Microbiology.pdf

MICROBIOLOGY And infection control Faculty of Nursing Sawsan A What are microbes? Definition By the name Micro = small, bios = life and ology = science. Thus, Microbiology is the science that study the living organisms that are individually t...

MICROBIOLOGY And infection control Faculty of Nursing Sawsan A What are microbes? Definition By the name Micro = small, bios = life and ology = science. Thus, Microbiology is the science that study the living organisms that are individually too small to be seen by the necked eye. Disciplines of Microbiology Basic microbiology; deals with fundamental and basic studies of microorganisms. Medical microbiology; concern the study of organisms which can produce different forms of infections and also deals with the study of the host reaction to such infections. Pharmaceutical microbiology; dealing with development of drugs, and their activities on M.O, production of certain pharmaceutical products by using microorganisms such as antibiotics, organic acids, amino acids, immunological and diagnostic agents. Veterinary microbiology; study of animal infectious microorganisms. Agriculture microbiology; concerns microorganisms which significantly affect plants such as those causing plant infection. Food microbiology; deals with microorganisms affecting foods either causing spoilage or affecting fermentation or flavors. Also, concern with food borne diseases. Ecological microbiology; study of the distribution and the effect of microorganisms on the environment. Microbiology can also subdivide according to the type of microorganisms into:  Bacteriology; deals with the study of bacteria.  Virology; study of viruses.  Mycology; study of fungi.  Phycology; study of algae.  Protozoology; study of protozoa. Microbiology is closely associated with other sciences such as:  Immunology; the science that deals with the host and parasite relationship and in other words how the host response to a foreign body.  Parasitology; deals with parasites and parasitic insects.  Biotechnology; means the use of microorganisms in the industries.   Pathology; deals with the diseases  Genetic engineering; manipulation of microorganisms for the production of human proteins using recombinant DNA technology. Nomenclature &taxonomy: General speaking, microorganisms are classified into Kingdoms, Divisions (phylum), Classes, Orders, Family, Genera and Species. The species can be classified into strains, subspecies. The strain is a progeny of a single cell that are genetically identical  The first letter of the genus must be in uppercase and the species must in lowercase style.  The Genus and species should be underlined or italicized. In many cases the genus name could be abbreviated to one or more letters according the international system of nomenclature.  Linnaeus classification: Before the discovery of m.o, all living creatures were classified into two kingdoms acc. To physiological and anatomical characters. Kingdom animals and plants.  Haeckel classification: after the discovery of M,O, all M.O were grouped into kingdom Protista.  Whittaker classification: Living organisms have been classified into five kingdoms namely: 1. Kingdom Animalia; concerns animals. 2. Kingdom Plantae; plants and multicellular algae. 3. Kingdom Protista; protozoa and single-celled algae. 4. Kingdom Fungi; molds and yeasts. 5. Kingdom Monera; includes only bacteria (Eubacteria, Archaebacteria and Cyanobacteria). Microorganisms occupy three kingdoms: 1. Kingdom Protista; protozoa and single-celled algae. 2. Kingdom Fungi; molds and yeasts. 3. Kingdom Monera: only bacteria  Comparison between eukaryotes and prokaryotes: Character Prokaryotic cell Eukaryotic cell Size Smaller Larger Defined nucleus Absent Present Histone proteins Absent Present chromosomes Single/circular Multiple/linear Cytoplasmic membrane Contain no sterols Contains sterols ribosomes Small type Sedimentation Larger type Sedimentation coefficient of 70s coefficient of 80 s Distributed in cytoplasm Array on ER Endoplasmic reticulum Absent Present reproduction A sexual by binary fission Sexual and or Asexual Mitochondria Absent Present Golgi app. Absent Present Cell wall peptidoglycan Chitin /cellulose Gas vacuoles Present in cyanobacteria Absent Pioneers in Microbiology o Antonie Van Leeuwenhoeek He was the first who had seen and described microbes using simple microscope. He referred them as animalcules. o Joseph Lister He discovered antiseptics. o Tyndall He introduced Tyndallization “Intermittent sterilization” as a method of sterilization. o Louis Pasteur Known as the father of Bacteriology, his achievements were: *Disproving theory of spontaneous generation of life. *Discovery of anaerobic microorganisms. *Discovery of microorganisms as causative agents of diseases *Pasteurization *Fermentation *Vaccination o Robert Koch He was a contemporary of and second only to Pasteur as a great contributor to bacteriology. He was the first to isolate anthrax bacilli in pure cultures (1876). He perfected the techniques of staining of bacteria (1881). He introduced solid nutrient culture media for purification of bacteria. The first to use of oil immersion lens. o Loeffler He discovered the causative agent of Diphtheria. o Fleming He discovered penicillin in 1928, however, the true value of penicillin was determined only during the Second World War (1940), and many lives were saved that it was called Miracle drug. o Domagk He introduced the sulfonamides. Size of microbes: Virus 10 →1000 nanometers Bacteria 0.1 → 5 micrometers Tools of Microbiology Compound light Microscope - live specimens - Electron Microscope - non-living specimens - Incubator – keep microbes warm for growth Techniques of Microbiology Staining – to better see structures Microbial Culture: - growing Container for microbe culture - usually Petri dish Culture media: - Food for the microbes - E.g. Agar – (from red algae) - Others such as nutrient broths. Pure Culture Techniques -Inoculation. – Isolation. - Identification Classification of Microorganisms: Microbes can be classified into four major groups: 1- Protozoa. 