ࡱ> q` R`bjbjqPqPK::&: : : L280A48"ZZZCCC$hz<5: ^:( C^^<ZZ}q†††^:DZ: Z†^†† D: †Z( d}†0†††: ֆ$CfeH$†K NCCC<<CCC^^^^0A0A0Avb0A0A0AN " &H Introduction to Microbiology 1. What is the unique features of Microorganisms? 1.1 Small size, Simple structure Antoni Van Leeuwenhock: The word microorganism is used to describe an organism that is so small that, normally, it can not be seen without use of microscopy. The living organisms of microscopic size Most microorganisms are unicellular Some of them are non-cellular structure 1.2 High diversity species. Bacteria ( actinomyce, algae, cyanobacteria) archaea fungi ( yeast, mold ) protozoa viruse 1.3 Widely distributed in various environment with large numbers soil. 109 CFU/g water air plants, animals, human body extreme environment, e.g. hot springs 1.4 Fast growing, easy cultivation generation time, min.- hr. 1.5 Easy mutation 2. The place of microorganisms in the living world 2.1 whittakers five kingdom concept (1969) Kingdom plantae Kingdom animalia Kingdom fungi Kingdom protista Kingdom monera This classification system was based on nutrition, photosynthesis, absorption and ingestion. 2.2 Woeses three domains ( kingdoms) Domain bacteria Domain Archaea Domain eucarya 3. The scope of microbiology Microbiology is the study of living organisms of microscopic size, which include bacteria, fungi, algae, protozoa and viruses. It is concerned with their morphology, reproduction, physiology, metabolism, genetics and classification. It includes the study of their distribution and function in nature, their relationship to each other and to other living organisms, their effects on human beings, animals and plants. 4. Why do we study microbiology? Microorganisms affect the well-being of people in a great many ways. They occur in large numbers in most natural environments. Some of them are beneficial and others are detrimental. 4.1 The beneficial aspects In Agriculture Microbial fertilizer Biological control SCP Microbial energy: methane gas for rural consumption In food production yogurt, cheese, wine(alcohol fermentation) food ingredients In environment protection treatment of waste materials to decompose materials: pesticides, herbicides In Biochemical industry Antibiotics enzymes Amino acides organic acids in medicine Vaccine Antibiotics penicillin The potential applied areas of Microbiology GMO Recombinant DNA technology Microbial plastics Microbial pesticides Microbiosensor M OahV " Microbial fuel cells _uirqe5u`l " Microbial DNA chip _uirDNAGr " Exploitation of microorganisms in extreme environments 4.2 The detrimental aspects " Cause diseases: human beings animals Plants " spoil food " deteriorate materials: iron pipes, wood, cloth Objectives: make full use and exploitation of the beneficial aspects; avoid and control the detrimental aspects Chapter 1 The Morphology and Fine Structure of Microorganisms 1. Procaryotic Microorganisms 1.1 Features distinquishing procaryotic from Eucaryotic cells featureProkaryotic cells Bacteria ( archaea)Eucaryotic cells(fungi, protozoa, plant, animal)Cell wall Cell membrane Cytoplasmic structures Mesosome Ribosome Mitochondria Chloroplast Golgi structure Endoplosmic reticulum nucleusPeptidoglycan as component Generally do not contain sterol; contain part of respiratory and, in some, photosynthetic machinery invagination Present 70S Absent Absent Absent Absent Not bounded by nuclear membrane; one circular chromosome, Chromosome does not Contain histones; no mitotic divirsion Nucleolus absentAbsente of peptidoglycan Sterols present; do not carry out respiratory and photosynthesis non absent 80S present present present present Bounded by nuclear membrane; More than one chromosome Chromosomes have histones; Mitotic nuclear division Nucleolus present 1.2 Bacteria 1.2.1 The size, shape and arrangement of bacterial cell sphierical cocci, coccus um; diameter A: diplococci B: streptococci C: tetrads D: staphylococci E: Sarcinae straight rods: widnth x Length um, bacilli, bacillus, Lactobacillus helically curved rods: spirilla, spirillum width x Length um (No. of curve) filament: hyphae, hypha ! mycelium diameter of hypha m Actinoinyces: streptomyces species % some are not constant, but pleomorphic shape 1.2.2 Simple staining technique The coloration of bacterial cell by applying a single solution of stain to a fixed smear is termed simple staining. Steps: Smear bacterial cells on a slide dry and fix The fixed smear is flooded with a dye solution for a specified period of time Wash off with H2O and dry the clide exame the size. Shape and arrangement of bacterial cells under microscope 1.2.3 The structures and functions of bacterial cells 1.2.3.1 Structures external to the cell wall A. Flagella, Flagellum Bacterial flagella are hairlike, helical appendages that protrude through the cell wall and are responsible for swimming motility. chemical composition: protein subunits: flagellin Location: polar: at one or both ends of the bacterium Lateral: alone the sides of the bacterium function: swimming motility gliding motility: some bacteria are motile when they are in contact with a solid surface. As they glide they exhibit a sinuous, flexing motion. This kind of movement is comparatively slow, only a few um per second. Bacterial chemotaxis many, perhaps most, bacteria are capable of directed swimming toward or away from various chemical compoundsa phemomenon called bacterial chemotaxis Bacterial phototaxis phototrophic bacteria move towards increasing light intensities. Pili, pilus( fimbriae) Pili are nonhelical, filamentous appendages that are thinner, shorter, and more numerous than flagella. The well known is the F pilus ( or sex pilus), it serves as the port of entry of genetic material during bacterial mating. B. Capsules Some bacterial cells are surrounded by a viscous substance forming a covering layer or envelope around the cell wall. If this layer can be visualized by light microscopy using special staining method, it it termed a capsule. If it is too thin to be seen by light microscopy, it is termed microcapsule. If is so abundant that many cells are embedded in a common matrix, the material is called slime. The conditions that some bacteria produce capsule High carbon content in media, low nitrogen The chemical composition of capsule polysaccharides polypeptides The functions of capsule They may provide protection against temporary drying by binding water molecules They may block attachment of bacterophages They may promote attachment of bacteria to surfaces, e.g. Streptococcus mutans, a bacterium associated with producing dental caries, firmly adheres to the smooth surfaces of teeth because of its secretion of a water-insoluble capsular polysaccharide. C. spore and cyst defination: under the undesirable growth conditions,e.g. Lack of