or Eubacteriales are prokaryotes which are built on three general geometric forms: spheres or cocci, rods, and spirals (including spi- ral helices). All true bacteria have rigid cell walls. They are either permanently immotile or move by means of one to many flagella. They may be aerobes or anaerobes. Some of the anaerobes are photosynthetic. Their sizes and shapes are usually constant except among the rods, in which rapidly- multiplying cells may be somewhat smaller than usual. When the cells divide, they often remain attached to each other and form characteristic, multicellular clusters. The shape of the cluster is determined by the number of division planes. When cocci divide in only one plane, they form chains which may be as much as 20 cells long. Diplococcus pneumoniae forms chains only two cells long while Streptococcus is an example of the long-chain forming type. On the other hand, cocci which divide along two planes, at right angles to each other, form sheets of cells, and cocci which divide in three planes form cube-shaped packets. If there is no regu- lar pattern of the orientation of successive division planes, a randomly-shaped cluster is formed. Staphylococcus is an example of a coccus which forms random clusters. A typical coccus is in the size range of 0.15–1.5 m in diameter. Rods always divide in only one plane. They may appear as single cells or groups of only two when they separate rap- idly. The common intestinal bacterium Escherichia coli (size 0.5 ϫ 2.0 m) is an example of this type. Frequently rods form long chains or streptobacilli. Bacillus megaterium (size 1.35 ϫ 3.0 m), the organism responsible for the “bloody bread” of ancient times, is an example of a chain forming rod. Some basically rodshaped bacteria are either curved or helical rods. Their sizes range from almost as small as the smallest straight rod shaped form to close to twice the length of the largest straight rod. True bacteria always divide by binary fi ssion after their single circular chromosome replicates in a semiconservative fashion. Some true bacteria have complicated life cycles which includes spore-formation. Spore-formers are all rods but belong to diverse genera. They are ecologically related in that they are found primarily in soil. Since that natural TABLE 2 Some characteristics of prokaryotic and eukaryotic cells Structure Prokaryote Eukaryote Weight Chromosome 0.001–1.0 pg one, single circular DNA double helix not complexed with histones 10–10,000 pg several linear DNA double helices (several chromosomes usually complex with histones) Nucleus No true nucleus. Chromosomes not separated from cytoplasm by a membrane True nucleus. Chromosomes enclosed in a nuclear membrane Reproduction Usually asexual, conjugation takes place rarely, no mitosis or meiosis Asexually by mitosis or sexually after meiosis Membranes Only cell limiting membrane present. Usually lacks sterols (except for mycoplasma) Cell limiting membrane plus membrane limited organelles present. Composition includes sterols Organelles None Many including mitochondria, chloroplasts (plants only), Golgi apparatus, lysosomes, etc. Apparatus for protein synthesis Ribosomes, 70 S type usually not associated with membranes Ribosomes, 80 S type in cytoplasm associated with endoplasmic reticulum. 70 S type in mitochondria and chloroplasts not associated with membranes C013_004_r03.indd 685C013_004_r03.indd 685 11/18/2005 10:41:57 AM11/18/2005 10:41:57 AM © 2006 by Taylor & Francis Group, LLC 686 MICROBIOLOGY environment is rather variable in that it can range from very hot to very cold and from very wet to very dry, the heat- and cold-resistant dormant spores offer the bacteria a means of surviving adverse environmental conditions for months or even years. Many important pathogens and commercially important organisms are spore formers, e.g. Bacillus anthra- cis which causes anthrax, Clostridium tetani which causes tetanus and Clostridium acetobutylicum which can ferment corn or potato mash into acetone, ethanol and butanol. Corynebacteria are also rod-shaped bacteria but they are pleomorphic and often look club-shaped. One of the best known members of the genus is C. diphtheriae, which causes diphtheria. Other members of the genus are commer- cially important as producers of the vitamin folic acid. Arthrobacter species are found widely in soil and water. Depending upon the nutrients supplied, they can appear as cocci or pleomorphic rods. 4) Spirochetes are NOT true bacteria though they resemble Eubacteriales in that they are spirally curved, unicellular and multiply by binary fission. They differ from eubacteria by the absence of a rigid cell wall which allows them to be quite flexible. They are all motile by means of axial filaments attached at the cell poles and spirally wrapped around the cell. The smallest spiro- chete is 0.1 ϫ 5 nm while the largest is 3.0 ϫ 120 m. One of the most important spirochetes is Treponema pallidum, which causes syphilis. 