2- Bacteria. 3- Fungi. 4- Viruses. 1- The Protozoa: These are unicellular organisms with protoplasm differentiated into nucleus and cytoplasm. Diameters in the range of 2-100 μm. The most important groups of medical protozoa are: A-Amoeba: Entamoeba species. Mode of Motility: pseudopodia. B- Mastigophora: Mode of Motility: the Flagella. Gastrointestinal flagellates: Giardia intestinalis Urogenital flagellates: Trichomonas vaginalis C- Ciliophora: motile by cilia. Example: Balantidium coli. D- Sporozoa: intracellular infection. Example: Plasmodium that cause Malaria. 2- The bacteria: Bacteria are unicellular prokaryotic microorganisms that multiply by binary fission. Bacteria can be classified according to morphology, arrangement, and staining reaction into the following groups: 1- Filamentous bacteria: Streptomyces: antibiotic producers. 2- True bacteria: Cocci: Gram positive: Staphylococcus, Streptococcus. Gram negative: Neisseria. Bacilli: Gram positive: Bacillus, Clostridum, Corynebacterium. Gram negative: Enterobacteriaceae, Brucella. 3- Spirochetes: Slender flexuous spiral bacteria. Borrelia, Treponema, Leptospira. 4- Mycoplasma: The Smallest bacteria that lack of a rigid cell wall. 5- Rickettsiae and Chlamydiae: intracellular parasites. 3- The Fungi: These are saprophytic or parasitic organisms possessing relatively rigid cell walls. Medical fungi can be divided into: 1- Mould: 2- True Yeasts 3- Dimorphic fungi 4- Yeast- like fungi 4- The viruses: Viruses consist of DNA or RNA enclosed in a simple protein shell known as a capsid. General properties of viruses: They are very small in size, from 20-300 m. They contain one kind of nucleic acid (RNA or DNA) as their genome. They are metabolically inert. They are obligate intracellular parasites. They are only seen by electron microscope. Depend on the parasitized cell for survival and multiplication Structure of bacterial cells: Size, Shape, and Arrangement of bacterial cells: Morphology and arrangement of bacterial cells are criteria used for classification of bacteria into following groups: 1. Cocci (Singular: coccus). 2. Rods (bacilli), (Singular: rod, bacillus). 3. Vibrios (Singular: vibrio). 4. Spirilla (singular :Spirillum) 5. Spirochetes. (Singular: Spirochaete). 1. Cocci: These are round or oval bacteria measuring about 0.5-1.0 micrometer in diameter. When they multiplying, cocci may form pairs, chains, or irregular groups. Cocci in pairs are called diplococci, for example, meningococci and gonococci. Cocci in chains are called streptococci, for example Streptococcus pyogens. Cocci in irregular groups are called Staphytococci, for example, Staphylococcus aureus. 2. Rods (bacilli): These are stick-like bacteria with rounded, square, or swollen ends. They measure 1- 10 micrometer in length by 0.3-1.0 micrometer in width. It may arrange in: A- Chains, for example, Streptobacillus species. B- Branching chains, for example, lactobacilli. C- Mass together, for example, Mycobacterium leprae. D- Remain attached at various angles resembling Chinese letters, for example, Corynebacterium diphtheria. 3-Vibrios: These are small slightly curved rods measuring 3-4 micrometer in length by 0.5 micrometers in width. Most vibrios are motile with a single flagellum at one end. They show a rapid darting motility. For example: vibrio cholerae. 4-Spirochetes: These are flexible, coiled, motile organism, 6-20 micrometer in length. They progress by rapid body movements. Spirochetes are divided into three main groups: A- Treponemes. B- Borreliae. C- Leptospires.  BACTERIAL Structures Outside the cell wall: Capsule or slime layer. Flagella. Pili. Capsule or (slime layer) A gelatinous layer covering the entire bacterium. It is composed of polysaccharide, except in the BACILLUS ANTHRAX which has a capsule of polymerized D-glutamic acid. if it tightly bound around the cell then it called capsule but if it appears unorganized and more loosely attached, then it called slime layer. Capsule or slime layer is one of the most virulence factors of bacteria, non-capsulated bacteria have lost their ability to produce the capsule are usually nonpathogenic. Capsulated or encapsulated bacteria limits the ability of phagocytes to engulf the bacteria, The glycocalyx also enables some bacteria to adhere to environmental surfaces. Streptococcus mutans, a normal flora bacteria responsible for initiating dental caries, breaks down sucrose into glucose and fructose, an enzyme called glucosyltransferase to convert the glucose to a sticky polysaccharide called dextran that forms its glycocalyx that mutans to adhere to the enamel of the tooth and form plaque. THE Surface layer It consists of a single molecular layer composed of identical proteins or glycoproteins, Act as a coarse molecular sieve. It provides a various selection including functioning as protective coats, molecular sieves and ion traps, involved in surface recognition and cell adhesion, Protect bacteria from harmful enzymes, from changes in pH, from the predatory bacterium B-dellovibrio. Flagella: Flagella are long , whiplike appendages that move the bacteria toward food and other attractants, a process called chemotaxis. The long filament , which acts as a propeller, is composed of many subunits of a single protein, flagellin, arranged in several intertwined chains. The energy for movement, the proton motive force, is provided by adenosine triphosphate (ATP), derived from the passage of ions across the membrane. Bacteria posing no flagella are nonmotile. A-Monotrichous (single flagellum) e.g., Vibrio cholers