nutrients, too acid, temperiture is too high, Some species of bacteria produce thick-walled structure which is metabolically dormant and can undergo germination and outgrowth to form a vegetative cell. This special structure is termed spore which is either within the cell (endospore) or external to the cell (exospore). shapes: function: spores are extremely resistant to desiccation, staining, disinfecting chemicals, radiation, and heat. Some of sproes can resist boiling for several hours, most of spores can resist heat treatment at 80! for at least 10 minutes. Dormant structure. " Cyst: cyst resembles endospore, especially desiccation-resistant. But it does not resist to heat treatment. Genus Azotobacter cyst 1.2.3.2 The cell wall " defination: A very rigid layer external to the cytoplasmic membrane. The main function is to give shape to the cell and prevent the cell from expanding and eventually bursting because of uptake of water. peptidoglycan: an insoluble, porous, cross-linked polymer of enormous strength and rigidity. Differential staining: staining procedures that make visible the differences between bacterial cells or parts of a bacterial cell are termed differential staining technique. The cells may be exposed to more than one dye solution or staining reagent. Gram staining: one of the most important and widely used differential staining techniques in microbiology is Gram staining. This technique was introduced by Christian Gram in 1884. In this process the fixed bacterial smear is subjected to the following reagents in the order listed: crystal violet, iodine solution, 95% alcohol (decolorizing agent), and safranin or some other suitable counterstain. Bacteria stained by the gram method fall into two groups: Gram positive bacteria (G+), which retain the crystal violet and hence appear deep violet in color; and Gram negative bacteria (G-), which lose the crystal violet, are counterstained by safranin, and hence appear red in color. Why does this proceduce stain some bacteria purple-violet and others red? % The wall of Gram positive bacteria % the structure and chamical composition % The wall of Gram negative bacteria % The structure and chemical composition 1.2.3.3 The cytoplasmic Membrane Immediately beneath the cell wall is the cytoplasmic membrane. This structure is approximately 7.5nm thick and is composed mainly of phospholipids (20~30%) and proteins (60~70%). % Mesosome many bacteria, especially G+ bacteria, possess membrane invaginations in the form of convoluted and vesicals termed mesosome. % Functions of mesosome: DNA replication cell division export of exocellular enzyme % The functions of cytoplasmic membrane % the CM is a barrier to penetration by most molecules; % specific proteins in the memtrane allow and facilitate the passage of small molecules (e.g. nutrients, waste products); % The CM contains various enzymes involved in respiratory metabolism and phtosynthesis. % CM is the site of ATP generation, synthesis of capsular and cell wall components. 1.2.3.4 The cytoplasm % Ribosome In the cytoplasmic area, granular in appearance and rich in the macromolecular RNA-protein bodies known as Ribosomes, on which proteins are synthesized. In the prokaryotic cells, the sedimentation coefficient of ribosome is 70S. % PHB: poly  hydroxybutyrate 1.2.3.5 The genetic materials % The nucleus % not bounded by nuclear membrane % one circular chromosome % chromosome does not contain histones % nucleolus absent % Plasmid In addition to the normal DNA chromosome, extrachromosomal genetic elements are offen found in bacteria. These elements are called plasmids, and are capable of autonomous replication in the cytoplasm in the bacterial cell. Plasmids are circular pieces of DNA that are extra genes. 1.2.2 Bacterial Reproduction and colony 1.2.2.1 Reproduction % Transverse Binary Fission The most common mode of bacterial cell division in the usual growth cycle of bacterial populations is transverse binary fission, in which a single cell divides into two cells after developing a transverse septum. Transverse binary fission is an asexual reproductive process. % Budding some bacteria reproduce by budding, a process in which a small protuberance ( bud ) develops at one end of the cell. This enlarges and eventually develops into a new cell which separates from the parent. % Fragmentation Bacteria that produce extensive filaments, such as Nocardia species, reproduce by fragmentation of the filaments into small bacillary or coccoid cells, each of which gives rise to a new cell. % Formation of conidiospores or sporangiospores Species of the genes streptomyces and related bacteria produce many spores by developing crosswalls (septation) at the hyphal tips. Each spore gives rise to a new cell. 1.2.2.2 Colony formation % Colony: visible aggregate of bacterial growth on a solid culture medium. It is a important characteriastics for identifying a certain bacterial species. % Colony-forming unit: aggregate of cells gives rise to a single colony in the plate count technique. Abbreviation:cfu 1.2.3 Actinomycetes 1.2.3.1 The characteristics of Actinomyctes % G+ bacteria % Filamentous  substrate mycelium hyphae!mycelium aerial mycelium conidiospores  forming spores sporangiospores % Reproduction % Fragmentation % Formation of spores % colony % Size small, round shape, tight, dry, diverse color, smell soil. % nutrient and environmental factors required for growth % chemoorganotroph % aerobe % neutral pH % optimal growth temperature: 23~37! 50~65! % They are distributed mainly in soils. They can grow in soils which have less water content than that needed for most other bacteria, because of their spores that can survive well in dry soil. % denifination: Actinomycetes: Gram positive bacteria that are characterized by the formation of branching filaments and spores. 1.2.3.2 The roles of Actinomycetes in nature % Many actinomycetes, such as, streptomycetes, can degrade polymeric organic substances in soil that are refractory to being decomposed by many other microorganism, e.g., starch, pectin, chitin % produce antibiotics, antifungal antibiotics % Fix N2: Frankia, These group of bacteria are highly efficient microaerophilic N2 fixers that occur within the root nodules of plants. Unlike Rhizobium spcies, however, they infect nonleguminous woody plants, such as alders. % some species are pathogenic to plants and humans. 1.2.4 Cyanobacteria % prokaryotic oxygenic photolithotrophs containing chlorophyll a and phycobilins % Algae: phototrophic eukaryotic microorganisms 1.3 Archaea An evolutionarily distinct domain of prokaryotes consisting of the methanogens, most extreme halophiles and hyperthermorphiles, and Thermoplasma. 