ميكروبيولوجيا الطلبة - الكورس الأول - الجامعة التقنية الوسطى PDF

‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫المرحلة ‪:‬الثانية‬ ‫المادة ‪:‬األحياء المجهرية‬ ‫قسم تقنيات المختبرات الطبية‬ ‫‪Training package in theory lecture‬‬...

‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫المرحلة ‪:‬الثانية‬ ‫المادة ‪:‬األحياء المجهرية‬ ‫قسم تقنيات المختبرات الطبية‬ ‫‪Training package in theory lecture‬‬ ‫‪Medical Microbiology‬‬ ‫‪For‬‬ ‫‪The students of second class in Medical laboratory department‬‬ ‫‪By‬‬ ‫م‪.‬د استبرق علي السوداني‬ ‫ا‪.‬د محمد فليح طريف‬ ‫ا‪.‬م‪.‬د عصام جمعة‬ ‫ا‪.‬م‪.‬د جليل نجاح جليل‬ ‫‪2022A.D‬‬ ‫‪1444A.H‬‬  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Title: Introduction and Classification of bacteria Lec 1 ‫العنوان‬ Name of the instructor: :‫اسم المحاضر‬ Professor Dr. Mohammed Flaih Target population: :‫الفئة المستهدفة‬ Second stage students Introduction: :‫المقدمة‬ Microbiology is a specialized area of biology* that deals with living things, ordinarily too small to be seen without magnification. Such microscopic* also study of microorganism (M.O) which are unicellular or cell-cluster microscopic organisms, microbiology involves study in numerous areas involving cell structure, function, genetics, immunology, biochemistry, epidemiology, and ecology. Microorganisms, also called microbes, are organisms that require a microscope to be readily observed. It be found in every ecosystem and in class association with every type of multicellular organisms. M.O are the oldest organisms, having evolved over the 4 billion years of earth’s history to the modern varieties we now observe. Microbes are classified into groups according to evolutionary relationships, provided with standard scientific names, and identified by specific characteristics.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ They populate the healthy human and animals body by billions as Normal flora and even participants in body functions, for ex. Bacteria play a role in the degradation in intestinal contents. These M.O consist of archaea, bacteria, viruses, fungi, protozoa, algae, and helminthes, which caused infection and spread of human diseases. Few species of M.O that harmful to human either by production toxic compounds and enzymes or direct infection by their virulence factors are characterized as pathogens. Pre test: :‫االختبار القبلي‬ Q\ What is meaning of microbiology and microorganism Q\ Microorganism consist of ………,…………,…………..,……….,………….,…………..,  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Scientific content: :‫المحتوى العلمي‬ Microbiology is a specialized area of biology* that deals with living things, ordinarily too small to be seen without magnification. Such microscopic* also study of microorganism (M.O) which are unicellular or cell-cluster microscopic organisms, microbiology involves study in numerous areas involving cell structure, function, genetics, immunology, biochemistry, epidemiology, and ecology. Microorganisms, also called microbes, are organisms that require a microscope to be readily observed. It be found in every ecosystem and in class association with every type of multicellular organisms. M.O are the oldest organisms, having evolved over the 4 billion years of earth’s history to the modern varieties we now observe. Microbes are classified into groups according to evolutionary relationships, provided with standard scientific names, and identified by specific characteristics. They populate the healthy human and animals body by billions as Normal flora and even participants in body functions, for ex. Bacteria play a role in the degradation in intestinal contents. These M.O consist of bacteria, viruses, fungi, protozoa, algae, and helminthes, which caused infection and spread of human diseases. Few species of M.O that harmful to human either by production toxic compounds and enzymes or direct infection by their virulence factors are characterized as pathogens. Classification of M.O Classification of M.O Eukaryotic Prokaryotic Protozoa Helminth Bacteria Bacteria is an unicellular m.o which have a rigid cell wall surrounding the cell membrane that determine the shape of bacteria.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Viruses are obligate intracellular parasite that don't have a cellular structure. Fungi are non photosynthetic, generally saprophytic eukaryotic m.o some fungi are multicellular filaments called molds where as other unicellular called yeast. Protozoa are single called non photosynthetic E. organism that found in different sizes and shapes many protozoa are among the most clinically important parasite of human. Helminthes are groups of worms that live as parasites they are multicellular E. organisms with complex body organization. Eukaryotic Cell and Prokaryotic Cell  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Eukaryotic Cell Nucleus Present Absent Number of chromosomes More than one One--but not true chromosome: Plasmids Cell Type Usually Usually unicellular multicellular (some cyanobacteria may be multicellular) True Membrane bound Nucleus Present Absent Example Animals and Bacteria and Archaea Plants Genetic Recombination Meiosis and Partial, un directional fusion of transfers DNA gametes Lysosomes and peroxisomes Present Absent Microtubules Present Absent or rare Endoplasmic reticulum Present Absent Mitochondria Present Absent Cytoskeleton Present May be absent DNA wrapping on proteins. Eukaryotes Multiple proteins act wrap their DNA together to fold and around proteins condense prokaryotic called histones. DNA. Folded DNA is then organized into a  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Eukaryotic Cell variety of conformations that are supercoiled and wound around tetramers of the HU protein. Ribosomes larger smaller Vesicles Present Present Golgi apparatus Present Absent Chloroplasts Present (in Absent; chlorophyll plants) scattered in the cytoplasm Flagella Microscopic in Submicroscopic in size, size; membrane composed of only one bound; usually fiber arranged as nine double surrounding two single Permeability of Nuclear Membrane Selective not present Plasma membrane with steroid Yes Usually no Cell wall Only in plant Usually chemically cells and fungi complex (chemically simpler)  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Eukaryotic Cell Vacuoles Present Present Cell size 10-100 um 1-10 um Classification of bacteria Classification is a method for organizing microorganisms into groups or taxa based on similar morphologic, physiologic, and genetic traits. The hierarchical classification system consists of the following taxa designations: Domains (Bacteria, Archaea, and Eukarya) Kingdom (contains similar divisions or phyla; most inclusive taxa) Phylum (contains similar classes; equivalent to the Division taxa in botany) Class (contains similar orders) Order (contains similar families) Family (contains similar genera) Genus (contains similar species) Species (specific epithet; lowercase Latin adjective or noun; most exclusive taxa) Historically, bacteria or prokaryotes (prenucleus) were included in a single domain. However, with the more detailed analysis using modern techniques, this domain has now been separated into the Bacteria and the Archaea (ancient bacteria). The Bacteria contain the environmental prokaryotes (blue green or cyanobacteria) and the heterotrophic medically relevant bacteria. The Archaea are environmental isolates that live in extreme environments such as high salt concentrations, jet fuel, or extreme temperatures. The third domain, Eukarya, eukaryotes (true nucleus), also contains medically relevant organisms, including fungi and parasites. There are several other taxonomic sublevels below the domains, as noted previously; however the typical application of organism classification in the diagnostic microbiology laboratory primarily uses the taxa beginning at the family designation.