5) Actinomycetes are NOT true bacteria. Rather, they are naturally-branching, filamentous, spore- forming organisms which have a mycelial struc- ture similar to that of filamentous fungi. Many actinomycetes, especially those from the genus Streptomyces, are commercially important sources of antibiotics. 6) Mycobacteria are rods which can form a rudimen- tary mycelium which resembles actinomycetes, but they differ in that their cell walls are particu- larly rich in waxes, which allows them to retain stain imparted by such dyes as basic fuchsin even after treatment with dilute acid. This property, called acid fastness, is characteristic of myco- bacteria. Many species occur in soil but the best known are the human pathogens M. tuberculosis and M. leprae , which cause tuberculosis and lep- rosy respectively. 7) Budding bacteria are NOT true bacteria. They possess a complicated life cycle which includes multiplication by budding rather than binary fis- sion. Their type of budding can be readily dis- tinguished from that of true fungi such as yeast. The budding bacterium Hyphomicrobium exists for part of its life cycle as a flagellated, slightly curved rod. For multiplication, the flagellum is lost, the chromosome replicates, and one chro- mosome migrates to one end of the cell where a hypha-like lengthening takes place. When the hyphal extension ceases, it becomes a rounded bud which contains the chromosome. The bud grows in length and diameter until it reaches the size of the mother cell, grows a new flagellum, and separates from the hyphal extension. 8) Gliding bacteria are diverse group of prokaryotes which are motile without having flagella. They have very close affinities to blue-green algae although gliding bacteria are not themselves pho- tosynthetic. They may be unicellular rods, helical or spiral-helical, or filamentous. 9) Blue-green algae or Cyanophyta are the only prokaryotic algae. They are a diverse group that include both unicellular and filamentous forms. They have cell walls that resemble Gram-negative bacteria but their photosynthesis more closely resembles that of eukaryotes in that it is aerobic rather than anaerobic (as in photosynthetic bacte- ria). They are among the most complex prokary- otes. Even though they lack defined organelles, e.g. they lack chloroplasts, many species have complex membranous or vesicular substructures which are continuous with the cell membrane. Some fi lamentous forms contain specialized structures such as gas vacuoles, heterocysts, or resting spores ( akinetes ). Gas vacuoles are frequently found in planktonic species, i.e. those which live in open water. These vacuoles are thought to provide the algae with a means of fl oating and sinking to the depth most appropriate to support photosynthesis. Heterocysts arise from vegetative cells and are thought to function in N 2 fi xation. Some blue-green algae show gliding motility. None are fl agellated. They are very widely distrib- uted either in terrestrial or aquatic habitats from the arctic to the tropics. Some forms are found in hot springs. Other Cyanophyta are symbionts in a variety of plants and animals. For example a species of Anabaena fi xes N 2 for its host the water fern, Azolla. Many blue-green algae form especially luxuriant mats of growth called blooms which clog water- ways and limit their use for navigation, etc. FIGURE 1 Animal viruses are often grown in embry- onated eggs. The position of the hypodermic needles indicates three common inoculation places. C013_004_r03.indd 686C013_004_r03.indd 686 11/18/2005 10:41:57 AM11/18/2005 10:41:57 AM © 2006 by Taylor & Francis Group, LLC MICROBIOLOGY 687 The prokaryotic blue-green algae, Cyanophyta, are usually divided into 5 groups: Chooccocales are unicellular. They sometimes occur in irregular packets or colonies. Cells multiply by binary fi ssion. Chamaesiphonales are unicellular, fi lamentous, or colo- nial epiphytes or lithophytes. Cells show distinct polarity from apex to base. The base usually has a holdfast which permits attachment to the substrate. Cells multiply by inter- nal septation or by formation of spherical cells ( gonidia ) at the ends of fi laments. Pleurocapsales are fi lamentous with differentiation into aerial and nonaerial elements. Cells multiply by crosswall formation or by internal septation. Nostocales are fi lamentous without differentiation into aerial and nonaerial elements. They are unbranched or falsely branched and frequently have pale, empty-looking cells called heterocysts and resting spores ( akinetes ). Reproduction is by liberation of a short fi lament only a few cells long, called a hormogonium, which then elongates. a) Nostacaceae are