B-Lophotrichous (multiple flagella drive in a single direction) e.g. Helicobacter pylori C-Amphitrichous (single flagellum on each of two opposite ends) D-Kophotrichous (more than one flagellum on each of two opposite ends) E-Peritrichous (have flagella projecting in all directions) e.g. Escherichia coli Axial filaments (spirochetes motility) Spirochetes move by using a flagellum-like structure called axial filament, produce an undulating motion (snake like movement). Pili “pilus” (fimbriae) Extend from the cell surface. They are shorter and straighter than flagella and are composed of subunits of protein called pilin, it mediate the attachment of bacteria to specific receptors on the human cell surface, which is a necessary step in the initiation of infection for some bacteria. A specialized kind of pilus, the sex pilus, forms the attachment between the male (donor) and the female (receptor) bacteria during conjugation (play a role in DNA transfer). Special Bacterial Structures (Endospores or spores) All Cocci bacteria are non-Spore forming bacteria unlike Bacilli (rod-shaped) bacteria, it has spore forming and non-spore forming bacilli. Sporulation (Spore forming bacilli) An endospore is a dormant, tough, and non-reproductive structure produced by bacteria; it ensures the survival of a bacterium through periods of environmental stress. This resistance may be mediated by dipicolinic acid, a calcium chelator found only in spores. Once it starts the sporulation it can live for a long time but cannot perform any activity or reproduction. Germination a specific enzymes degrade the coat, water and nutrients enter then germination into a metabolizing reproducing bacterial cell (vegetative forms) occurs. This differentiation process is not a means of reproduction, since one cell produces one spore that germinate into one cell. Positions of Spores The position of spores in relation to the body of the bacillus may be: central, terminal or subterminal. They differ in shape they may be oval or rounded. The position and shape of spores are characteristic of the species. Bacterial Call Wall The vast majority of bacteria have a cell wall containing a special polymer called peptidoglycan. it is important in defining the shape of the cell, and giving the cell mechanical strength. peptidoglycan layer contains of alternating units of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM). The NAM residues are cross-linked with oligopeptides that end with D-alanine. Gram Reaction (Gramstain named after Christian Gram, who developed the staining protocol) This reaction reveals fundamental differences in the structure of bacteria. Gram's method: crystal violet-iodine complex when treated with Alcohol then washing then adding Safranin red. Gram-positive bacteria appear blue-black or purple The cell wall of GPB lies beyond the cell membrane and is largely made up of peptidoglycan up to 40 layers of this polymer, conferring enormous mechanical strength on the cell wall. Other polymers including teichoic and lipoteichoic acids also lie in the cell walls of GPB. These act as surface antigens. Gram-negative bacteria appear red the outer membrane of GNB lies beyond a One or two layers of peptidoglycan lie beyond the periplasm. It is full of proteins including enzymes. GNB are thus mechanically much weaker than GP cells. Lipopolysaccharides (LPS) Also known as lipoglycans, extending from the outer membrane of the gram- negative bacteria Contains of inner core, outer core and O-antigen repeat. It acts as endotoxins and elicit strong immune responses. O-antigen is also a polysaccharide chain that extends from the core polysaccharide. The presence or absence (more hydrophobic) of O-chains determine whether the LPS is considered smooth or rough. Core oligosaccharide Lipid A Contains unusual fatty acids, it component of an endotoxin held responsible for the toxicity of gram-negative bacteria. When bacterial cells are lysed by the immune system, fragments of membrane containing lipid A are released into the circulation, causing fever, diarrhea, and possible fatal endotoxic shock (also called septic shock). Cell Wall of Acid-Fast Bacteria It has an unusual cell wall “Acid-fast” resulting in their inability to be Gram stained. (Zeihl-Neelsen Stain) method The TB resist decolorization with (stains blue) after being stained with carbolfuchsin (red dye) so, it appears as a red thin line surrounded by the stain blue in the background. And thus, they are stained red. This property is related to the high lipid content of the cell wall (mycolic acids). e.g. Mycobacterium tuberculosis (TB) A highly infectious and highly resistant microorganism slow grower up to 24h.  The peptidoglycan layer is linked to arabinogalactan (D-arabinose and D-galactose) which is linked to high-molecular weight mycolic acids. Mycoplasma A genus of bacteria which naturally lacks the cell wall. they are unaffected by many common antibiotics such as penicillin or other beta- lactam antibiotics that target cell wall synthesis. Several species are pathogenic in humans, including M. pneumoniae, which is an important cause of atypical pneumonia and other respiratory disorders, and M. genitalium, which is believed to be involved in pelvic inflammatory diseases L-form Bacteria Cell wall deficient (CWD) or L-form bacteria Partial or complete loss of the bacterial cell wall artificially. Named “L” for Lister Institute lap where first described by malassez & vignal. Bacteria that have some cell-wall remaining (partial removal of the cell wall) are termed spheroplasts. Bacteria that completely loss all of the bacterial cell wall are termed protoplasts. The cell wall can be removed by the action of cell wall synthesis inhibitors (e.g. penicillin), or by the action of some enzymes that degrade polysaccharides of the cell wall (e.g. lysozyme). Cell