2. Eucaryotic Microorganisms 2.1 Features distinguishing Eucaryotic fro prokaryotic cells, see 1.1 2.2 Fungi Molds and yeasts 2.2.1 Characteristics of fingi 2.2.1.1 Morphology 2.2.1.1.1 Yeast yeast cells are larger than most bacteria. Yeasts vary considerably in size, ranging from 1~5 m in width and from 5~30m or more in length. They are commonly egg shaped, but some are spherical. Each species has a characteristic shape, but even in pure culture there is considerable variation in size and shape of individual cells, depending on age and environment. 2.2.1.1.2 Mold The thallus of a mold consists essentially of two parts: the mycelium (mycelia) and the spores ( resistant, dormant cells) % The mycelium The mycelium is a complex of several filaments called hyphae (singular, hypha), New hyphae generally arise from a spore germination. Each hypha is about 5~10m wide. There are three types of hyphae:  Yeasts~bacteria 2.2.1.1.3 Colony molds~Actinomycetes 2.2.1.2 Reproduction 2.2.1.2.1 Asexual Reproduction Fungi reproduce naturally by a varity of means. Asexual reproduction (also called somatic or vegetative reproduction) does not involve the union of nuclei, sex cells, or sex organs. % Budding of somatic cells or spores % Fragmentation of the hyphal cells, each fragment becoming a new cell % Formation of asexual spores. Asexual spores are produced in large numbers. There are many kinds of asexual spores: 2.2.1.2.2 Sexual reproduction sexual reproduction is carried out by fusion of the compatible nuclei of two parent cells. The process of sexual reproduction begins with the joining of two cells and fusion of their protoplasts (plasmogamy), thus enabling the two haploid nuclei of two mating types to fuse together (karyogamy) to form a diploid nucleus. This followed by meiosis, which again reduces the number of chromosomes to the haploid number. Sexual spores are produced by the fusion of two nuclei. There are several types of sexual spores 2.2.1.3 Classification of Fungi Ainsworth classification system Myxomycota   Mastigomycotina: nonseptate mycelium, sporangiospores, flagella, oospores Zygomycotina: nonseptate mycelium, sporangiospores, no flagella, zygospores Eumycota Basidiomycotina: septata mycelium, Basidiospores Ascomycotina: septate mycelium, Ascospores Deuteromycotina: septate mycelium, conidiospores, sexual reproduction has not been found 2.2.1.4 Physiology and ecology % Fungi are chemoorganotrophic organisms that have no chlorophyll. They require organic compounds for energe source and nutrition. % The saprophytes Some fungi feed on dead organic matter, they are known as saprophytes. Saprophytes decompose complex Plant and animal remains, breaking them down into simpler chemical substances that are returned to the soil, thereby increasing soil fertility. These simpler chemical substances can be used by other organisms to produce useful products. This is very important to reduce organic waste in environment. % The parasites Some fungi are living in or on another organism. They are called parasites.Parasites cause diseases in plants, humans and other animals. Although fungal diseases are less commonly encountered than bacterial or virus diseases in humans and other animals, they are of great importance in causing diseases of plants. % Fungi are better able to withstand extreme environmental conditions than most other microorganisms. For example, yeasts and molds can grow in a substrate or medium containing high concentrations of sugars that inhibit most bacteria; this is why jams may be spoiled by molds but not by bacteria. Yeast and mold generally can tolerate more acidic conditions than other microbes. % Some yeasts are facultative; that is they can grow under both aerobic and anaerobic conditions. Molds are aerobic microorganisms. % Fungi grow over a wide range of temperature the optimal growth temperature: 22~30! saprophytic species 30~37! pathogenic species Some fungi can grow at or near 0! and thus can cause spoilage of meat and vegetables in cold storage. % There are many differences between fungi and bacteria. A summary is listed in the following table. Comparative features of fungi and bacteria CharacteristicFungiBacteriacell type optimum pH optimum temperature oxgen requirement Light requirement Sugar concentration in media Carbon requirement Cell-wall stralctural components Antibiotic susceptibilityEucaryotic 3.8~5.6 22~30!(saprophytes) 30~37!(parasites) strictly aerobic(molds) facultative (some yeasts) none 4~5% organic chitin, cellulose, hemicellulose resistant to penicillins, tetra- cyclines, chloramphenical; sen- sitive to griseofulvinProkaryotic 6.5~7.5 20~37!(mesophiles) aerobic, anaerobic facultative some photosynthetic bacteria 0.5~1% inorganic or/and organic peptidoglycan resistant to griseofulvin; sensitive to penicillins, te- tracyclines, chloramphenical 2.2.1.5 Molds and their associations with other organisms % There are some interesting partnerships in nature involving a mold and some other organism. In some of these associations the partners are dependent on each other and can not survive alone. In others, The individuals can survive independently. % Lichens Lichens are composite organisms composed of fungi and algae, each contributing to the benefit of both. The algae synthesize carbohydrates by photosynthesis and obtain other nutrients from the fungi; the fungi depend on the algae for the organic carbon. % Mycorrhizas Mycorrhiza is a symbiotic association of a fungus with the roots of a higher plants. This association is usually beneficial to the host plant as well as the symbiont, and sometimes the host can not thrive without the benefits derived from the fungus (e.g. orchid). Mycorrhizas enhance mineral absorption by the plants. 2.3 Algae 2.4 Protozoa 3 Viruse 3.1 The general characteristics of viruses % noncellullar structure Virion: The structurally complete mature and infectious virus is called the virion. % Small size Viruses are so small that they can only be seen at magnifications provided by the electron microscope. They are 10~100 times smaller than most bacteria, with an approximate size range of 20~300nm. Thus they pass through the pores of filters which do not permit the passage of most bacteria. % Obligate intracellular parasites Viruses are incapable of independent growth in artificial media. They can grow only in animal0plant cells or microorganicms, because viruses lack metabolic. Machinery of their own to generate energy or to synthesize proteins. They depend on the host cells to carry out these vital functions. % Replication Viruses reproduce in the host cells by replication that is a process in which many copies are made of each viral component and are then assembled to produce progeny virus. During reproduction in the host cells viruses may cause diseases. % Definition of viruses Viruses are noncellular infectious entities whose genomes are a nucleic acid, either DNA or RNA; which reproduce only in living cells by replication; and which use the host cells biosynthetic machinery to direct the synthesis of specialized particles (Virions), which contain the viral genomes and transfer them efficiently to other cells. 