unbranched and produce hetero- cysts. They frequently produce akinetes. b) Rivulariaceae are unbranched or falsely branched. Filaments taper from base to tip. Heterocysts are usually present at the base. There is some akinete formation. c) Scytonemataceae are false branched. Heterocysts are frequently found at branch points. d) Stigonematalis are filamentous with aerial and nonaerial differentiation. Hormogonia and hetero- cysts are present. They often show true branching and have pit connections between cells. Akinetes are rare. Eukaryotes Eukaryotic microorganisms include all the algae (except the Cyanophyta ), all the protozoa, and most fungi. All are microscopic in size. The eukaryotic algae are separated into nine divi- sions based upon their pigment and carbohydrate reserves (Table 3). They are all photosynthetic and, like higher plants, evolve oxygen during photosynthesis. Many algae are obli- gate phototrophs. That is, they are completely dependent upon photosynthesis: they can not use exogenously supplied organic compounds for growth in either the dark or light. Some algae are facultative phototrophs; they are able to uti- lize organic compounds for growth in the dark but fi x carbon dioxide photosynthetically in the light. Occasionally algae, especially unicellular forms, per- manently lose their chloroplasts by exposure to any one of several adverse conditions, e.g. heat or chemicals. If the organism had been a facultative phototroph, before the loss of the chloroplasts, it has the enzymatic machinery necessary to survive except that now, in its chloroplastless state, it is indis- tinguishable from certain other unicellular organisms more commonly called protozoa. The ease with which an organ- ism at this primitive level of evolution may be interchanged between groups containing a preponderance of plant-like or animal-like attributes underlines the need for the term protist rather than plant or animal to describe them. Indeed both botanists and zoologists claim the protists. Some algae e.g. Euglena spp., normally only form chloroplasts when they grow in the light while others e.g. Chlorella spp. form chlo- roplasts regardless of the presence of absence of light. There is great diversity in size, shape, presence or absence of life cycles, type of multiplication, motility, cell wall chemistry, and chloroplast structure. Although these parameters are of great assistance in defi ning affi nities among algae, there are still groups whose proper place is debated. Many algae are important as sources of food, chemi- cal intermediates of industrial and medical importance, and research tools. Others are nuisances which clog waterways or poison other aquatic life with their potent toxins. Eukaryotic Algal Groups The eight groups are: 1) Chlorophyta (green algae) are either marine or fresh- water forms. This large and diverse group includes forms which are either unicellular, colonial, filamen- tous, tetrasporal (cells separated but held together in groups of four in a mucilaginous material), coe- nobial (cells more or less attached to each other in an aggregate), or siphonaceous (simple, nonseptate filaments). They frequently have life cycles which RELATIVE SIZE OF BACTERIA Clostridium 1x3.10m Salmonella 0.6x2.3m Hemophilus 0.3x0.6–1.5m Pseudomonas 0.5x1.3m Fusibacterium 0.75–1.5x8.80m Neisseria 0.6x0.8m Streptococcus 0.5–0.75 m Staphylococcus 0.8–1m Erythocyte 7m diameter FIGURE 2 Relative sizes of bacteria. C013_004_r03.indd 687C013_004_r03.indd 687 11/18/2005 10:41:57 AM11/18/2005 10:41:57 AM © 2006 by Taylor & Francis Group, LLC 688 MICROBIOLOGY include motile, flagellated stages. Both asexual and sexual reproduction occurs. 2) Euglenophyta differ from the other algae by pos- sessing a rather flexible cell wall which allows con- siderable plasticity of form. They are either fresh water or marine forms. They all have two flagella but in some genera the second flagellum is often rudimentary. Many forms are phagotrophic (can ingest particles). Chloroplastless forms are fairly common. Multiplication is only by asexual means. 3) Xanthophyta are mostly freshwater forms. They may be unicellular, colonial, filamentous or siphonaceous. Some forms have life cycles which include both asexual and sexual reproduction. Motile anteriorly flagellated cells are found. 4) Chrysophyta are mainly freshwater forms but important marine forms are known. Most genera are unicellular but there are some colonial forms. Cell walls are often composed of siliceous or cal- careous plates. Some form siliceous cysts. They are mainly found in fresh water but some impor- tant marine forms exist. Reproduction is asexual. 5) Phaeophyta (diatoms) are unicellular or colonial forms with distinctly patterned siliceous cell walls. Both asexual and sexual multiplication is found. Freshwater, marine, soil and aerial forms exist. 