membrane (plasma membrane or cytoplasmic membrane) It is the boundary layer of the bacteria cell, it functions in: Transporting nutrients into the cell and waste materials out of the cell. Selective permeability the entry of organic and inorganic substances inside, and prevents the escape of cellular material outside the cell. Anchors DNA during replication. A site for enzymes that function in the cell wall synthesis. A location of enzymes that used for energy production. Secretion of enzymes and toxins. Composition of the cell membrane - 60% protein as a globules like an icebergs “Model: fluid mosaic”, 40% lipid, mainly phospholipid. -When antimicrobial substances act on cell membrane, bacterial death usually follows. - Phospholipid molecules arranged in two parallel layers (Phospholipid bilayer) one inside and one outside of the membrane. - outer and inner hydrophilic surface and hydrophobic layer in btw. Cytoplasm The internal matrix of the cell. It is a gelatinous mass of proteins, carbohydrates, lipids, nucleic acids, salts, inorganic ions and all dissolved in water. Thick, semitransparent and elastic. It is the foundation substance of the cell. It has two distinct areas :1- amorphous matrix. 2- inner nucleoid. Ribosomes -Differ in size and chemical composition from eukaryotic. -The site of protein synthesis. -In prokaryotic: in the cytoplasm, eukaryotic: on endoplasm reticulum. Inclusion bodies Globules of starch glycogen or lipid, store nutrients for later use during periods of starvation. serve as phosphate depots. known as volutin. These bodies stain red with dyes such as methylene blue instead of blue and thus known as “metachromatic”. Their Presence in diphtheria bacilli assists in identification procedures Bacterial Chromosome The bacterial chromosome is a single, in a discrete areas known as the nucleoid. Histones are not required to maintain the conformation of DNA, and the DNA does not form nucleosomes.  Plasmids: are extrachromosomal, double-stranded, circular DNA molecules that are capable of replicating independently of the bacterial chromosome. Plasmids can be integrated into the bacterial chromosome. Plasmids often provide a selective advantage such as conferring resistance to one or more antibiotics. Plasmids are usually found in Gram-negative bacteria. Mesosomes It is important during the cell division. It functions as the origin of the transverse septum that divides the cell in half and as the binding site of the DNA which will become the genetic material of each daughter cell. Bacterial Metabolism Metabolic processes are concerned with all those biological or chemical reactions which can be carried out by microorganisms. Since microorganisms are capable of carrying out chemical reactions, the processes involved must follow the basic lows of thermodynamics and catalysis. In Catabolism 1. Oxidation or destruction of large substrates into smaller units (building units) such as amino acids, monosaccharide, fatty acids… these units will then be used in biosynthesis. 2. Energy will be elaborated and will be stored in adenosine triphosphate (ATP) molecules. In Anabolism 1. The building blocks produced by catabolism are converted to activated monomers and coenzymes this step require energy in the form of ATP. 2. The activated monomers are then polymerized into macromolecule (polysaccharides) Energy in the form of ATP is also required in this step. 3. The polymerized molecules transported to the appropriate area of the Cell and assembled to form the cell structures. The net result will be cell growth. Regulation of Metabolism A large number of microbial enzymes participate in the conversion of carbon and energy source into small building unites and liberation of energy (Cb)  These enzymes are generally synthesized inside the M cell irrespective of the presence of their substrates and “constitutive enzymes”. Some other enzymes, usually concerned in the breakdown of a particular substrate, are not produced by the organism except in the presence of the substrate itself or other compounds of a close chemical structure (inducers). The majority of enzymes are in fact produced only when needed and, in the mount needed. This is generally referred to as enzyme induction, i.e formation of an enzyme only upon stimulation which is triggered by a need. Once the need disappears, synthesis of the enzyme stops. The trigger is often the product of the reaction catalyzed by the enzyme. When this reaches a certain concentration (above what the cell needs) This product represses further synthesis of the enzyme and the phenomenon is called end product repression. The enzymes catalyzing biosynthesis “anabolism” are subject to control by feedback inhibition and repression just as the enzymes concerned in “catabolism” of specific compounds are controlled by induction. In feedback inhibition other end product in the pathway inhibits the action of the first enzyme in the pathway. in repression the end product if it already presents in the growth medium or when it is formed in sufficient quantities. The product influences the extent of activity of the enzyme. High concentration of the product immediately inhibits the activity of enzyme molecules already synthesized and thus the rate of product formation decreases this phenomenon is called “end product inhibition”. Both end product inhibition and repression act only in the first specific Enzyme reaction in a sequence of divergent reactions so that the product does not inhibit formation of other products in the same pathway. High energy transfer compounds It is essential for the life of bacteria that the energy released from Exergonic reactions be used to derive endergonic of coupling exergonic reactions with endergonic reactions. The common reactants of greatest use to the cell are those capable of transferring large amounts