3.2 Bacteriophage Virus that infects bacteria. Phage, bacterial virus % The shape of bacteriophage % Lytic bacteriophage When lytic phages infect cells, the cells respond by producing large numbers of new viruses. That is, at the end of the incubation period the host cell bursts or lyses, releasing new phages to infect other host cells. This process is called a lytic cycle. % Temperate bacteriophage The viral nucleic acid is replicated in the host bacterial cells from one generation to another without any cell lysis. However, temperate phages may spontaneously become virulent at some subsequent generation and lyse the host cells. Chapter 2 Nutritional Types and Enery production 1 Nutritional Requirements 1.1 Source of Energy All microorganisms require a source of energy. Some rely on chemical compounds for their energy and are designated as chemotrophs. Others can utilize radiant energy (light) and are called phototrophs. 1.2 Source of electrons All microorganisms require a source of electrons for their metabolism. Some can use reduced inorganic compounds as electron donors and are termed lithotrophs. Others use organic compounds as electron donors and are called organotrophs. 1.3 Carbon source All microorganisms require carbon in some form for use in synthesizing cell components. All require at least small amount of CO2. However, some can use CO2 as their major, or even sole, source of carbon; such microorganisms are termed autotrophs. Others require organic compounds as their carbon source and are termed heterotrophs. 1.4 Nitrogen source All microorganisms require nitrogen in some form for cell components. Bacteria are extremely versatile in this respect. Unlike eucaryotes, some bacteria can use atmospheric nitrogen. Others thrive on inorganic nitrogen compounds such as nitrate, or ammonium salts, and still others derive nitrogen from organic compounds such as amino acids. 1.5 Oxygen sulfur and phosphorus All microorganisms require oxygen, sulfur and phosphorus for cell components. Oxygen is provided in various forms, such as water, component atoms of various nutrients, or molecular oxygen. Sulfur is needed for synthesis of certain amino acids. Some bacteria require organic sulfur compounds, some are capable of utilizing inorganic sulfur compounds, and some can even use elemental sulfur. Phosphorus, usually supplied in the form of phosphate, is an essential component of nucleotides, nucleic acids, phospholipids, and other compounds. 1.6 Mineral elements All microorganisms require metal ions, such as K+, Ca2+, Mg2+, and Fe2+ for nomal growth. Other metal inos are also needed but usually only at very low concentrations, such as Zn2+, Cu2+, Mo6+, and Co2+. These are often termed trace elements. Not all the biological functions of metal ions are known, but Fe2+, Mg2+, Zn2+, Ca2+, Mo6+, and Cu2+, are known to be cofactors for various enzymes. Most bacteria do not require Na+, but certain marine bacteria, cyanobacteria, and photosynthetic bacteria do require it. For those members of archaeobacteria known as the extreme halophiles, the requirement is astonishing. They can not grow with less than 12~15% NaCl. They require this high level of NaCl for maintenance of the integrity of their cell walls and for the stability and activity of their certain enzymes. 1.7 Vitamins All microorganisms need vitamins and vitaminlike compounds. Some bacteria are capable of synthesizing vitamins from other compounds in the culture media, but others can not do so and will not grow unless the required vitamins are supplied in the media. 1.8 Water All microorganisms require water. Nutrients must be in aqueous solution before they can enter the cells. Water is to dissolve and disperse nutrients and to provide a suitable milieu for the various metabolic reactions of a cell. Moreover, the high specific heat of water provides resistance to sudden, transient temperature changes in the environment. 2 Nutritional Types of Microorganisms Although microorganisms have great diversity of nutritional requirement, they can be divided into four major groups on the basis of their utilization of energy and carbon sources. 2.1 Photolithotroph or photoautotroph They utilize light as energy source, and CO2 as major or even sole source of carbon.   2.2 Photoorganotroph or photoheterotroph They require light as energy source and organic compounds as carbon source.  CH3 Light CO2 + 2 CHON [ CH2O] + 2CH3COCH3 + H2O CH3 Rhodospirillum 2.3 Chemolithotroph or Chemoautotroph They rely on inorganic chemical compounds for their energy and use CO2 as their major, or even sole source of carbon.    2.4 Chemoorganotroph or Chemoheterotroph They utilize organic compounds as their energy and carbon Source. Most bateria and all fungi belong to this group. 3 Medium, Media or culture mediaum, culture media 3.1 definition: an aqueous solution containing various nutrients suitable for the growth of microorganisms. 3.2 Types of media 3.2.1 Based on the chemical composiion % Synthetic media The chemical composition of every ingredients in the medium is clear. Medium for cultivation of Actinomycetes Starch 20g KNO3 1.0g K2HPO4 0.5g MgSO4%7H2O 0.5g NaCl 0.5g FeSO4%7H2O 0.01g d-H2O 1000ml % Natural media The media containing complex natural raw materials surch as peptones, meat broth, yeast extract, plants and animal materials. Medium for cultivation of fungi Potato 200g sucrose 20g H2O 1000ml Medium for cultivation of bacteria Beef extract 5.0g peptone 10.0g NaCl 5.0g H2O 1000ml 3.2.2 Based on physical status % Broth media: liquid media % Solid media: The solidifying agent is added to the liquid media. The solidifying agent is usually agar, which at concentrations of 1.5~2.0% forms firm, transparent gels that are not degraded by most bacteria. Silica gel is sometimes used as an inorganic solidifying agent for cultivation of autotrophic bactoria. % Semisolid media: Agar concentration: 0.2~0.5% 3.2.3 Based on special purpose % Selective media These media provide nutrients that enhance the growth of a particular type of microorganism and do not enhance (and may even inhibit) other types of microorganisms that may be present. For instance, nitrogen-free medium (Ashby) will specifically select the growth of nitrogen-fixing bacteria. % Enriched media These media contain a certain nutrient that promotes and enriches a special type of microorganism. For example, a medium in which cellulose is the only carbon source will enrich the growth of cellulose-utilizing microorganism. During the process of cultivation, the noncellulose-utilizing microorganisms are eliminated gradually. This is especially useful for isolation of a bacterium when the number is very low in the sample. % Differential media The media that contain some kinds of ingredients which can differentiate various kinds of microorganisms. For example, if a mixture of bacteria is inoculated onto a blood-containing agar medium, some of the bacteria may hemolyze the red blood cells; others do not. Thus one can distinquish between hemolytic and nonhemolytic bacteria on the same medium. 