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Family A family encompasses a group of organisms that may contain multiple genera and consists of organisms with a common attribute. The name of a family is formed by adding the suffix -aceae to the root name of one of the group’s genera, called the type genus; for example, the Streptococcaceae family type genus is Streptococcus. One exception to the rule in microbiology is the family Enterobacteriaceae; it is named after the “enteric” group of bacteria. Bacterial (prokaryotic)-type species or strains are determined according to guidelines published by the International Committee for the Systematics of Prokaryotes. Species definitions are distinguished using DNA profiling, including a nearly complete 16S rRNA sequence with less than 0% to 5% ambiguity in combination with phenotypic traits. Genus Genus (plural, genera), the next taxon, contains different species that have several important features in common. Each species within a genus differs sufficiently to maintain its status as an individual species. Placement of a species within a particular genus is based on various genetic and phenotypic characteristics shared among the species. Microorganisms do not possess the multitude of physical features exhibited by higher organisms such as plants and animals. Species Species (abbreviated as sp., singular, or spp., plural) is the most basic of the taxonomic groups and can be defined as a collection of bacterial strains that share common physiologic and genetic features and differ notably from other microbial species. Occasionally, taxonomic subgroups within a species, called subspecies, are recognized. Nomenclature Nomenclature is the naming of microorganisms according to established rules and guidelines set forth in the International Code of Nomenclature of Bacteria (ICNB) or the Bacteriological Code (BC). It provides the accepted labels by which organisms are universally recognized. Because genus and species are the groups commonly used by microbiologists, the discussion of rules governing microbial nomenclature is limited to these two taxa. In this binomial (two name) system of nomenclature, every organism is assigned a genus and a species of Latin or Greek  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ derivation. Each organism has a scientific “label” consisting of two parts: the genus designation, in which the first letter is always capitalized, and the species designation, in which the first letter is always lowercase. The two components are used simultaneously and are printed in italics or underlined in script. For example, the streptococci include Streptococcus pneumonia. Post test: :‫االختبار البعدي‬ Q\ Enumerate classification of microorganism Q\ Compare between Eukaryotic cell and prokaryotic cell References Foundations in Microbiology 4th Edition Kathleen Park Talaro Pasadena City College Arthur Talaro Pasadena City College 2001 2- Todar's Online Textbook of Bacteriology Dedication to Hans Zinsser 2005 3- Different size, shape and arrangement of bacterial cell 2022 4- Prokaryotic cells structure, function, and definition 2019 5- Bailey & Scott’s Diagnostic Microbiology Fourteenth Edition Patricia M. Tille, PhD, BS, MT(ASCP), FACSc 2017 6- Jawetz, Melnick, & Adelberg’s 2019 Medical Microbiology Twenty-Eighth Edition  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Title: The Structure of a Generalized Procaryotic cell Lec2,3 :‫العنوان‬ Cell Name of the instructor: :‫اسم المحاضر‬ Estabraq Ali AL-Sodani ‫محمد فليح‬.‫ استبرق علي السوداني د‬.‫د‬ Target population: :‫الفئة المستهدفة‬ Second stage students Introduction: :‫المقدمة‬ Procaryotic cells are the smallest, simplest, and most abundant cells on earth. Representative procaryotes include bacteria and archaea, both of which lack a nucleus and organelles but are functionally complex. The structure of bacterial cells is compact and capable of adaptations to a myriad of habitats. The cell is encased in an envelope that protects, supports, and regulates transport. Bacteria have special structures for motility and adhesion to the environment. Bacterial cells contain genetic material in a single chromosome, and ribosomes for synthesizing proteins. Bacteria have the capacity for reproduction, nutrient storage, dormancy, and resistance to adverse conditions. Shape, size, and arrangement of bacterial cells are extremely varied. Bacterial taxonomy and classification is based on their structure, metabolism, and genetics. Bacterial cells appear featureless and two-dimensional when viewed with an ordinary microscope. Not until they are subjected to the scrutiny of the electron microscope and biochemical studies does their intricate and functionally complex nature become evident. The descriptions of bacterial structure, except where otherwise noted, refer to the bacteria,* a category of procaryotes with  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ peptidoglycan in their cell walls. presents a three dimensional anatomical view of a generalized (rod-shaped) bacterial cell. As we survey the principal anatomical features of this cell, we will perform a microscopic dissection of sorts, following a course that begins with the outer cell structures and proceeds to the internal contents. Pre test: :‫العنوان‬ Q\ Enumerate shapes of bacteria? Scientific content: :‫المحتوى العلمي‬ Bacterial Morphology Most clinically relevant bacterial species range in size from 0.25 to 1 µm in width and 1 to 3 µm in length, thus requiring microscopy for visualization. Just as bacterial species and genera vary in their metabolic processes, their cells also vary in size, morphology, and cell-to-cell arrangements and in the chemical composition and structure of the cell wall. The bacterial cell wall differences provide the basis for the Gram stain, a fundamental staining technique used in bacterial identification schemes. This staining procedure separates almost all medically relevant bacteria into two general types: gram-positive bacteria, which stain a deep blue or purple, and gram-negative bacteria, which stain a pink to red.This simple but important color distinction is the result of differences in the constituents of bacterial cell walls that influence the cell’s ability to retain differential dyes after treatment with a decolorizing agent. Common bacterial cellular morphologies include cocci (circular), coccobacilli (ovoid), and bacilli (rod shaped), as well as fusiform (pointed end), curved, or  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ spiral shapes. Cellular arrangements are also noteworthy. Cells may characteristically occur singly, in pairs, or grouped as tetrads, clusters, or in chains. The determination of the Gram stain reaction and the cell size, morphology, and arrangement are essential aspects of bacterial identification. Parts of prokaryotic cell procaryote cell Appendages cell envelope cytoplasm A. C.E C. Flagella/periplasmic Glycocalyx (capsules, slime Cell pool flagella layers) Ribosomes Pili, fimbriae Cell wall Granules Cell membrane Nucleoid/chromosome  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫المرحلة ‪:‬الثانية‬ ‫المادة ‪:‬األحياء المجهرية‬ ‫قسم تقنيات المختبرات الطبية‬  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ 1-THE CELL ENVELOPE: THE OUTER WRAPPING OF BACTERIA The majority of bacteria have a chemically complex external covering, termed the cell envelope, that lies outside of the cytoplasm. It is composed of three basic layers known as the glycocalyx, the cell wall, and the cell membrane. The layers of the envelope are stacked one upon another and are often tightly bonded together like. Although each envelope layer performs a distinct function, together they act as a single protective unit. The envelope is extensive and can account for one-tenth to one-half of a cell’s volume. A-The Bacterial Surface Coating, or Glycocalyx The bacterial cell surface is frequently exposed to severe environmental conditions. The glycocalyx develops as a coating of macromolecules to protect the cell and, in some cases, help it adhere to its environment. Glycocalyces differ among bacteria in thickness, organization, and chemical composition. Some bacteria are covered with a loose, soluble shield called a slime layer that evidently protects them from loss of water and nutrients. Other bacteria produce capsules of repeating polysaccharide units, of protein, or of both. Acapsule is bound more tightly to the cell than a slime layer is, and it has a thicker, gummy consistency that gives a prominently sticky (mucoid) character to the colonies of most encapsulated bacteria. Specialized Functions of the Glycocalyx Capsules are formed by a few pathogenic bacteria, such as Streptococcus pneumonia (a cause of pneumonia, an infection of the lung),Haemophilus influenzae (one cause of meningitis), and Bacillus anthracis (the cause of anthrax). Encapsulated bacterial cells generally have great pathogenicity because capsules protect the bacteria against white blood cells called phagocytes. B-Peptidoglycan (PG) Cell wall Immediately below the glycocalyx lies a second layer, the cell wall. This structure accounts for a number of important bacterial characteristics. In general, it determines the *shape of a bacterium, and it also provides the *kind of strong structural support necessary to keep a bacterium from bursting or collapsing because of changes in osmotic pressure.