of free energy called “high energy transferring compounds”. A variety of such compounds exist in cells. E. ATP, GTP, UTP, PEP. ATP is by far the most important. All the compounds listed before can transfer their energy directly or indirectly to ATP synthesis. Energy Released from ATP by hydrolysis ATP + H2O → ADP + H3PO4; ΔG = -7300 cal. ADP is also a high energy transfer compound, since its hydrolysis yields a large quantity of energy. ADP + H2O → AMP + H3PO4; Δ G = - 7300 cal. AMP however is a low – energy compounds, its hydrolysis liberates only a small quantity of energy. AMP + H2O → Adenosine + H3PO4; Δ G = - 2000 cal. Several types of chemical reactions are involved in energy production, but OXIDATION – REDUCTION is probably the commonest. Carbon Requirement Microorganisms are classified into groups according to CR: 1. Lithotrophic or autotrophic: which are able to utilize CO2 as the main source of carbon. They obtain their energy either by oxidation of simple inorganic compounds (and hence are called chemosynthetic autotrophs) or from sunlight (and hance are called photosynthetic autotrophs). 2. Organotrophic or heterotrophic: which require complex organic compounds as a source of carbon. This group of microorganisms also obtain their energy from oxidation of suitable organic compounds. Energy requirement Only chlorophyll containing organisms can utilize sun light as a source of energy. A few bacteria do, and since they derive the energy required for synthetic reactions from photons. They are described as photosynthetic while most bacteria and all fungi are chemosynthetic since they derive energy from chemical bonds of inorganic compounds or more commonly, of organic compounds. Several groups of bacteria “green and purple bacteria” can perform photosynthesis. In presence of light and green pigment (bacterial chlorophyll) Chemosynthetic bacteria derive energy from chemical bonds by oxidizing (releasing electrons) from nutrients in their environment. Different types of bacteria may oxidize specific substances, E.g. Hydrogen bacteria convert hydrogen to water, Methane bacteria oxidize methane to CO2, Nitrifying bacteria oxidize ammonia to nitrite or nitrate, Sulphur bacteria oxidize H2S to sulphate. However, the most common are the bacteria which oxidize various organic compounds most notably sugars. Models of energy generation in biological system Energy generating reactions involve, formation of high energy bonds, usually phosphates, and we can thus refer to it as phosphorylation. This occurs in two types : (1) Substrate Phosphorylation Where a phosphorylated organic substrate donates the phosphate directly to ADP forming ATP without electron donation. 1,3 diphosphoglycerate ------------→ 3 phosphoglycerate ADP to ATP (2) Oxidative Phosphorylation Where ADP is phosphorylated by inorganic phosphate. A membrane associated redox process in which electrons flow through electron transport chain result in phosphorylation of ADP to ATP by membrane bound enzyme ATPase. the electrons reached the final electron acceptor by electro motive force. Bacteria growth Microorganisms are classified according to their requirements for oxygen into 4 g: 1- obligate aerobes: they fail to grow in absence of oxygen since only oxygen can act a terminal electron acceptor. They have the following reaction: a. O2+E===H2O+E by cytochrome oxidase system. b. O2+E===H2O2+E by flavoprotein system. Since hydrogen peroxide is highly toxic to the cell. c. H2O2===H2O+O2 by catalase system. 2- Micro-aerophilic: these grow in presence if little amount of O2 They possess reaction (a) but the amount of cytochrome oxidase is limited. They also possess reaction (b) but not (c) accordingly, in high oxygen tension, both reaction (a) and (b) operate and thus H2O2 accumulates and the cells die. This happens in the top of the tube of liquid medium in which they grow. At lower levels, reaction (a) operates but (b) does not ( insufficient oxygen for both ) and the bacteria grow. At the bottom of the tube there is no oxygen and both reaction (a) and (b) fail hence they do not grow. 3- Obligate anaerobes: They possess (b) but not (a) or (c) thus in presence of any oxygen they accumulate H2O2 and thus die off instead they operate reaction (d) where organic or inorganic compounds are reduced by electrons released from nutritive substrates, e.g.: Reduction of nitrate and sulfate. 4- facultative anaerobes: all of reaction a,b,c and d operate and thus they grow in presence or absence of oxygen. The toxicity of O2 results from its reduction by enzymes found in the cell as “flavoproteins” to hydrogen peroxide and the more toxic free radical superoxide “O2” Aerobes and facultative anaerobes are protected from these products by the present of superoxide-dismutase and catalase enzymes that catalyzes the reaction. 20¯2 + 2 H → superoxide dismutase → O2 + H2O2 2H2O2 → catalase → H2O + O2 All obligate anaerobes lack both superoxide dismutase and catalase enzyme. Many methods are available for exclusion of O2 in case of anaerobic cultivation e.g: reducing agents such as sodium thioglycolate can be added to liquid cultures. Tubes of agar can be sealed with layer of paraffin. Culture vessels can be placed in a container form which O2 is removed by evacuation or chemical means. Nitrogen requirements Nitrogen is required by living beings for the synthesis of amino acids and proteins, purines and pyrimidines and nucleic acid, vitamins and coenzymes, etc. Some bacteria can utilize inorganic nitrogen compounds ammonia, nitrates, nitrites,.. etc. as the sole source of nitrogen. These bacteria are simple in their growth requirements (since it requires only a few simple nitrogen compounds) but complex in its metabolism (since it possesses a large number of enzymes which continually synthesize a large number of organic nitrogen