3.3 Preparation of media 3.3.1 The principles % The nutrients in the medium with suitable concentration and ratia must satisfy the normal growth of microorganisms. % Optimum pH for growth % In large scale protuction, the source and price of the ingredients in the medium must be considered. 3.3.2 The procedures % Each ingredient with correct amount is dissolved in the appropriate volume of distilled H2O; % The pH of the fluid medium is determined with a pH meter and adjusted if necessary; % If a solid medium is desired, agar is added and the medium is boiled to dissolve the agar; % The medium is sterilized, generally by autoclaving. Some specific ingredients that are heat-labile are sterilized by filtration. 4 Transport of nutrients 4.1 Passive diffusion Passive diffusion is the process that solute molecules cross the membrane as a result of a difference in concentration of the molecules across the membrane. The difference in concentration ( higher outside the membrane than inside) governs the rate of inward flow of the solute molecule. With time, this concentration gradient diminishes until equilibrium is reached. Except for water and some lipid-soluble molecules, few compounds can pass through the cytoplasmic membrane by passive diffusion. In passive diffusion, no substance in the membrane interacts specifically with the solute molecule, and energy is not needed. 4.2 Facilitated diffusion This process is similar to passive diffusion in that the solute molecule also flows from a higher to lower concentration. But it is different from passive diffusion because it involves a specific protein carrier molecule called permease located in the cytoplasmic membrane. The carrier molecule combines reversibly with the solute molecule, and the carrier-solute complex moves between the outer and inner surfaces of the membrane, releasing one solute molecule on the inner surface and returning to bind new one on the outer surface. This type of transportation is common in eucaryotic cells. Sugars enter them by this way. No ATP is needed. There is specific interactions between the solute and protein carrier in the membrane. 4.3 Group translocation Group translocation is the process in which the solute is chemically altered in the course of passage across the membrane. 4.4 Active transport Almost all solutes are taken up by cells through active transport. The three steps of active transport are: % Binding of solute to a receptor site on a membrane-bound carrier protein. % Translocation of the solute-carrier complex across the membrane. % Carrier protein release solute to the cell interior " Group translocation and active transport require energy and accumulate substrates against concentration gradient.Solutes can be concentrated within the cell several thousand times greater than outside the cell. There is specific interaction between the solute and protein carrier in the membrane. 5 Energy production(ATP) 5.1 Energy production by aerobic process % Substrate-level phosphorylation: production of ATP by the direct transfer of a high-energy phosphate molecule from a phosphorylated organic compound to ADP. % Oxidative phosphorylation: production of ATP at the process of electron transport. 5.2 Energy production by anaerobic process % Fermentation Fermentation is a process of anaerobic oxidation of organic compounds. Neither gaseous oxygen nor respiratory chain is involved in this energy-yielding process. ATP is produced by substrate-level phosphorylation. An organic compound is the electron acceptor. The lactic fermertation is a typical example. Streptococcus lactis, the bacterium responsible for the souring of milk, dissimilates glucose to lactic acid, which accumulates as the sole fermentation product. 5.3 Energy production by photosynthesis % Photophosphorylation: Synthesis of high-energy phosphate bonds as ATP, using light energy. Chapter 3 pure culture and Growth 1. Pure culture A culture containing only one strain of microorganism, which is usually derived from a single colony. 1.1 Methods of isolating pure cultures 1.1.1 The streak-plate technique By means of a transfer loop, a loop of the mixed culture is placed on the surface of an agar medium and streaked across the surface. This manipulation thins out the bacteria on the agar surface so that some individual bacteria are separated from each other. 1.2 The pure-plate and spread plate Techniques Notes: The pure-plate technique has certain disadvantages: 1) Some of the microorganisms are trapped beneath the surface of the medium when it gels. 2) The microorganism being isolated must be able to withstand temporary exposure to the 45! temperature of the liquid agar medium, For instance, the pure-plate method would be unsuitable for isolating psychrophilic microorganisms. In the spread-plate technique, I ml of the diluted sample is placed onto the surface of an agar plate, and spread evenly over the surface by means of a steril, bent glass rod. In contrast to the pure-plate method, only surface colonies develop; moreover, the microorganism being isolated is not required to withstand the 45! temperature of liquid agar. 1.1.3 Micromanipulator technique A device called the micromanipulator can be used in conjunction with a microscope to pick up a single cell ( or spore) from a mixed culture. This technique permits the operator to control the movements of a micropipette or a microprobe (a fine needle) so that a single cell (or sproe) can be isolated. This technique requires a skilled operator. 