6) Pyrrophyta are unicellular flagellates with cel- lulose cell walls which are sometimes formed in plates. Reproduction is asexual. Sexual reproduc- tion is rare. 7) Cryptophyta are unicellular, usually flagellated forms which produce asexually. 8) Rhodophyta (red algae) are unicellular, filamentous or leafy forms with complex sexual cycles. Most are marine but there are a few freshwater forms. Fungi The “true” fungi or Eumycota are eukaryotes which are related to both protozoa and algae. They are divided between Reserve material (cont.) b-1,3 glucans Sugars Sugars alcohols Mannitol Division Laminarin Paramylon Chrysolamainarin Floridoside Sucrose Lipid Chlorophyta (green algae) ϩ Euglenophyta Xanthophyta ϩ ϩ Chrysophyta Phaeophyta (brown algae) ϩ o ϩϩ Bacillariophyta (diatoms) ϩϩ Pyrrophyta ϩ Cryptophyta Rhodophyta (red algae) ϩ TABLE 3 Divisions and characteristics of the eukaryotic algae Pigments Reserve material Chlorophyll Biliproteins Starches (a-1,4-glucans) a b c d e Phyco-cyanin Phyco-erythrin True starch Floridian starch Chlorophyta (green algae) ϩϩϪϪϪ ϩ Euglenophyta ϩϩϪϪϪ Ϫ Ϫ Xanthophyta ϩϪϪϪϩ Ϫ Ϫ Chrysophyta ϩϪ Ϫ Ϫ Phaeophyta (brown algae) ϩϪϩϪϪ Ϫ Ϫ Bacillariophyta (diatoms) ϩϪϩϪϪ Ϫ Ϫ Pyrrophyta ϩϪϩϪϪ Ϫ Ϫ ϩ Cryptophyta ϩϪϩϪϪ ϩ ϩ ϩ Rhodaphyta (red algae) ϩϪϪ ? Ϫϩ ϩ ϩ C013_004_r03.indd 688C013_004_r03.indd 688 11/18/2005 10:41:58 AM11/18/2005 10:41:58 AM © 2006 by Taylor & Francis Group, LLC MICROBIOLOGY 689 microscopic and macroscopic and macroscopic groups. In general, they have rigid cell walls, lack chlorophyll, and are usually immotile. Most fungi reproduce asexually or sexu- ally by means of spores though important budding groups such as yeasts are well known. Since fungi are classifi ed by the pattern of their sexual structures, fungi whose sexual stages are unknown are placed into a group called Fungi Imperfecti and assigned genera on the basis of their asexual structures. They are further subdivided into the so-called lower and higher fungi. The lower fungi, Phycomycetes, are also called water molds but not all are aquatic (e.g. black bread molds). Some species multiply by means of fl agellated gametes or fl agellated spores i.e. more like certain green algae than other fungi; Most, but not all, Phycomycetes have COCCI BACILLI VIBRIOS SPIRILLA SPIROCHAETES ACTINOMYCETALES (A) MORPHOLOGICAL CHARACTERIZATION OF BACTERIA (B) (C) FIGURE 3 A. General morphological characteristics of bacteria; B. Variety of morphological types among the cocci; C. Variety of morphological types among the bacilli (rods). C013_004_r03.indd 689C013_004_r03.indd 689 11/18/2005 10:41:58 AM11/18/2005 10:41:58 AM © 2006 by Taylor & Francis Group, LLC 690 MICROBIOLOGY ELEVATION EDGE Flat Raised Low Convex High Convex Entire Umbonate Convex with papillate surface Erose Crenated Undulate Lobate Rhizoid FIGURE 5 Diagrammatic representa- tion of types of bacterial colonies. These shapes are specific for individual types and are therefore quite useful as a step in the process of identification of unknown organisms. SPORE FORMS FIGURE 4 Diagrammatic represen- tation of spores (clear areas) inside rod-shaped bacteria. Note (bottom row) that free spores may be ball or egg-shaped. hyphae, microscopic cytoplasm-fi lled tube-like branches (lacking crosswalls), which together make a felty mat called a mycelium. Individual hyphae are microscopic but the mycelium, equivalent to a bacterial colony, is macroscopic. Growth takes place by extension of the hyphae. Specialized spore-containing bodies called sporangia can form at the ends of some hyphae. Sexual reproduction requires fusion of hyphae from two different mycelia to form a specialized zygospore. It is more common now to discard the term Phycomyetes and instead subdivide the group into 4 classes in which affi n- ities are much clearer. However, at present, the literature is divided in its use of the older and newer terminology. As with bacteria, chemical analyses of structures and metabolic pathways followed are important in defi ning the classes. These four classes are: 1) Chytridiomycetes lack true mycelia. They are aquatic, have posteriorly uniflagellated zoospores and cell walls composed of chitin. 2) Hyphochytridiomycetes have true mycelia. They are aquatic, have anteriorly uniflagellated zoospores and cell walls composed of chitin. 3) Oomycetes have true well developed mycelia and cell walls composed of cellulose. a) Saprolegniales are generally aquatic and have asexual spores on specialized mycelear structures. Only male gametes are motile. b) Peronosporales are generally terrestrial. Sporan- gia either produce asexual zoospores or may germinate directly to form hyphae. Both gametes are nonmotile. 