compounds). Some bacteria require e.g. only one vitamin or amino acid and synthesize the rest of organic nitrogen compounds from inorganic nitrogen. The growth of such bacteria would be proportional to the amount of the required vitamin or amino acid in their food and hence are used for the assay of such compound in pharmaceutical preparations. Othe bacteria are more complex and usually require a partially hydrolyzed well-balanced protein diet. Others are strict in their requirement and need specific material in their food e.g: haemophiles influenze which requires the “X” factor (haematin) and /or “V” factor (coenzyme I or II) to grow. Such organic nitrogen compounds required for growth are called growth factors; growth-promoting compounds or accessory growth substances. A few species of bacteria can utilize atmospheric nitrogen and these are called nitrogen-fixing bacteria and grow in symbiosis with the roots of legumes. Requirement Of Other Elements Sulfur is required since it is a constituent of certain amino acids, e.g. cystine. Except for microorganisms which require these amino acids, sulfates fill the need for this element. From is required for the synthesis of cytochrome. Many enzymatic reactions require certain heavy metal ions such as Mg ( which is also especially required for the important reactions in which ATP participates ), Zn. Co. Cu. Mn. Mb. and many other metal ions required in trace amounts.  In addition, sodium, potassium and calcium ions are generally required for the proper functioning of enzymatic reactions. Large quantities of phosphorous are required for the synthesis of ATP, nucleic acids and lipids. All these elements can be utilized by microorganisms when they are supplied as inorganic salt. In environments used for the cultivation of microorganisms ( microbiological media ) it is customary to include in addition to water, carbon and nitrogen sources, significant quantities of phosphates and magnesium sulfate. For other elements, requirements are so small that their need can be satisfied from the impurities present in other constituents of the medium. PH Requirements The best growth for most bacteria is obtained when the pH of the environment is close to neutrality ( 6.5 to 7.8 ). A few species can tolerate acid environments, e.g. species of Lactobacillus and Acetobacter and a few can tolerate high pH e.g. vibrio cholera. Molds tolerate a wider range of pH ( 2-8.5) and tend to grow at pH slightly below neutrality ( about 5.5) while yeasts usually grow at the low pH of 3.5 to 4.5 ( range about 3 to 6.5). When microorganisms reproduce, they release waste products that may change the PH of the environment. If the pH change is extensive, their environment become unsuitable and this can be avoided by adding buffers ( chemicals that resist change in pH ). Food can be preserved by lowering their PH and so we avoided spoilage because their low pH inhibit microbial growth. Moisture Requirements Water in the environment is required to dissolve foods and wastes, as a medium for biochemical reactions and in way as a source of hydrogen and oxygen. Only free and not bound moisture is considered, e.g. a 4% aqueous agar gel contain  about 94% moisture but very few bacteria can grow in it while milk, with about 87 % moisture is suitable for bacterial growth since it contains more free moisture. Molds can generally grow in the least amount of moisture thus can be seen on almost dry surface e.g. on leather, while yeasts more moisture. Bacteria require much more moisture than fungi and among them some require more moisture than others, e.g. Neisseria gonorrhoeae and Mycobacterium tuberculosis respectively. A dry environment results in the loss of the cell ‘s water to its surrounding and this disrupt cellular activities. The available water surrounding the organisms is exposed as water activity (aw), the aw is the relative humidity of the air space in the immediate environment as broth RH. aw = RH/100 = 98.5/100 = 0.985 Microorganisms commonly require water activities above 0.90 in order to grow and multiply The optimal is 0.95. Some fungi can grow in 0.60. The water activity of foods can be used to predict how rapidly food spoilage occur. Food with high water activity spoil more rapidly. In some cases, the water activities of foods can be artificially lowered in order to prevent food spoilage e.g freeze-dried foods are resistant to spoilage by microorganisms because the foods have been desiccated in vacuum and so have very low water activity. The Osmotic Pressure Osmotic pressure is defined as the minimum amount of pressure that must be applied to solution in order to prevent the inward flow of water across asemipermeable membrane within the solution. Moisture affects the osmotic pressure and the ionic strength of the environment. In a hypotonic medium, water passes freely through the cytoplasmic membrane and build up some pressure inside the cell in attempting to balance the osmotic pressure inside and outside the cell. Unless the cell wall is intact and strong the cytoplasmic membrane would eventually rupture and the cell dies. This happens in the presence of penicillin which prevents the formation of cell walls. In an isotonic environment penicillin does not kill bacteria, which can grow and multiply without their protective cell walls.  