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ The cell walls of most bacteria gain their relatively rigid quality from a unique macromolecule called peptidoglycan (PG). Peptidoglycan is only one of several materials found in cell walls, and its amount and exact composition vary among the major bacterial groups. Because many bacteria live in aqueous habitats with a low solute concentration, they are constantly absorbing excess water by osmosis. Some factors effect in C.W 1-Several types of drugs used to treat infection (penicillin, cephalosporins) are effective because they target the peptide cross-links in the peptidoglycan. lysis*. 2-Some disinfectants (alcohol, detergents) also kill bacterial cells by damaging the cell wall. 3-Lysozyme, an enzyme contained in tears and saliva, provides a natural defense against certain bacteria by hydrolyzing the bonds in the glycan chains and causing the wall to breakdown. Differences in Cell Wall Structure The Gram-Positive Cell Wall The bulk of the gram-positive cell wall is a thick, homogeneous sheath of peptidoglycan ranging from 20-80 nm in thickness. It also contains tightly bound acidic polysaccharides, including teichoic acid and lipoteichoic acid. Teichoic acid is a polymer of ribitol or glycerol and phosphate embedded in the peptidoglycan sheath. Lipoteichoic acid is similar in structure but is attached to the lipids in the plasma membrane. These molecules appear to function in cell wall maintenance and enlargement during cell division, and they also contribute to the acidic charge on the cell surface. The Gram-Negative Cell Wall The gram-negative cell wall is more complex in morphology because it contains an outer membrane OM, has a thinner shell of peptidoglycan, and has an extensive space surrounding the peptidoglycan. The outer membrane is somewhat similar in construction to the cell membrane, except that it contains specialized types of polysaccharides and proteins. The uppermost layer of the OM contains lipopolysaccharide (LPS). The polysaccharide chains extending off the surface function as antigens and receptors. The innermost layer of the OM is another lipid layer anchored by means of lipoproteins to the peptidoglycan layer below. The outer membrane serves as a partial chemical sieve by allowing only relatively  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ small molecules to penetrate. Access is provided by special membrane channels formed by porin proteins that completely span the outer membrane. The size of these porins can be altered so as to block the entrance of harmful chemicals, making them one defense of gram-negative bacteria against certain antibiotics. The bottom layer of the gram-negative wall is a single, thin (1–3 nm) sheet of peptidoglycan. Although it acts as a somewhat rigid protective structure as previously described, its thinness gives gram-negative bacteria a relatively greater flexibility and sensitivity to lysis. There is a well-developed periplasmic space surrounding the peptidoglycan. This space is an important reaction site for a large and varied pool of substances that enter and leave the cell.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ C- CELL MEMBRANE STRUCTURE Appearing just beneath the cell wall is the cell, or cytoplasmic, membrane, a very thin (5–10 nm), flexible sheet molded completely around the cytoplasm. Its general composition was described as a lipid bilayer with proteins embedded to varying degrees. Bacterial cell membranes have this typical structure containing primarily phospholipids (making up about 30–40% of the membrane mass) and proteins (contributing 60–70%). Oligosaccharide Integral protein Glycolipid Hydrophobic helix Hopanoid l Peripheral Phospholipid protein In some locations, the cell membrane forms internal pouches in the cytoplasm called mesosomes*. These are prominent in gram-positive bacteria but are harder to see in gram negative bacteria because of their relatively small size. Mesosomes presumably increase the internal surface area available for membrane activities. Functions of the Cell Membrane Since bacteria have none of the eucaryotic organelles, the cell membrane provides a site for functions such as 1-Energy reactions. 2-Nutrient processing, and synthesis. 3-A major action of the cell membrane is to regulate transport, that is, the passage of nutrients into the cell and the discharge of wastes. 4- Although water and small uncharged molecules can diffuse across the membrane unaided.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ 5-membrane is a selectively permeable structure with special carrier mechanisms for passage of most molecules. 6-The cell membrane is also involved in secretion, or the discharge of a metabolic product into the extracellular environment. 7-Most enzymes of respiration and ATP synthesis reside in the cell membrane since procaryotes lack mitochondria. 8-Other products (enzymes and toxins) are secreted by the membrane into the extracellular environment. D-The appendages of bacteria Pili(Fimbriae) Many Gram-negative bacteria possess rigid surface appendages called pili (L “hairs”) or fimbriae (L “fringes”). They are shorter and thinner than flagella; similar to flagella, they are composed of structural protein subunits termed pilins. Some pili contain a single type of pilin, others more than one. Minor proteins termed adhesins are located at the tips of pili and are responsible for the attachment properties. Two classes can be distinguished: ordinary pili, which play a role in the adherence of symbiotic and pathogenic bacteria to host cells; and sex pili, which are responsible for the attachment of donor and recipient cells in bacterial conjugation a means of DNA transfer (sex pili). provide motility (flagella) Bacterial flagella are thread-like appendages composed entirely of protein called flagellin , approximately 20 nm in diameter. They are the organs of locomotion for the forms that possess them. motility is restored within 3–6 minutes. The flagellins of different bacterial species presumably differ from one another in primary structure. They are highly antigenic (H antigens), and some of the immune responses to infection are directed against Flagella vary in number and arrangement as well as in the type and rate of motion they produce.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ a. b. c. d. Electron micrographs depicting types of flagellar arrangements. (a) Monotrichous flagellum (b) Lophotrichous flagella (c) amphitrichous (and lophotrichous) in arrangement and coil up into tight loops. (d) peritrichous flagella 2- CONTENTS OF THE CELL CYTOPLASM Encased by the cell membrane is a dense, gelatinous solution referred to as cytoplasm, which is another prominent site for many of the cell’s biochemical and synthetic activities. Its major component is water (70–80%), which serves as a solvent for the cell pool, a complex mixture of nutrients including sugars, amino acids, and salts. The components of this pool serve as building blocks for cell synthesis or as sources of energy. The cytoplasm also contains larger, discrete cell masses such as the chromatin body, ribosomes, mesosomes, and granules. A-Bacterial Chromosomes and Plasmids: The Sources of Genetic Information The hereditary material of bacteria exists in the form of a single circular  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ strand of DNA designated as the bacterial chromosome.* By definition, bacteria do not have a nucleus; that is, their DNA is not enclosed by a nuclear membrane but instead is aggregated in a dense area of the cell called the nucleoid.* The chromosome is actually an extremely long molecule of DNA that is tightly coiled around special basic protein molecules so as to fit inside the cell compartment. many bacteria contain other, nonessential pieces of DNA called plasmids.* These tiny, circular extra-chromosomal strands can be free or integrated into the chromosome; they are duplicated and passed on to offspring. They are not essential to bacterial growth and metabolism, but they often confer protective traits such as resisting drugs and producing toxins and enzymes. B-Ribosomes: Sites of Protein Synthesis A bacterial cell contains thousands of tiny, discrete units called ribosomes.