1.2 Miaintenance and preservation of pure culture Most microbiology laboratories maintain a large collection of strains, frequently referred as a stock-culture collection. These sterains are needed for research work and for practical usage. The two international famous culture collections are ATCC and USDA. 1.2.1 The objective of maintenance and preservation of pure culture % Preservation at 4! The strains are stored in nomal refrigerator. Periodic transfer to fresh media is needed. Advantage: simple, convenient to use. Disadvantage: It can not prevent changes in their characteristics due to genetic mutations. % Freeze-Drying method Most microorganisms die if cultures become dry, although spore-and cyst-formers can remain viable for many years. However, freeze-drying can satisfactorily preserve many kinds of microorganisms that would be killed by ordinary drying. In this process a dense cell suspension is placed in small vials and frozen at  60~78!. The vials are then connected to a high-vacuum line for dehydration. The vials are sealed off under a vacuum and stored in a refrigerator. The freeze-drying cultures can be revived by opening the vials, adding liquid medium and transferring to a suitable growth medium. Disadvantage: The method is complicate. There may be small damage to cell structure. Advantage: The storage time can be very long. Many species preserved by this method have remained viable and unchanged in their characteristics for more than 30 years. It is convenient for exchange between different laboratories and organizations. % storage at -70!~80! 20% glycerol is added to the liquid medium to prevent cell damage due to ice crystal formation % storage using liquid nitrogen (-150!) % storage using sand % storage using oil 2. Quantitative measurement of microbial growth 2.1 Enumeration of cell numbers 2.1.1 Total count % Direct Microscopic Count % Turbidity 2.1.2 Viable count % The plate-count method % Most probable number, MPN 2.2 Determination of cell biomass and cell activity % Determination of the dry weight of cells % Determination of nitrogen content, DNA, ATP % Determination of metabolic activity. 3 Growth curve of bacteria The most common means of bacterial reproduction is binary fission, one cell divides, producing two cells. When we inoculate a fresh medium with a given number of cells, determine the viable bacterial population intermittently during an incubation period of 24 hours ( more or less), and plot the logarithms of the number of cells versus time, we obtain a curve of the type that is called bacterial growth curve. 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A. The lag phase The addition of inoculum to a new medium is not followed immediately by a doubling of the population. The population remains temporarily unchanged. There is a lag in cell division. But this does not mean that the cells are dormant. On the contrary, during this stage the individual cells are very active, and synthesizing enzymes and molecules required for cell division. Time for adjustment in the new environment is needed. At the end of the lag phase, cells begin to divide. However, since not all cells complete the lag phase simultaneously, there is gradual increase in the population until the end of this period. B. The logarithmic or exponential phase During this period the cells divide steadily at a constant rate, and the log of the number of cells plotted against time results in a straight line. The increase in population is by geometric progression. Moreover, the population is most nearly uniform in terms of chemical composition of cells, metabolic activity, and their physiological characteristics, so log-phase cultures are commonly used for studies of microbial metabolism. Generation time (G): the time required for the population to double. The generation time can be calculated by the following formula:  N: the number of generations; T: time; No: the viable number of bacterial cells at time o; Nt: the viable number of bacterial cells at log phase and at time t. Growth rate ( R ): the number of generations per hour.  C. The stationary phase The viable number of bacterial cells remains constant for a time. The reproduction rate is balanced by an equivalent death rate. This can be attributed to a variety of circumstances, particularly the exhaustion of some nutrients, and the production of toxic products during growth. D. The decline or death phase Following the stationary phase the bacteria die faster than new cells are produced, the number of viable cells decreases exponentially, essentially the inverse of growth during the log phase. A variety of conditions contribute to bacterial death, but the most important are the depletion of essential nutrients and the accumulation of inhibitory products, such as acids. Batch culture: A closed-system microbial culture of fixed volume. 4 Synchronous Growth " Growth in a cell population in which all cells divide at the same time. " The significance It is not feasible to analyze a single bacterium because of its small size. However, if all the cells in a culture were to be in the same stage of the division cycle, the result from analysis of the cell crop could be interpreted as that for a single cell. " Methods to obtain synchronous culture `$ The most common method of synchronization takes advantage of the fact that the smallest cells in a log-phase culture are those which have just divided. When those cells are separated out by filtration or by differential centrifugation, they are reasonably well synchronized with each other. a$ A cell population can be synchronized by manipulating the physical environment or the chemical composition of the medium. For example, the cells may be inoculated into a medium at a suboptimal temperature. If they are kept in this condition for some time, they will metabolize slowly but will not divide. When the temperature is subsequently raised, the cells will undergo a synchronized division. 5 Continuous culture " In both experimental research and in industrial processes, it is often desirable to maintain a bacterial population growing at a particular rate in the exponential or log phase. This condition is known as steady-state growth. The culture volume and the cell concentration are both kept constant by allowing fresh sterile medium to enter the culture vessel at the same rate that  spent  medium, containing cells, is removed from the growing culture. Under these conditions, the rate at which new cells are produced in the culture vessel is exactly balanced by the rate at which cells are being lost through the overflow from the culture vessel. " One type of system that is widely emploged for continous cultivation is the chemostat. Another type is the turbidostat. " The batch culture and continous culture. Charpter 4 Soil Microbiology Few environments on earth have as great a variety of microorganisms as soil. Bacteria, fungi, algae, protozoa and viruses make up this microscopic menagerie, which may reach a total of billions per gram. The great diversity of the microbial flora makes it extremely difficult to determine accurately the total number of microorganisms present. Cultural methods will reveal only those physiological and nutritional types compatible with the cultural environment. Direct microscopic counts theoretically should permit enumeration of all except the viruses, but this technique also has its limitations, especially, in distinguishing living from dead microorganisms. Very often the microbiological analysis of soil is concerned with the isolation and identification of specific physiological types of microorganisms. For this purpose sective media, differential media, and enriched media are appropriate. 