In a hypertonic environment water passes from the cell and since the cell wall is rigid while the cytoplasmic membrane is not, plasmolysis occurs with the cytoplasmic membrane shrinking inward. This hinders the growth of the cell and may cause death. Some bacteria and many yeasts and molds are osmophilic i.e. can grow easily in very hypertonic environments with very low moisture content. In addition, there are certain ocean bacteria which are halophilic i.e. can grow in high sodium chloride concentration while most bacteria and fungi can tolerate only low salt concentration. Some bacteria can tolerate up to 6.5 % or 9 % sodium chloride, this being a characteristic of the species. Foods fruit preserves and salted fish are resistant to spoilage by microorganisms because of their high osmotic pressure ( hypertonic ). Microorganisms present are unable to grow because the hypertonic environment draws out the cells water, thus inhibiting metabolism. Temperature Requirements The optimum temperature for growth is that at which the microorganisms multiplies fastest and produces the highest yield. For each species there are also maximum and minimum temperatures for growth, beyond which the organism does not multiply. According to Temperature Requirements, Bacteria are divided into: 1- Mesophilic bacteria : Growth temperature ranges between 10oC, and 42oC, the optimum being 35o C to 37oC for pathogenic bacteria and about 22-25oC for non-pathogenic bacteria. Most pathogenic bacteria are mesophilic. 2-Thermophilic bacteria : Have an optimum temperature of 45o C and capable of growing at 65o C or higher. 3-Psychrophilic bacteria : Few bacteria have an optimum temperature of 15oC and can grow near zero C. However, in the frozen state no microorganisms can grow. Most fungi tend to have an optimum temperature of 20-25o C Bacterial growth Increase in number of cells, not cell size. One cell becomes colony of millions of cells. Asexual reproduction------ Two identical daughter cells 1- doubling of bacterial chromosome---2/3 of the cycle of bacterial growth. 2- bacterial DNA attaches to cell membrane (or mesosome) during replication. 3-segregation of two DNA copies takes place. 4-two daughter cells detach from each other or remain. Factors regulating growth: - Nutrients - Environmental conditions: (temperature, pH, osmotic pressure) - Generation time Population growth: Increase number or cell mass per unit time. Generation time, G, (doubling time): - Time required for a number or mass of cells to double. Minute range ---- rapid growers Hours range (24hr) --- slow growers e.g: M. tuberculosis Lag phase: (Adaptation, preparation for division, increase in size and density) - Bacteria are becoming "adapted" to the new environmental conditions (pH, temperature, nutrients, etc.). - Enzymes and intermediates are formed and accumulate until they are present in concentrations that are permit growth. - An increase in bacterial mass per unit of volume, but no increase in cell count. - The metabolism of the bacteria adapts to the conditions of the nutrient medium. Log phase (logarithmic or exponential) - Growth yield and growth rate - Conditions are optimal for growth. - The living bacteria population increases rapidly with time at an exponential growth in numbers, and the growth rate increasing with time.  Stationary phase - Depletion of nutrient, accumulation of toxic materials, cell crowding With the exhaustion of nutrients and accumulation of metabolic wastes, the growth rate has slowed to the point where the growth rate equals the death rate. - - Effectively, there is no net growth in the living bacteria population. Decline phase - The living bacteria population decreases with time, due to a lack of nutrients and toxic metabolic by-products. - Number of cells that are capable of division decreases progressively. Continuous growth - Constant volume flow system, fresh media, excess cells are removed. A “continuous culture” is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a chemostat. Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained. Synchronous growth - Filtration, nutrient removal. Mother cells are larger in size than newly formed daughter cells. Special bacterial filters were used to allow larger cells to retain and smaller cells to pass through. Addition of fresh nutrient medium to the filters allow the entrapped cells to divide at the same time. - heating spores Hot/cold brings cells to same metabolic state - Starvation (nutrient removal). Nutrient removal from the medium will result in failure of cells to divide. Already dividing cells can complete their division then stop further division. Addition of nutrients to non dividing cells all cells will divide at the same time.  Measurement of bacterial growth. - Total count Breed slide method Counting chambers (Petroff-Hauser counter or hemocytometer) Electronic counters (Coulter counter) - Viable count Spread plate and pour plate Measurement of bacterial mass. Dry weight, turbidimetic method Nitrogen content methods Metabolic methods Breed slide method a known volume of bacterial suspension is taken up and delivered onto a known area of microscope slide. The suspension is spread over the slide with a straight inoculating wire. If the area of microscopical field is known the number of cells in the original culture can be calculated using simple equation Counting chambers (Petroff-Hauser counter or hemocytometer) Direct Microscopic Counts – Petroff-Hausser counting chamber. A special counting slides as Petroff-Hauser counter or hemocytometer are used. Grid marked on these slides are divided into squares of known areas that receive known volume of bacterial suspension. Counting the number of bacteria per area under the microscope allows the determination of total number of cells per ml by multiplication of microscopic count per unit area. Electronic counters: Coulter counter microbial suspension forced through small orifice in the machine. movement of microbe through orifice impacts drop in voltage.  