* ribosomes show up as fine, spherical specks dispersed throughout the cytoplasm that often occur in chains (polysomes). Chemically, a ribosome is a combination of a special type of RNA called ribosomal RNA, or rRNA (about 60%), and protein (40%). The essential function of ribosomes is protein synthesis. C-Inclusions, or Granules: Storage Bodies Most bacteria are exposed to severe shifts in the availability of food. During periods of nutrient abundance, they compensate by laying down nutrients intracellularly in inclusion bodies, or inclusions,* of varying size, number, and content. Other inclusions, also called granules, contain crystals of inorganic compounds and are not enclosed by membranes. Sulfur granules of photosynthetic bacteria polyphosphate granules of Corynebacterium and Mycobacterium are of this type. The latter represent an important source of building blocks for nucleic acid and ATP synthesis. They have been termed metachromatic* granules because they stain a contrasting color (red, purple) in the presence of methylene blue dye. D- BACTERIAL ENDOSPORES: Endospores are dormant bodies produced by the grampositive genera Bacillus, Clostridium, and Sporosarcina. These bacteria have a two-phase life cycle—a vegetative cell and an endospore.The depletion of nutrients, especially an adequate carbon or nitrogen source, is the stimulus for a vegetative cell to begin spore formation.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Post test Compare between Gram positive and Gram negative cell wall? References 1- Foundations in Microbiology 4th Edition Kathleen Park Talaro Pasadena City College Arthur Talaro Pasadena City College 2001 2- Todar's Online Textbook of Bacteriology Dedication to Hans Zinsser 2005 3- Different size, shape and arrangement of bacterial cell 2022 4- Prokaryotic cells structure, function, and definition 2019 5- Bailey & Scott’s Diagnostic Microbiology Fourteenth Edition Patricia M. Tille, PhD, BS, MT(ASCP), FACSc 2017 Jawetz, Melnick, & Adelberg’s 2019 Medical Microbiology Twenty-Eighth Edition  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Title: :‫العنوان‬ Microbial Growth and Metabolism Lec 4 Name of the instructor: :‫اسم المحاضر‬ Dr. Mohammed Flaih ‫د محمد فليح طريف‬.‫ا‬ Target population: :‫الفئة المستهدفة‬ Second stage students Introduction: :‫المقدمة‬ The division of a bacterial cell occurs mainly through binary, or transverse, fission; binary means that one cell becomes two, and transverse refers to the division plane forming across the width of the cell. During binary fission, the parent cell enlarges, duplicates its chromosome, and forms a central transverse septum that divides the cell into two daughter cells. This process is repeated at intervals by each new daughter cell in turn, and with each successive round of division, the population increases in size, number, and mass. Pretest: :‫االختبار القبلي‬ Q\ Enumerate factors effecting in bacterial growth? Scientific Content: :‫المحتوى العلمي‬ Microbial Growth and Metabolism The division of a bacterial cell occurs mainly through binary, or transverse, fission; binary means that one cell becomes two, and transverse refers to the division plane forming across the width of the cell. During binary fission, the parent cell enlarges, duplicates its chromosome, and forms a central transverse septum that divides the cell into two daughter cells. This process is repeated at intervals by each new daughter cell in turn, and with each successive round of division, the population increases in size, number, and mass.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Pretest: :‫االختبار القبلي‬ Q\ Enumerate steps of binary fission of a rod shaped bacterium? STAGES IN THE NORMAL GROWTH CURVE The system of batch culturing described as closed, meaning that nutrients and space are finite and there is no mechanism for the removal of waste products. Data from an entire growth period of 3 to 4 days typically produce a curve with a series of phases termed 1- The lag phase 2- The exponential growth (log) phase 3- The stationary phase 4- The death phase 1- The lag phase is a relatively “flat” period on the graph when the population appears not to be growing or is growing at less than the exponential rate. Growth lags primarily because: (a) The newly inoculated cells require a period of adjustment, enlargement, and synthesis; (b) the cells are not yet multiplying at their maximum rate; and (c) the population of cells is so sparse or dilute that the sampling misses them. The length of the lag period varies somewhat from one population to another. The cells reach the maximum rate of cell division during the  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ 2-exponential growth (log) phase, a period during which the curve increases geometrically. This phase will continue as long as cells have adequate nutrients and the environment is favorable. 3-stationary growth phase, the population enters a survival mode in which cells stop growing or grow slowly. The curve levels off because the rate of cell inhibition or death balances out the rate of multiplication. The decline in the growth rate is caused by depleted nutrients and oxygen, excretion of organic acids and other biochemical pollutants into the growth medium, and an increased density of cells. As the limiting factors intensify, cells begin to die in exponential numbers and they are unable to multiply. 4-death phase The curve now dips downward as the death. The speed with which death occurs depends on the relative resistance of the species and how toxic the conditions are, but it is usually slower than the exponential growth phase. Viable cells often remain many weeks and months after this phase has begun. Surface growth If a single bacterial cell is placed on a solid nutrient agar surface progressive of this cell remain close to the site of deposition and eventually form a compact macroscopic mass of cells called colony. For rapidly growing species over night  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ incubation at 30 to 37 is sufficient to produce millions of cells. The colonies morphology include color, shape, adherence, smell, and surface texture can used guide for identification of the bacterial species. Cell numbers can be counted directly by 1- a microscope counting chamber. 2- Coulter counter or flow cytometer. 3- Cell growth can also be determined by turbidometry. 4- total cell count. The Meaning of Bacterial Death For a microbial cell, death means the irreversible loss of the ability to reproduce (grow and divide). the empirical test of death is culture of cells on solid media: A cell is considered dead if it fails to give rise to a colony on appropriate medium. Obviously, then, the reliability of the test depends on the choice of medium and conditions: For example, a culture in which 99% of the cells appear “dead” in terms of the ability to form colonies on one medium may prove to be 100% viable if tested on another medium. Furthermore, the detection of a few viable cells in a large clinical specimen may not be possible by directly plating a sample because the sample fluid itself may be inhibitory to microbial growth. In such cases, the sample may have to be diluted first into liquid medium, permitting the outgrowth of viable cells before plating. The conditions of incubation in the first hour after treatment are also critical in the determination of “killing.” For example, if bacterial cells are irradiated with ultraviolet light and plated immediately on any medium, it may appear that 99.99% of the cells have been killed. If such irradiated cells are first incubated in a suitable medium for 20 minutes, plating may indicate only 10% killing. In other words, irradiation determines that a cell will “die” if plated immediately but will live if allowed to repair radiation damage before plating. A microbial cell that is not physically disrupted is thus “dead” only in terms of the conditions used to test viability. Post test Q\ Enumerate steps of growth curve?  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ References 1- Foundations in Microbiology 4th Edition Kathleen Park Talaro Pasadena City College Arthur Talaro Pasadena City College 2001 2- Todar's Online Textbook of Bacteriology Dedication to Hans Zinsser 2005 3- Different size, shape and arrangement of bacterial cell 2022 4- Prokaryotic cells structure, function, and definition 2019 5- Bailey & Scott’s Diagnostic Microbiology Fourteenth Edition Patricia M. Tille, PhD, BS, MT(ASCP), FACSc 2017 6-Jawetz, Melnick, & Adelberg’s 2019 Medical Microbiology Twenty-Eighth Edition  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Title: :‫العنوان‬ Bacterial Cultivation Lec 5 Name of the instructor: :‫اسم المحاضر‬ Dr. Mohammed Flaih ‫د محمد فليح طريف‬.