4) Zygomycetes are terrestrial and have large and well developed mycelia and nonmotile spores. Asexual spores are produced in sporangia. Cell walls are made of chitosan or chitin. There are two classes included in the higher fungi. 1) The Ascomycetes are the best known and largest class of fungi. Ascomycetes have hyphae divided by porous crosswalls. Each of these hyphal compart- ments usually contains a separate nucleus. Asexual spores called conidia, form singly or in chains at the tip of a specialized hypha. The sexual structure called ascus, is formed at the enlarged end of a spe- cialized fruiting structure and usually contains eight ascospores. Some important microscopic members of this group include yeasts, mildews, the common red bread mold and many species which produce antibiotics. On the other hand macroscopic forms include Morchella esculenta or morels which are highly regarded as a delicacy by gourmets. 2) The Basidiomycetes are entirely macroscopic and are commonly known as mushrooms and toadstools. Slime Molds The slime molds, Myxomycetes, are at times classifi ed with either true fungi or protozoa or, as here, treated separately. They produce vegetative structures which look like ameboid C013_004_r03.indd 690C013_004_r03.indd 690 11/18/2005 10:41:59 AM11/18/2005 10:41:59 AM © 2006 by Taylor & Francis Group, LLC MICROBIOLOGY 691 protozoa and fruiting bodies which produce spores with cell walls like fungi. There are two major subdivisions (a) Cellular and (b) Acellular. They both primarily live on decaying plant material and can ingest other microorganisms, such as bacte- ria, phagocytically. Both have life cycles, but that of the acel- lular slime molds is more complicated. Cellular slime molds have vegetative forms composed of single ameboid cells. Cyclically, ameboid cells aggregate to form a slug-shaped pseudoplasmodium that begins to form fruiting bodies when the slug becomes immotile. Spores are fi nally produced by the fruiting bodies. Acellular slime molds have vegetative forms called plas- modia which are composed of naked masses of protoplasm of indefi nite size and shape and which travel by ameboid movement (protoplasmic streaming). Two kinds of nesting structures are produced: fruiting bodies (part of the sexual cycle) and sclerolia. Protozoa The last major group of microorganisms are the protozoa. As already stated, it is very hard to distinguish plants from animals at this primitive stage in evolution where organisms have some attributes of each. Most workers therefore are less interested in whether protozoa should be claimed by bota- nists or zoologists as they are in studying the group as the root of a phylogenetic tree which gave rise to clearly sepa- rable plants and animals. Protozoa range in size from that of large bacteria to just visible without a microscope. They have a variety of shapes, multiplication methods and associ- ations which range from single cells to specialized colonies. They are variously found in fresh water, marine, terrestrial, and occasionally, aerial habitats. Both freeliving and para- sitic forms are included. Most are motile but there are also important nonmotile forms. The protozoa are divided into four subphyla (I–IV). I . Sarcomastigophora include forms which have either fl a- gella, pseudopodia or both. Usually a single-type of nucleus (though opalinids contain multiples of this one type) is pres- ent except in development stages of a few forms. Asexual reproduction by binary fi ssion is common. One whole class contains chloroplasts and are claimed by both protozoolo- gists and algologists (they are considered here in detail with the eukaryotic algae). Many important parasites of diverse animal and some plant groups are found here. Sexual repro- duction is present in a few forms. The Sarcomastigophora are divided into three super- classes. A. Mastigophora ( fl agellates ) Are further sub-divided into Phytomastigophorea or plant-like fl agellates (see eukaryotic FIGURE 6 Bacterial motility. Motility is tested by stabbing an inoculated needle into a tube of very vis- cous growth medium. The motile organisms (S. typhi and P. vulgaris) grow away from the stab mark. 4 3 5 1 2 FIGURE 7 Isolation of single bacterial colonies on agar plates by dilution streaking. A diagrammatic representation of method of streaking inoculated needle across nutrient-containing plate. Stippled area is the primary inoculation. The inoculation needle is then flamed to sterilize and is then drawn across the stippled areas as indicated for area 1. The needle is then resterilized and drawn across area 2, etc. FIGURE 8 Isolation of single colonies by pour plate technique. C013_004_r03.indd 691C013_004_r03.indd 691 11/18/2005 10:41:59 AM11/18/2005 10:41:59 AM © 2006 by Taylor & Francis Group, LLC 692 MICROBIOLOGY algae) and Zoomastigophorea or animal-like fl agellates which are divided into nine orders. 