drop in voltage is detected every time a bacterial cell passes through. Each drop in voltage is recorded as a digit corresponding to a single cell through electronic recording machine. Viable count Concentrated samples are diluted by serial dilution. The diluted samples can be either plated by spread plating or by pour plating. Diluted samples are spread onto media in Petri dishes and incubated. Colonies are counted. The concentration of bacteria in the original sample is calculated (from plates with 25 – 250 colonies, from the FDA Bacteriological Analytical Manual). It is more appropriate to express the result of viable count as Colony Forming Unit/ml. Dry weight method Washed bacterial growth is oven dried or freeze dried then weighed on precision balance. Suitable only when a good quantity of bacterial growth could be obtained. Inaccurate results obtained when: 1- growth condition are not well controlled. 2-bacteria are actively storing heavy reserve materials. Turbidimetric method When a beam of light is directed to a cell suspension a part of it is absorbed and a part is scattered in proportion to the degree of turbidity of the suspension. The amount of light absorbed is measured on a colorimeter and is expressed as absorbance (A) or optical density (O.D). O.D represents the population size. O.D values can be converted to cell mass by construction of standard calibration curve. This method is rapid and reproducible. Nitrogen content method Nitrogen content of a bacterial population is proportional to cell mass. Washed population is subjected to chemical method for determination of total nitrogen by special equipment. This method is tedious and require well defined growth conditions. Metabolic methods The rate of consumption or accumulation of a certain metabolite in the culture medium is proportional to the number of cells in the culture. For ex. The quantity of glucose consumed is high when the number of cells is high and amount of lactic acid accumulates in the culture is proportional to the number of cells. Bacteria enzymes Bacteria are such active metabolic units and so they are especially rich in enzymes. Enzymes are functional proteins that catalyze metabolic reactions enhancing their rates. They may act alone or may require one or more of the following :- - Prosthetic groups ( not easily dissociable from the protein apoenzyme ). - Conzymes ( usually vitamins that are easily dissociable from the protein ). - Various metal ions. (1) Synthetic enzymes: These participate in anabolism, or synthesis of various molecules and structures required by the bacteria. They are intracellular. (2) Respiratory enzymes: These are intracellular enzymes concerned with the release of energy from nutrients in the process of catabolism. If in oxidation, oxygen is the final oxidizer ( terminal electron acceptor ) respiration is aerobic and if it is not, respiration is anaerobic, and usually resulting in the formation of acids and possibly gases ( mainly CO2 and hydrogen ). (3) Hydrolytic Enzymes : Certain nutrients are so complex that they can not diffuse from the medium into the protoplasm ( across the cytoplasimc membrane ) unless they are hydrolyzed to smaller diffusible molecules by hydrolytic enzymes that are secreted by the organism into the medium and hence are extracellular. Some of the extracellular enzymes may not serve food- hydrolyzing enzymes but basically to digest a dense medium so that the bacteria can diffuse more rapidly and thoroughly throughout the medium. This is common with pathogens which secrete enzymes that destroy the tissues or that kill various defense bodies or substances ( e.g. leucocytes) which the body sends out to the site of infection to fight the bacteria. The ability of bacteria to produce various extracellular enzymes is characteristic of the species( or even the strain ) and is enzymes that act upon : - Lipids and fats, by lipases ( mainly estruses ). - Carbohydrates, by enzymes that hydrolyses starch ( amylases ). - Pectins ( pectinases ) : cellulose ( celluloses ) disaccharides ( e.g. lactase, maltase and sucrase ) … etc. Bacteria which produce such enzymes are called saccharolytic bacteria. proteins, by proteinases and polypeptides that produce polypeptides and amino acids respectively. Natural protein hydrolysis ( proteolysis ) is called purification and is carried out by proteolytic bacteria which are commonly anaerobic. Example of extracellular enzymes which enhance the pathogenicity of bacteria are: Hyaluronidase : which dissolves hyaluronic acid. Collagenases : which dissolve collagen. Gelatinase : which dissolves gelatin. Fibrinolysin : which dissolves fibrin. Haemolysins : which dissolves RBC’s.  Coagulases : which dissolves plasma. Leucocidins : which kill leucocytes. Other Bacterial Products: Pigments: Some bacteria characteristically produce specific pigments when they are cultivated under specific conditions. These pigments may by intracellular and cannot diffuse into the medium and thus only the cells are colored or they may be extracellular and diffuse into the medium coloring it and these may be soluble in water only or may also dissolve in one or more organic solvent. Toxins: These are poisons which are produced as a result of the growth of the bacteria or may be part of the bodies of the bacteria. They are usually responsible for the characteristic lesions which pathogenic bacteria produce in their hosts. They may be extracellular ( exotoxins ) or intracellular ( endotoxins ). Many of the non- digestive extracellular enzymes mentioned above may be also considered bacterial toxins. Pyrogenic materials: These are special endotoxins which produce a febrile reaction upon injection in minute doses in a subject. These pyrogens are apparently very potent lipopolysaccharide complexes produced by certain gram-negative bacteria. They are dangerous when present in parenteral preparations and may be removed by adsorption on ion- exchange resins. They may be inactivated only by heating at temperatures over 300 ºC but not by normal sterilization processes. The best method of eliminating them from pharmaceutical preparations is to avoid their presence in the first place.