‫ا‬ Target population: :‫الفئة المستهدفة‬ Second stage students Introduction: :‫المقدمة‬ Biologists studying large organisms such as animals and plants can, for the most part, immediately see and differentiate their experimental subjects from the surrounding environment and from another. In fact, they can use their senses of sight, smell, hearing, and even touch to detect and evaluate identifying characteristics and to keep track of growth and developmental changes. First, most habitats (such as the soil and the human mouth) harbor microbes in complex associations, so it is often necessary to separate the species from one another. Second, to maintain and keep track of such small research subjects, microbiologists usually have to grow them under artificial conditions. A third difficulty in working with microbes is that they are invisible and widely distributed, and undesirable ones can be introduced into an experiment and cause misleading results.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Scientific content: :‫المحتوى العلمي‬ Microbiologists use five basic techniques to manipulate, grow, examine, and characterize microorganisms in the laboratory these are : 1-inoculation 2- incubation 3- isolation 4-inspection 5-identification  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫المرحلة ‪:‬الثانية‬ ‫المادة ‪:‬األحياء المجهرية‬ ‫قسم تقنيات المختبرات الطبية‬  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Types of culture media used in microbiology Media contains:  carbon source e.g., Glucose  various salts and minerals  amino acids and nitrogen source e.g., beef, yeast extract  water Media are of different types on consistency and chemical composition. A. On Consistency: 1. Solid Media. Advantages of solid media: (a) Bacteria may be identified by studying the colony character, (b) Mixed bacteria can be separated. Solid media is used for the isolation of bacteria as pure culture. 'Agar' is most commonly used to prepare solid media. Agar is polysaccharide extract obtained from seaweed. Agar is an ideal solidifying agent in 1.5-2.5% percentage as it is : (a) Bacteriologically inert, i.e. no influence on bacterial growth, (b) It remains solid at 37°C, and (c) It is transparent.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ 2. Liquid Media. It is used for profuse growth, e.g. blood culture in liquid media, without agar. Mixed organisms cannot be separated. 3. Semi-solid. it is used for detected motile bacteria, in 0.5% agar B. On Chemical Composition : 1. Routine Laboratory Media 2. Synthetic Media. These are chemically defined media prepared from pure chemical substances. It is used in research work. ROUTINE LABORATORY MEDIA These are classified into six types: (1) Basal media, (2) Enriched media, (3) Selective media, (4) Indicator media, (5) Transport media, and (6) Storage media. 1. BASAL MEDIA. Basal media are those that may be used for growth (culture) of bacteria that do not need enrichment of the media. Examples: Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media. 2. ENRICHED MEDIA. The media are enriched usually by adding blood, serum or egg. Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar media. 3. SELECTIVE MEDIA. These media favour the growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey agar, Lowenstein-Jensen media, tellurite media (Tellurite inhibits the growth of most of the throat organisms except diphtheria bacilli). Antibiotic may be added to a medium for inhibition.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ 4. INDICATOR (DIFFERENTIAL) MEDIA. An indicator is included in the medium. A particular organism causes change in the indicator, e.g. blood, neutral red, tellurite. Examples: Blood agar and MacConkey agar are indicator media. 5. TRANSPORT MEDIA. These media are used when specie-men cannot be cultured soon after collection. Examples: Cary-Blair medium, Amies medium, Stuart medium. 6. STORAGE MEDIA. Media used for storing the bacteria for a long period of time. Examples: Egg saline medium, chalk cooked meat broth, Brain heart infusion broth. COMMON MEDIA IN ROUTINE USE A- Nutrient Broth. Uses: (1) As a basal media for the preparation of other media, (2) To study soluble products of bacteria. B- Nutrient Agar. It is solid at 37°C. C- Peptone Water. Peptone 1% and sodium chloride 0.5%. It is used as base for sugar media and to test indole formation. D- Blood Agar. Most commonly used medium. 5- 10% defibrinated sheep or horse blood is added to melted agar at 45-50°C. Blood acts as an enrichment material and also as an indicator. Certain bacteria when grown in blood agar produce haemolysis around their colonies. Certain bacteria produce no haemolysis. Types of changes : (a) beta β-haemolysis. The colony is surrounded by a clear zone of complete haemolysis, e.g. Streptococcus pyogenes is a beta haemolytic streptococci, (b) Alpha α-haemolysis. The colony is surrounded by a zone of greenish discolouration due to formation of biliverdin, e.g. Viridans streptococci, (c) Gamma γ-haemolysis, or, No haemolysis. There is no change in the medium surrounding the colony, E- Chocolate Agar or Heated Blood agar: Prepared by heating blood agar. It is used for culture of pneumococcus, gonococcus, meningococcus and Haemophilus. Heating the blood inactivates inhibitor of growths.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ F- MacConkey Agar. Most commonly used for enterobacteriaceae. It It is a selective and indicator medium : (1) Selective as bile salt does not inhibit the growth of enterobactericeae but inhibits growth of many other bacteria. (2) Indicator medium as the colonies of bacteria that ferment lactose take a pink colour due to production of acid. Acid turns the indicator neutral red to pink. These bacteria are called 'lactose fermenter', e.g. Escherichia coli. Yellow or Colorless colony indicates that lactose is not fermented, e.g. Salmonella. Shigella, Vibrio. G- Mueller Hinton Agar. Disc diffusion sensitivity tests for antimicrobial drugs should be carried out on this media as per WHO recommendation to promote reproducibility and comparability of results. Post test: :‫االختبار البعدي‬ Q/ Write types of media classified depended consistency? References 1- Foundations in Microbiology 4th Edition Kathleen Park Talaro Pasadena City College Arthur Talaro Pasadena City College 2001 2- Todar's Online Textbook of Bacteriology Dedication to Hans Zinsser 2005 3- Different size, shape and arrangement of bacterial cell 2022 4- Prokaryotic cells structure, function, and definition 2019 5- Bailey & Scott’s Diagnostic Microbiology Fourteenth Edition Patricia M. Tille, PhD, BS, MT(ASCP), FACSc 2017 6-Jawetz, Melnick, & Adelberg’s 2019 Medical Microbiology Twenty-Eighth Edition  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Title Metabolism (energy production ) Lec 6,7 Name of the instructor: :‫اسم المحاضر‬ Estabraq Ali AL-Sodani ‫ استبرق علي السوداني‬.‫د‬ Target population: :‫الفئة المستهدفة‬ Second stage students Introduction: :‫المقدمة‬ 1-Aerobic Respiration Aerobic respiration is a series of enzyme-catalyzed reactions in which electrons are transferred from fuel molecules such as glucose to oxygen as a final electron acceptor. This pathway is the principal energy-yielding scheme for aerobic heterotrophs, and it provides both ATP and metabolic intermediates for many other pathways in the cell, including those of protein, lipid, and carbohydrate synthesis Aerobic respiration in microorganisms can be summarized by an equation: Glucose (C6H12O6) _ 6 O2 _ 38 ADP _ 38 Pi →6 CO2 _ 6 H2O _ 38 ATP 2-Anaerobic respiration Some bacteria have evolved an anaerobic respiratory system that functions like the aerobic cytochrome system except that it utilizes oxygen-containing salts, rather than free oxygen, as the final electron acceptor. Of these, the nitrate (NO3_) and nitrite (NO2_) reduction systems are best known. The reaction in species such as Escherichia coli is represented as: TABLE 8.4 Nitrate reductase NO3+NADH → NO2+H2O +NAD_2+ 3-Fermentation Of all the results of pyruvate metabolism, probably the most varied is fermentation. Technically speaking, fermentation* is the incomplete oxidation of glucose or  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ other carbohydrates in the absence of oxygen, a process that uses organic compounds as the terminal electron acceptors and yields a small amount of ATP. Fermentation is also what bacteriologists call the formation of acid, gas, and other products by the action of various bacteria on pyruvic acid. The process is a common metabolic strategy among bacteria. Scientific content: :‫المحتوى العلمي‬ The Metabolism of Microbes Metabolism, from the Greek term metaballein, meaning change, pertains to all chemical reactions and physical workings of the cell. Although metabolism entails thousands of different reactions, most of them fall into one of two general categories. Anabolism,* sometimes also called biosynthesis, is any process that results in synthesis of cell molecules and structures. It is a building and bond making process that forms larger molecules from smaller ones, and it usually requires the input of energy. Catabolism* is the opposite, or complement, of anabolism. Catabolic reactions are degradative; they break bonds, convert larger molecules into smaller components, and often produce energy.The linking of anabolism to catabolism ensures the efficient completion of many thousands of cellular processes. Metabolism is a self-regulatory process that maintains the stability of the cell. Metabolism of nutrients can extract energy in the form of adenosine triphosphate (ATP), or other high energy compounds, that can be channeled into such processes as biosynthesis, transport, growth, and motility.