1) Choanoflagellida have a single anterior flagellum surrounded posteriorly by a collar. Some forms are attached to substrates. They are solitary or colonial and are all free-living. 2) Bicosoecida have 2 flagella (one free, the other attached to the posterior of the organism). They are free-living. 3) Rhizomastigida have pseudopodia and 1–4 or more flagella. Most species are free-living. 4) Kinetoplastida have 1–4 flagella and all have a kinetoplast (specialized mitochondrion). Many important pathogens (e.g. trypanosomes) and some free-living genera are included. 5) Retortamonadida have 2–4 flagella. The cytostome is fibril-bordered. All are parasitic. 6) Diplomonadida have 2 karyomastigonts, each with 4 flagella and sets of accessory organelles. Most species are parasitic. 7) Oxymonadida have one or more karyomastigonts, each with 4 flagella. All species are parasitic. 8) Trichomonadida have mastigont systems with 4–6 flagella. Some have undulated membranes. Many important pathogens (e.g. Trichomonas ) are included. 9) Hypermastigida have mastigont systems with numerous flagella and multiple parabasal apparatus. All are parasitic. Some forms reproduce sexually. B. Opalinata Are an intermediary group related to both ciliates and fl agellates and are entirely parasitic. Opalinics have many cilia-like organelles arranged in oblique rows over their entire body surface. They lack cytosomes (oral open- ings). They have multiple nuclei (ranging from 2 to many) which divide acentrically. The whole organism divides by binary fi ssion. Life cycles are complex. C. Sarcodina Or ameboid organisms have Pseudopodia which are typically present but fl agella may be present during certain restricted developmental stages. Some forms have external or internal tests or skeletons which vary widely in type and chemical composition. All reproduce asexually by fi ssion but some also reproduce asexually. Most species are free-living (in both aquatic and terrestrial habitats) but some are important pathogens; for example, Entameba his- tolytica , which causes amebic dysentary. The sarcodinids are further divided into three classes. 1) Rhizopodae, a free-living, mostly particle-eating (phagotrophic) group which includes both naked and shelled species. The specialized pseudopodia are called lobopodia, filopodia, or reticulopodia. 2) Piroplasmea. These parasitic small, piriform, round, rod-shaped or ameboid organisms do not form spores, flagella or cilia. Locomotion is by body-flexing or gliding. They reproduce by binary fission or schizogony. 3) Actinopodea are free-living, spherical, typically floating forms with typically delicate and radiose pseudopodia. Forms may be naked or have mem- braneous, clutenoid, or silicated tests. Both asex- ual and sexual reproduction occurs. Gametes are usually flagellated. II. Sporozoa typically form spores without polar fi laments and lack fl agella or cilia. Both asexual and sexual reproduc- tion takes place. All species are parasitic. Some have rather complicated life cycles. The Sporozoa are divided into three classes: A. Telesporea Can reproduce sexually or asexually, have spores, move by body fl exion or gliding and generally do not have pseudopodia. B. Toxoplasmea Reproduce asexually, lack spores, pseudo- podia or fl agella, and move by body fl exion or gliding. C. Haplosporea Reproduce asexually and lack fl agella. They have spores and may have pseudopodia. III. Cnidospora have spores with one or more polar fi laments and one or more sporoplasms. All species are parasitic. There are two classes. IV. Ciliophora have simple cilia or compound ciliary organ- elles in at least one stage of their life cycle. They usually have two types of nucleus. Reproduction is asexually by fi ssion or sexually by various means. Most species are free- living but parasitic forms are known. ENERGY AND CARBON METABOLISM All cells require a source of chemical energy and of carbon for building protoplasm. Regardless of whether the cell type is prokaryote or eukaryote or whether it is more plant-like or more animal-like, this basic requirement is the same. The most basic division relates to the source of carbon used to build protoplasm. Organisms which can manufacture all their carbon-containing compounds from originally ingested inor- ganic carbon (CO 2 ) are called autotrophs while those which require ingestion of one or several organic compounds for use in the manufacture of cellular carbon compounds are called heterotrophs. Some organisms are nutritionally versatile and may operate either as autotrophs or heterotrophs and are therefore referred to as facultative-autotrophs or facultative- heterotrophs (depending upon which mode of nutrition usu- ally predominates). Autotrophs are further divided according to the manner in which they obtain energy. Chemoautotrophs (also called chemotrophs or chemolithotrophs oxidize various