1. The Rhizosphere Rhizosphere: The region where the soil and roots make contact is designated the rhizosphere which is characterized by a zone of increased microbiological activity. The microbial population on and around roots is considerably higher than that of root-free soil. The differences are both quantitative and qualitative. Bacteria predominate, and their growth is enhanced by nutritional substances released from the plant roots, e.g., amino acids, vitamins, and other nutrients. The growth of the plant is influenced by the products of microbial metabolism that are released into soil. It has been reported that amino acid requiring bacteria exist in rhizosphere in larger numbers than in the root free soil. It has been demonstrated that the microbiota of the rhizoshere is more active physiologically than that of nonrhizosphere soil. The rhizosphere represents a tremendously complex biological system, and there is a great deal yet to be learned about the interactions which occur between the plant and the microorganisms intimately associated with its root system. Electron microscope techniques have been developed to observe microorganisms directly on the root surfaces. 2. Interactions among soil microorganisms The microorganisms that inhabit the soil exhibit many different types of associations or interactions. Some of the associations are neutral; some are beneficial or positive; others are negative or detrimental. 2.1 Neutral association Neutralism Two different species of microorganisms inhabit the same environment without affecting each other. For example, each could utilize different nutrients without producing metabolic end products that are inhibitory. 2.2 positive associations Mutualism mutualism is a symbiotic relationship in which each microorganism benefits from the association. Lichen: A mutalistic (or symbiotic) association of an alga and a fungus. Commensalism Commensalism refers to a relationship between microorganisms in which one species of a pair benefits; the other is not affected. For example, many fungi are able to dissimilate cellulose to glucose. Many bacteria are unable to utilize cellulose, but they can use the fungal breakdown products as energy and carbon sources. 2.3 Negative associations Antagonism The killing, injury, or inhibition of growth of one species of microorganism by another when one organism adversely affects the environment of the other. This kind of association is called antagonism. The antagonistic microorganisms may be of great practical importance in biological control, since they often produce antibiotics or other inhibitory substances which affect the normal growth or survival of other microorganisms (pathogenic microorganisms). Competition Competition is a negative association among species which compete for essential nutrients. In such situation the best adapted microbial species will predominate, or eliminate other species which are dependent upon the same limited nutrient substance. Parasitism Parasitism is defined as a relationship between microorganisms in which one lives in or on another. The parasite feeds on the cells, tissues, or fluids of another microorganism, the host, which is commonly harmed in the process. The parasite is dependent upon the host and lives in intimate physical and metabolic contact with the host. All major groups of plants, animals, and microorganisms are susceptible to attack by microbial parasites. Viruses that attack bacteria, fungi and algae are strict intracellular parasites since they can not be cultivated as free living forms. Predation Eating of an individual of one species by an individual of another species. Protozoa eat bacteria. 3. Biogeochemical role of soil microorganisms Soil microorganisms serve as biogeochemical agents for the conversion of chemical compounds. The conversion of complex organic compounds into simple inorganic compounds or elements provides for the continuity of elements as nutrients for plants, aminals and microorganisms. 3.1 Biochemical transformations of nitrogen compounds: The nitrogen cycle    3.1.1 Proteolysis The biochemical process Proteolysis is the process of enzymatic hydrolysis of proteins. This is accomplished by microorganisms capable of elaborating extracellular proteinase that convert the protein to smaller units (peptides). The peptides are than attacked by peptidases, resulting in the resease of individual amino acid. The overall reactions may be summarized:   Proteolytic microorganisms Bacteria: some bacterial species elaborate large amounts of proteolytic enzymes. Among the most active in this respect are some of the clostridia; a lesser degree of activity is found in species of the genera Proteus, Pseudomonas and Bacillus. Fungi: Rhizopus, Mucor, Aspergillus 3.1.2 Ammonification Many microorganisms can deaminate amino acids. The production of ammonia is referred as ammonification. Biochemical process  3.1.3 Nitrification Microorganisms (nitrifying bacteria) convert ammonia to nitrate. This process is called nitrification. The process occurs in two steps, each step performed by a different group of bacteria. `$Oxidation of ammonia to nitrite  a$Oxidation of nitrite to nitrate  Nitrifying bacteria Nitrifying bacteria, including ammonia oxidizers and nitrite oxidizers, are gram negative chemolithotrophs. Their main source of carbon is obtained through CO2 fixation; energy is derived by the oxidation of NH3 or NOdepending upon the group. 3.1.4 Denitrification The transformation of nitrate to gaseous nitrogen is accomplished by microorganisms in a series of biochemical reactions. This process is called denitrification. NO NO NO N2O N2 Nitrate nitrite nitric oxide Nitrous oxide nitrogen Denitrifying bacteria There are diverse groups of bacteria in soil, sewage and aquatic environments that can transform NOto N2. They include some chemotrophs, phototrophs, heterotrophs and autotrophs. Environmental conditions in a soil have a significant effect on the level of denitrification. The process is enhanced in soils by `$ oxygen supply is limited, a$ an abundance of organic matter, b$ elevated temperature (25~65!),c$ neutral or alkaline pH. From the standpoint of griculture and environment, both nitrification and denitrification are undesirable pocesses. 3.1.5 Biological Nitrogen Fixation A number of microorganisms are able to use molecular nitrogen in the atmosphere as their source of nitrogen. The conversion of molecular nitrogen into ammonia is known as biological nitrogen fixation. 