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ ENZYMES: CATALYZING THE CHEMICAL REACTIONS OF LIFE chemical reactions of life, even when highly organized and complex, cannot proceed without a special class of proteins called enzymes.*Enzymes are a remarkable example of catalysts,* chemicals that increase the rate of a chemical reaction without becoming part of the products or being consumed in the reaction. Do not make the mistake of thinking that an enzyme creates a reaction. Because of the great energy of some molecules, a reaction could occur spontaneously at some point even without an enzyme, but at a very slow rate. enzymes, which speed up the rate of reactions, are necessary to life. How Do Enzymes Work? We have said that an enzyme speeds up the rate of a metabolic reaction, but just how does it do this? During a chemical reaction, reactants are converted to products by bond formation or breakage. A certain amount of energy is required to initiate every such reaction, which limits its rate. This resistance to a reaction, which must be overcome for a reaction to proceed, is measurable and is called the energy of activation. At the molecular level, an enzyme promotes a reaction by serving as a physical site upon which the reactant molecules, called substrate. Enzyme Structure The primary structure of all enzymes is protein (with some exceptions— Microbits), and they can be classified as simple or conjugated.1- Simple enzymes consist of protein alone, whereas 2-conjugated enzymes contain protein and non protein molecules. A conjugated enzyme, sometimes referred to as a holoenzyme,* is a combination of a protein, now called the apoenzyme, and one or more cofactors. Cofactors are either organic molecules, called coenzymes, or inorganic  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ elements( metal ions). The Role of Microbial Enzymes in Disease Many pathogens secrete unique exoenzymes that help them avoid host defenses or promote their multiplication in tissues. Because these enzymes contribute to pathogenicity, they are referred to as virulence factors, or toxins in some cases. 1-Streptococcus pyogenes (a cause of throat and skin infections) produces a streptokinase that digests blood clots and apparently assists in invasion of wounds. 2-Pseudomonas aeruginosa, a respiratory and skin pathogen, produces elastase and collagenase, which digest elastin and collagen. These increase the severity of certain lung diseases and burn infections. 3-Clostridium perfringens, an agent of gas gangrene, synthesizes lecithinase C, a lipase that profoundly damages cell membranes and accounts for the tissue death associated with this disease. The Sensitivity of Enzymes to Their Environment The activity of an enzyme is highly influenced by the cell’s environment. In general, enzymes operate only under the natural temperature, pH, and osmotic pressure of an organism’s habitat. When enzymes are subjected to changes in these normal conditions, they tend to be chemically unstable, or labile. Low temperatures inhibit catalysis, and high temperatures denature theapoenzyme. Denaturation is a process by which the weak bonds that collectively maintain the native shape of the apoenzyme are broken. This disruption causes extreme distortion of the enzyme’s shape and prevents the substrate from attaching to the active site. Such nonfunctional enzymes block metabolic reactions and thereby can lead to cell death. Low or high pH or certain chemicals (heavy metals, alcohol) are also denaturing agents. CELL ENERGETICS Cells manage energy in the form of chemical reactions that change molecules. This often involves activities such as the making or breaking of bonds and the transfer of electrons. Not all cellular reactions are equal with respect to energy. Some release energy, and others require it to proceed. For example, a reaction that proceeds as follows: X +Y Enz Z+Energy exergonic.* Enz Energy +A +B C endergonic,*  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Exergonic and endergonic reactions are coupled, so that released energy is immediately put to use. Summaries of metabolism might make it seem that cells “create” energy from nutrients, but they do not. What they actually do is extract chemical energy already present in nutrient fuels and apply that energy toward useful work in the cell. At the simplest level, cells possess specialized enzyme systems that trap the energy present in the bonds of nutrients as they are progressively broken. During exergonic reactions, energy released by electrons is stored in various high-energy phosphate molecules such as ATP. As we shall see, the ability of ATP to temporarily store and release the energy of chemical bonds fuels endergonic cell reactions. Before discussing ATP, let us examine the process behind electron transfer: redox reactions.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Nutrient cycles and regulation Lec 7 ‫استبرق السوداني‬.‫محمد فليح د‬.‫د‬ Pathways of Bioenergetics One of the scientific community’s greatest achievements was deciphering the biochemical pathways of cells. Initial work with bacteria and yeasts, followed by studies with animal and plant cells, clearly demonstrated metabolic similarities and strongly supported the concept of the universality of metabolism. The study of the production and use of energy by cells is called bioenergetics, including catabolic routes that degrade nutrients and anabolic routes that are involved in cell synthesis. Acquisition of Nutrients Bacteria use various strategies for obtaining essential nutrients from the external environment and transporting these substances into the cell’s interior. For nutrients to be internalized, they must cross the bacterial cell wall and membrane. These complex structures help protect the cell from environmental insults, maintain intracellular equilibrium, and transport substances into and out of the cell. Although some key nutrients (e.g., water, oxygen, and carbon dioxide) enter the cell by simple diffusion across the cell membrane, the uptake of other substances is controlled by membrane selective permeability; still other substances use specific transport mechanisms. Active transport is among the most common methods used for the uptake of nutrients such as certain sugars, most amino acids, organic acids, and many inorganic ions. The mechanism, driven by an energy-dependent pump, involves carrier molecules embedded in the membrane portion of the cell structure. These carriers combine with the nutrients, transport them across the membrane, and release them inside the cell. Group translocation is another transport mechanism that requires energy but differs from active transport in that the nutrients being transported undergoes chemical modification. Many sugars, purines, pyrimidines, and fatty acids are transported by this mechanism. Production of Precursor Metabolites  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Once inside the cell, many nutrients serve as the raw materials from which precursor metabolites for subsequent biosynthetic processes are produced. These metabolites, listed in the Figure below, are produced through two central pathways: the Embden-Meyerhof-Parnas (EMP) pathway (glycolysis) and the tricarboxylic acid (TCA) cycle.