inorganic compounds to obtain energy while photoautotrophs (also called phototrophs or photolithotrophs ) convert light to chemical energy via the absorption of light energy by special pig- ments (chlorophylls and carotenoids). In both cases, chemical energy is stored in the form of chemical bond energy in the compound adenosine triphosphate (ATP). C013_004_r03.indd 692C013_004_r03.indd 692 11/18/2005 10:41:59 AM11/18/2005 10:41:59 AM © 2006 by Taylor & Francis Group, LLC MICROBIOLOGY 693 When bonds of ATP indicated by ~ are broken, a con- siderable amount of energy is released. This ~ bond cleav- age energy operates the biological engines: it is the universal chemical power which operates in all cells, autotroph or het- erotroph. Chemolithotrophic nutrition is only used by certain true bacteria. These bacteria are of ecological importance in that they are used to convert one form of nitrogen to another (i.e. in the nitrogen cycle) or industrially to oxidize low grade metallic or non-metallic ores. There are six bacterial groups which are chemolithotrophic. 1) The ammonia oxidizers such as Nitrosomonas, Nitrosococcus, Nitrosocystis, Nitrosogloea and Nitrosospira. One scheme for ammonia oxidation had hydrox- ylamine as an obligate intermediate and has been proposed for Nitrosomonas. 2) The nitrite oxidizers such as Nitrobacter and Nitrocystis. One proposed scheme for nitrite oxi- dation for Nitrobacter is: NO cytochrome reductase cytochrome C cytochrome oxi 2 Ϫ ⎯→⎯⎯⎯⎯→⎯⎯⎯ ddase O ATP ADP 2 3) Hydrogen oxidizers Hydrogenomonas. One pro- posed hydrogen oxidation scheme is: H 2 →2H ϩ ϩ 2e→unknown→fl avor protein compound →ubiquinone→O 2 cytochrome b compex Nicotinamide adenine→menadione →cytochrome C→cytochrome a →O 2 dinucleotide (NAD) 4) Ferrous compound oxidizing bacteria such as Ferrobacillus and Thiobacillus ferroxidans. One proposed ferrous oxidizing scheme for F. ferrooxidans is: 4FeCO 3 ϩ O 2 ϩ 6H 2 O→4Fe(OH) 3 ϩ 4CO 2 5) Methane oxidizers such as Methanomonas methano- oxidans and Pseudomonas methanica are common in the upper layers of marine sediments and soil. Methane is oxidized in the following manner: CH 4 →CH 3 OH→HCHO→HCOOH→CO 2 6) The sulfur-compound oxidizing bacteria Thioba- cillus. Four pathways for oxidation of thiosulfate (S 2 O 3 ϩ2 ) by different Thiobacillus species are known. These are: a) 6Na 2 S 2 O 3 ϩ SO 2 →4Na 2 SO 4 ϩ 2Na 2 S 4 O 6 2Na 2 S 4 O 6 ϩ 6H 2 O ϩ 7O 2 →2Na 2 SO 4 ϩ 6H 2 SO 4 b) Na 2 S 2 O 3 ϩ 2O 2 ϩ H 2 O→Na 2 SO 4 ϩ H 2 SO 4 c) 5Na 2 S 2 O 3 ϩ H 2 O ϩ 4O 2 →5Na 2 SO 2 ϩ H 2 SO 4 ϩ 4S 2S ϩ 3O 2 ϩ 2H 2 O→2H 2 SO 4 d) 2Na 2 S 2 O 3 ϩ H 2 O ϩ 1/2O 2 →Na 2 S 4 O 6 ϩ 2NaOH Photolithotrophic nutrition is used by photosynthetic bacteria, blue green algae and eukaryotic algae. The general reaction in which both utilization of CO 2 (carbon dioxide fi xation) and energy generation is summarized is: CO H A CH O A H O 22 2 ϩϩϩ nv ⎯→⎯ ()2 2 Where A is either oxygen for all eukaryotic algae and the prokaryotic blue-green algae (H 2 A = H 2 O), or sulfur for green sulfur bacteria, Chlorobacteriaceae, and purple sulfur bacte- ria, Thiorhodaceae (H 2 A = H 2 S) or any one of several organic compounds for nonsulfur purple bacteria, Athiorhodaceao (H 2 A = H 2 -organic compound which is oxidizable). Both green and purple sulfur bacteria are obligate anaerobes whereas the non-sulfur purple bacteria are facul- tative anaerobes (they are anaerobic when growing hetero- trophically). In all cases, photosynthetic organisms operate by the initial transduction of light to chemical energy. In this transduction, chlorophyll ϩ light quanta Ch1 ϩ (excited chlorophyll) ϩ e Ϫ (electron driven off of Ch1). Many such events take place simultaneously and electrons released during these reactions migrate through the photosynthetic unit to the reaction center and transfer energy to a special reaction-center chlorophyll. At the reaction center, a charge separation of the oxidant and reductant occurs. Electron fl ow after this event differs in photosynthetic bacteria as compared with algae and higher plants (Figures 9 and 10). In addition, differences in photosynthetic ability exist among organisms based upon the absorption maxima of their light-transducing pigments (primarily chlorophylls). The combination of light intensity, wavelength of available light, wavelength of operation of principal energy trans- ducing pigment, degree of aerobiasis, and availability of oxidizable compound (H 2 O, H 2 S, or H 2 -organic compound) all infl uence the effi ciency of photosynthesis. These factors should be borne in mind when one looks for the ecological niche occupied by these various organisms. Ecology of Microorganisms One should understand the physiological requirements of microorganisms before investigating the effects of environ- mental changes on the distribution and activity of diverse C013_004_r03.indd 693C013_004_r03.indd 693 11/18/2005 10:42:00 AM11/18/2005 10:42:00 AM © 2006 by Taylor & Francis Group, LLC [...]