3.1.5.1 Nitrogen fixing Microorganisms Two groups of microorganisms are involved in biological nitrogen fixation nonsymbiotic microorganisms, those living freely in the soil. e.g. Azotobacter. The process to fix nitrogen by this group of bacteria is called nonsymbiotic nitrogen fixation. Symbiotic microorganisms, those living in roots of plants. e.g. Rhizobium. The process to fix nitrogen by this group of bacteria is called symbiotic nitrogen fixation. 3.1.5.2 The essential reactants in the biological nitrogen fixation process The nitrogenase enzyme complex This has been characterized as two components, and neither is active without the other. Component I is known as the Mo-Fe protein. Component II, which is a smaller molecule is Fe protein. Both molecules contain sulfur. A strong reducing agent ATP A requlating system for NH3 production and utilization A system that protects the nitrogen-fixing system from inhibition by molecular oxgen. 3.1.5.3 The overall biochemical reaction for BNF  N2+6e-+12ATP+12H2O +12ADP+12Pi+4H+ 3.1.5.4 Nonsymbictic nitrogen fixation nonsymbiotic nitrogen fixation has been studied extensively with clostridium pasteurianum and species of Azotobacter. The former is an anaerobic bacillus, and the latter are aerobic spherical cells. Both are widely distributed in soils. 3.1.5.5 Symbiotic nitrogen fixation symbiotic nitrogen fixation is accomplished by the bacteria of the genus Rhizobium in association with legumes (e.g. soybean, peanut, clover, pea) Characteristics of Rhizobium  G-. small rod, chemoorganotroph ( YEM, TY), producing capsule, flagellum, no endospore, fast-growing Rhizobium, slow-growing Bradyrhizobium Symbiotic properties of Rhizobium `$ Infection: Rhizobia invade plant root, through root hairs and form nodules. a$ Host specificity: not all species of Rhizobium produce nodulation and fix nitrogen with any legume. There is specificity between the bacteria and legumes. b$ Effectiveness: The amount of nitrogen fixed. How to estimate the effectiveness? Plant growth: Dry weight, fresh weight, height. Nodulation: nodule number and weight, The Nitrogenase activity Acetylene reduction method  HC CH H2C=DH2 Acetylene Ethylene N N NH3 The amount of nitrogen fixed To demonstrate growth in jensen s N-free medium. Plant total nitrogen of inoculated  plant total nitrogen of uninoculated. 15N technique. c$ Competitiveness, one strain compete nodulation with other rhizobial strains. Methods to study rhizobial competiveness  marker techniques FA Melanin production Antibiotics resistant DNA hybridization Insertion of marker genes TN5 transposon Lux AB Cel B Gus A AFLP fingerprint technique. 3.1.5.6 Rhizobial phylogeny and taxonomy Chapter 5 Microbial Genetics 1 Inheritance (heredity) and Variability Inheritance is the exact transmission of genetic information from parents to their progeny. Variability is the differences between parents and their progeny. 2 Genotype and phenotype Genotype refers to the genetic constitution of the cell. Phenotype is the expression of the genotype in observable characteristics of the cell. 3 Gene mutation 3.1 concepts Gene mutation is a change in the nucleotide sequence of a gene. This gives rise to a changed genotype. Spontaneous mutation: mutation occurs under natural conditions. The mutation rate is low. Induced mutation: mutations obtained by use of a mutagen, mutagen: Any agent that increases the mutation rate (UV) mutant: A microorganism which shows the effects of a mutation is called a mutant. 3.2 Types of mutation Point mutation Point mutations occur as a result of the substitution of one nucleotide for another in the specific nucleotide sequence of a gene Frameshift mutation These mutations result from an addition or loss of one or more nucleotides in a gene and are termed insertion or deletion, respectively. 3.3 Phenotypes of microbial mutants mutants that exhibit an increased tolerance to inhibitory agents, particularly antibiotics. ( antibiotic-or drug- resistant mutants) Mutants that are nutritionally deficient, that is, they require a more complex medium for growth than the original culture which they were derived. (auxotrophic mutants) mutants that show changes in the surface structure and composition of the microbial cell ( antigenic mutants) mutants that demonstrate an altered fermentation ability or increased or decreased capacity to produce some end products. mutants that exhibit changes in colonial form or ability to produce pigments. mutants that exhibit some changes in morphological changes, for example, the loss of ability to produce spores, capsule, or flagellum. 3.4 The practical implications of mutation It is important to isolate mutants capable of producing high yield of an end product. For example, the yield of penicillin in commercial production has been dramatically increased through selection of mutant. Some microorganisms are known to develop resistante to certain antibiotics because of mutation. This fact is of great importance in the treatment of disease, since antibiotics originally effective for the control of a bacterial infection become less effective or ineffective as antibiotic-resistant mutants appear. Microbial mutants have been extensively used in microbial research. % Many mutants are able to revert to the original Phenotype (wild-type) by reverse mutation. 4 Bacterial gene transfer and Recombination Recombinant DNA: A DNA molecule containing DNA originationg from two or more sources. Recombination: Process by which genetic elements in two separate genomes are brought together in one unit. Recombinant: A microorganism resulting from recombination. In bacterial recombination the cells do not fuse, and usually only a portion of the chromosome from the donor cell is transfered to the recipient cell. There are three types of gene transfer in bacteria. 4.1 Conjugation Transfer of genes between bacterial cells that are in physical contact with one another. (Fig.A) 4.2 Transduction Transfer of genes from one bacterial cell to another by a bacteriaphage. (Fig.B) 4.3 Transformation  naked donor DNA passes into recipient and is integrated into the chromosome of recipient cell. (Fig.C.D) 4.4 The Griffith experiment, in 1928, English health officer He found the  transforming principle Avery, Macleod and McCarty in 1944 proved this transforming principle is DNA, and defined DNA as the chemical substance responsible for inheritance. Fig.E 5 Genetic Engineering Genetic engineering refers to the development of microorganisms with genetic structure altered by biochemical manipulation. This kind of biochemical procedure is termed recombination DNA technology, and involves the use of plasmids and certain bacteriophages. This technology consists of isolating, purifying, and identifying genetic material from one source, tailoring it for insertion into a new host; and isolating a colony of cells with the desired new genes (Fig.F).  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