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫المرحلة ‪:‬الثانية‬ ‫المادة ‪:‬األحياء المجهرية‬ ‫قسم تقنيات المختبرات الطبية‬  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ The two major pathways and their relationship to one another are shown in this Figure, also the figure not shown are the alternative pathways (e.g., the Entner- Doudoroff and the pentose phosphate pathway) that play key roles in redirecting and replenishing the precursors as they are used in subsequent processes. The Entner-Doudoroff pathway catalyzes the degradation of gluconate and glucose. The gluconate is phosphorylated, dehydrated, and converted into pyruvate and glyceraldehyde, leading to ethanol production. Alternatively, the pentose phosphate pathway uses glucose to produce reduced nicotinamide adenine dinucleotide phosphate (NADPH), pentoses, and tetroses for biosynthetic reactions such as nucleoside and amino acid synthesis. The production efficiency of a bacterial cell resulting from these precursor- producing pathways can vary substantially, depending on the growth conditions and availability of nutrients. This is an important consideration, because the accurate identification of medically important bacteria often depends heavily on methods that measure the presence of products and byproducts of these metabolic pathways.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Energy Production The third type of fueling pathway is one that produces the energy required for nearly all cellular processes, including nutrient uptake and precursor production. Energy production is accomplished by the breakdown of chemical substrates (i.e., chemical energy) through the degradative process of catabolism coupled with oxidation-reduction reactions. In this process, the energy source molecule (i.e., substrate) is oxidized as it donates electrons to an electronacceptor molecule, which is then reduced. The transfer of electrons is mediated through carrier molecules, such as nicotinamide-adenine- dinucleotide (NAD_) and nicotinamide- adenine-dinucleotide-phosphate (NADP_). The energy released by the oxidation-reduction reaction is transferred to phosphate-containing compounds, where highenergy phosphate bonds are formed. ATP is the most common of such molecules. The energy contained in this compound is eventually released by the hydrolysis of ATP under controlled conditions. The release of this chemical energy, coupled with enzymatic activities, specifically catalyzes each biochemical reaction in the cell and drives cellular reactions. The two general mechanisms for ATP production in bacterial cells are substrate- level phosphorylation and electron transport, also referred to as oxidative phosphorylation. In substrate-level phosphorylation, high-energy phosphate bonds produced by the central pathways are donated to adenosine diphosphate (ADP) to form ATP directly from the substrate as opposed to generation via the electron transport chain. In addition, pyruvate, a primary intermediate in the central pathways, serves as the initial substrate for several other pathways to generate ATP by substrate- level phosphorylation. These other pathways constitute fermentative metabolism, which does not require oxygen and produces various end products, including alcohols, acids, carbon dioxide, and hydrogen.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ The specific fermentative pathways and the end products produced vary with different bacterial species. Detection of these products is an important basis for laboratory identification of bacteria. Oxidative Phosphorylation Oxidative phosphorylation involves an electron transport system that conducts a series of electron transfers from reduced carrier molecules such as NADH2, NADPH2 and FADH2 (flavin adenine dinucleotide), produced in the central pathways (as shown in the Figure below), to a terminal electron acceptor.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫المرحلة ‪:‬الثانية‬ ‫المادة ‪:‬األحياء المجهرية‬ ‫قسم تقنيات المختبرات الطبية‬  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫المرحلة ‪:‬الثانية‬ ‫المادة ‪:‬األحياء المجهرية‬ ‫قسم تقنيات المختبرات الطبية‬  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ There are several important facets of aerobic respiration: 1. The total yield of ATP is 40: 4 from glycolysis, 2 from the TCA cycle, and 34 from electron transport. However, since 2 ATPs were expended in early glycolysis, this leaves a maximum of 38 ATPs.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ 2. Six carbon dioxide molecules are generated during the TCA cycle. 3. Six oxygen molecules are consumed during electron transport. 4. Six water molecules are produced in electron transport and 2 in glycolysis,but because 2 are used in the TCA cycle, this leaves a net number of 6. The energy produced by the series of oxidation-reduction reactions is used to generate ATP from ADP. When oxidative phosphorylation uses oxygen as the terminal electron acceptor, the process is known as aerobic respiration. Anaerobic respiration refers to processes that use final electron acceptors other than oxygen. A knowledge of which mechanisms bacteria use to generate ATP is important for designing laboratory protocols for cultivating and identifying these organisms. For example, 1- some bacteria depend solely on aerobic respiration and are unable to grow in the absence of oxygen (strictly aerobic bacteria). 2- Others can use either aerobic respiration or fermentation, depending on the availability of oxygen (facultative anaerobic bacteria). 3- For still others, oxygen is absolutely toxic (strictly anaerobic bacteria). Biosynthesis The fueling reactions essentially bring together all the raw materials needed to initiate and maintain all other cellular processes. The production of precursors and energy is accomplished through catabolic processes and the degradation of substrate molecules. The three remaining pathways for biosynthesis, polymerization, and assembly depend on anabolic metabolism. In anabolic metabolism, precursor compounds are joined for the creation of larger molecules (polymers) required for assembly of cellular structures (Figure 2-11). Biosynthetic processes use the precursor products in dozens of pathways to produce a variety of building blocks, such as amino acids, fatty acids, sugars, and nucleotides (Figure 2-11). Many of these pathways are highly complex and interdependent, whereas other pathways are completely independent. In many cases, the enzymes that drive the individual pathways are encoded on a single mRNA molecule that has been transcribed from contiguous genes in the bacterial chromosome (i.e., an operon).  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ As previously mentioned, bacterial genera and species vary extensively in their biosynthetic capabilities. Knowledge of these variations is necessary to use optimal conditions for growing organisms under laboratory conditions. For example, some organisms may not be capable of synthesizing an essential amino acid necessary as a building block for proteins. Without the ability to synthesize the amino acid, the bacterium must obtain the building block from the environment. Thus if the organism is cultivated in the microbiology laboratory, the amino acid must be provided in the culture medium. Polymerization and Assembly Various anabolic reactions assemble (polymerize) the building blocks into macromolecules, including lipids, lipopolysaccharides, polysaccharides, proteins, and nucleic acids. This synthesis of macromolecules is driven by energy and enzymatic activity in the cell. Similarly, energy and enzymatic activities also drive the assembly of various macromolecules into the component structures of the bacterial cell. Cellular structures are the product of all the genetic and metabolic processes discussed. Post test: :‫االختبار البعدي‬ Reference: :‫المصادر‬  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Title: :‫العنوان‬ Bacterial genetics Lec 8 Name of the instructor: :‫اسم المحاضر‬ Estabraq Ali AL-Sodani ‫ استبرق علي السوداني‬.‫د‬ Target population: :‫الفئة المستهدفة‬ Second stage students Introduction: :‫المقدمة‬ Genetics* is the study of the inheritance, or heredity,* of living things. It is a wide-ranging science that explores the transmission of biological properties (traits) from parent to offspring, the expression and variation of those traits, the structure and function of the genetic material, and how this material changes. Organism genetics observes the heredity of the whole organism or cell; chromosome genetics examines the characteristics and actions of chromosomes; and molecular genetics deals with the biochemistry of the genes. All of these levels are useful areas of exploration, but in order to understand the expressions of microbial structure, physiology, mutations, and pathogenicity, we need to examine the operation of genes at the cellular and molecular levels. The study of microbial genetics provides a greater understanding of human genetics and an increased appreciation for the astounding advances in genetic engineering we are currently witnessing. Pre test: :‫االختبار القبلي‬ Q\ Define Genetics and gene?  