... principle of revealing (selectively enriching for) a minor population of desired microbial type among a multitude of undesirable organisms makes use of an enrichment medium in which the weed-killer is used as either (a) the only source of organic carbon and nitrogen, (b) the only source of organic carbon though other sources of nitrogen are present, or (c) the only source of nitrogen though other sources of. .. indicative of toxicity to one or more types of microorganisms The profound changes in populations of higher plants and animals due to disruption of the balance of microbial life can be readily appreciated when the cyclic nature of nitrogen and sulfur dissimilation is considered (Figures 12 and 13) These interdependences emphasize the key role played by microorganisms in maintaining the balance of soil... Xanthophyta Chla Chle 682 Cyano-, Chryso- and Rhodophyta Chla 683 Non sulfur purple bacteria Green algae and Euglenids, higher plants classical microbiology These laboratory methods, pioneered by Winogradsky (1856–1953) and Beijerinck (1851–1931) and refined by others, utilize specialized, restrictive, physical and chemical conditions to select and cause to predominate one or few types of organisms from a highly... as a weed-killer consists of (a) preparing a standard column composed of soil or water from the body in question, and (b) after the column has been allowed to develop its natural population (c) an isotopically labelled version of the weed-killer can be added in the concentration (and 10 ϫ the concentration that the weed-killer is to be used) After a time equivalent to that in which the weed-killer is... understands how to manipulate these laboratory systems, it is easier to analyze field observations in which specific conditions which result in microbial changes can be recognized and, if necessary, altered We will first explain the principles of selective enrichment techniques and then look at the natural distribution of microorganisms and their relationship to higher plants and animals Highlights of microbial... contained in the liquid and solid phases, and, of course, (c) the initial populations of microorganisms In the examples shown, light is provided to ensure growth of photosynthetic organisms If the basic principles of the Winogradsky column are to be used to reveal the microbial population in a particular soil or water sample, then the column and all its components are first sterilized and then inoculated... in question Determination of Gross Populations of Microorganisms Quantitative sampling techniques are required for the determination of the microbial population in air, water, or soil samples The method used for sample collection must ensure against (a) loss of more than a trivial number of microorganisms and (b) cross-contamination from other sources during sample transport and laboratory manipulation... putrefaction bacteria SH of amino acids to make protoplasm FIGURE 13 The sulfur cycle Microorganisms are becoming increasingly useful for processing low-grade sulfur-containing ores Sometimes overlooked is the natural cyclic distribution of sulfur-containing compounds in which microorganisms play principle roles membrane is exposed and the desired amount of air passed through it Air-borne microorganisms... soil samples are handled aseptically and weighed amounts are tested for their microbial population REFERENCES 1 Environmental Microbiology, in The Natural Environment and the Bioecochemical Cycles, O Hutzinger, ed., Springer Verlag, Heidelberg, 1985 2 Saunders, V.A and Saunders, J.R., Microbial Genetics Applied to Biotechnology, Croom-Helm, London, 1987 3 Zehnder, A.J.B., Biology of Anaerobic Microorganisms,... pure or axenic (a = absence of, xenos = strangers) culture The common nutritional requirements of microorganisms, the quantities in which they must be supplied, and the biological uses of each substance are shown in Table 5 The selective enrichment techniques to be described are most frequently used for the isolation of bacteria, yeast, and certain prokaryotic algae An outline of selecting properties is . some of the properties of each type. Viruses, on the other hand, are not cells at all. Instead of arguing endlessly about whether a micro- organisms is more plant-like or more animal-like and. The combination of light intensity, wavelength of available light, wavelength of operation of principal energy trans- ducing pigment, degree of aerobiasis, and availability of oxidizable compound. planes form cube-shaped packets. If there is no regu- lar pattern of the orientation of successive division planes, a randomly-shaped cluster is formed. Staphylococcus is an example of a coccus