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ Scientific content: :‫المحتوى العلمي‬ THE NATURE OF THE GENETIC MATERIAL For a species to survive, it must have the capacity of self replication. In single- celled microorganisms, reproduction involves the division of the cell by means of binary fission, budding, or mitosis, but these forms of reproduction involve a more significant activity than just simple cleavage of the cell mass. Because the genetic material is responsible for inheritance, it must be accurately duplicated and separated into each daughter cell to ensure its normal function. The Levels of Structure and Function of the Genome The genome is the sum total of genetic material of a cell. Although most of the genome exists in the form of chromosomes, genetic material can appear in non chromosomal sites as well. For example, bacteria and some fungi contain tiny extra pieces of DNA (plasmids), and certain organelles of eucaryotes (the mitochondria and chloroplasts) are equipped with their own genetic programs. Genomes of cells are composed exclusively of DNA, but viruses contain either DNA or RNA as the principal genetic material. Although the specific genome of an individual organism is unique, the general pattern of nucleic acid structure and function is similar among all organisms. In general, a chromosome is a discrete cellular structure composed of a neatly packaged elongate DNA molecule. The chromosomes of eucaryotes and bacterial cells differ in several respects. 1- The structure of eucaryotic chromosomes consists of a DNA molecule tightly wound around histone proteins, whereas a bacterial chromosome (chromatin body) is condensed and secured into a packet by means of histone like proteins. 2-Eucaryotic chromosomes are located in the nucleus; they vary in number from a few to hundreds; they can occur in pairs(diploid) or singles (haploid); and they appear elongate. In contrast, most bacteria have a single, circular chromosome, although exceptions exist in a few bacteria that have linear or multiple chromosomes. The chromosomes of all cells are subdivided into basic informational packets called genes.  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ A gene can be defined from more than one perspective. In classical genetics, the term refers to the fundamental unit of heredity responsible for a given trait in an organism. In the molecular and biochemical sense, it is a site on the chromosome that provides information for a certain cell function. More specifically still, it is a certain segment of DNA that contains the necessary code to make a protein or RNA molecule. For an analogy that clarifies the relationship of the genome, chromosomes, genes, and DNA. Nonchromosomal Elements Although the bacterial chromosome represents the majority of a cell's genome, not all genes are confined to the chromosome. Many genes may also be located on plasmids and transposable elements. Both of these extrachromosomal elements are able to replicate and encode information for the production of various cellular products. Many of these elements replicate by integration into the host chromosome, whereas others, referred to as episomes, are capable of replication independently of the host chromosome. Although considered part of the bacterial genome, they are not as stable as the chromosome and may be lost during cellular replication, often without any detrimental effects on the viability of the cell. Plasmids exist as double-stranded, closed, circular, autonomously replicating extrachromosomal genetic elements ranging in size from 1 to 2 kilobases up to 1 megabase or more. The number of plasmids per bacterial cell varies extensively, and each plasmid is composed of several genes. Some genes encode products that mediate plasmid replication and transfer between bacterial cells, whereas others encode products that provide a specialized function, such as a determinant of antimicrobial resistance or a unique  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ metabolic process. Unlike most chromosomal genes, plasmid genes do not usually encode for products essential for viability. Plasmids, in whole or in part, may also become incorporated into the chromosome. Transposable elements are pieces of DNA that move from one genetic element to another, from plasmid to chromosome or vice versa. Unlike plasmids, many are unable to replicate independently and do not exist as separate entities in the bacterial cell. The two types of transposable elements are the simple transposon or insertion sequence (IS) and the composite transposon. Insertion sequences are limited to containing the genes that encode information required for movement from one site in the genome to another. Composite transposons are cassettes (grouping of genes) flanked by insertion sequences. The internal gene embedded in the insertion sequence encodes for an accessory function, such as antimicrobial resistance. Plasmids and transposable elements coexist with chromosomes in the cells of many bacterial species. These extrachromosomal elements play a key role in the exchange of genetic material throughout the bacterial microbiosphere, including genetic exchange among clinically relevant bacteria. Replication and Expression of Genetic Information Replication Bacteria multiply by binary fission (a form of cell division), resulting in the production of two daughter cells from one parent cell. As part of this process, the genome must be replicated so that each daughter cell receives an identical copy of functional DNA. Replication is a complex process mediated by various enzymes, such as DNA polymerase and cofactors; replication must occur quickly and accurately. For descriptive purposes, replication may be considered in four stages as show in figure  ‫الجامعة التقنية الوسطى‬ ‫كلية التقنيات الصحية والطبية \بغداد‬ ‫الثانية‬: ‫المرحلة‬ ‫األحياء المجهرية‬: ‫المادة‬ ‫قسم تقنيات المختبرات الطبية‬ 1. Unwinding or relaxation of the chromosome’s supercoiled DNA 2. Separation of the complementary strands of the parental DNA so that each may serve as a template (i.e., pattern) for synthesis of new DNA strands, referred to as semiconservative replication 3. Synthesis of the new (i.e., daughter) DNA strands 4. Termination of replication, releasing two identical chromosomes, one for each daughter cell. Relaxation of supercoiled chromosomal DNA is required so that enzymes and cofactors involved in replication can access the DNA molecule at the site where the replication process will originate (i.e., origin of replication). The 1-origin of replication (a specific sequence of approximately 300 base pairs). 2-replication fork two bidirectional forks are involved in the replication process. 3-DNA polymerase playing a central role.Using each parental strand as a template, DNA polymerase adds nucleotide bases to each growing daughter strand in sequence that is complementary to the base sequence of the template (parent) strand. The process generally takes approximately 20 to 40 minutes in rapidly growing bacteria such as E. coli. The replication time for a particular bacterial strain can vary depending on environmental conditions, such as the availability of nutrients or the presence of toxic substances (e.g., antimicrobial agents). Genetic alterations and diversity in bacteria are accomplished by three basic mechanisms: mutation, genetic recombination, and genetic exchange between bacteria, with or without recombination. Mutation Mutation is defined as an alteration in the original nucleotide sequence of a gene or genes within an organism’s genome; that is, a change in the organism’s genotype. This alteration may involve a single DNA base in a gene, an entire gene, or several genes. Mutational changes in the sequence may arise spontaneously, perhaps by an error made during DNA replication. Alternatively, mutations may be induced by mutagens (i.e., chemical or physical factors) in the environment or by biologic factors, such as the introduction of foreign DNA into the cell. Alterations in the DNA base sequence can re