DOC-20240902-WA0042..pptx

The Scope of Bacteriology  The Bacteria are a group of single-cell microorganisms with procaryotic cellular configuration.  The genetic material (DNA) of procaryotic cells exists unbound in the cytoplasm of the cells.  There is...

The Scope of Bacteriology  The Bacteria are a group of single-cell microorganisms with procaryotic cellular configuration.  The genetic material (DNA) of procaryotic cells exists unbound in the cytoplasm of the cells.  There is no nuclear membrane, which is the definitive characteristic of eucaryotic cells such as those that make up plants and animals.  Until recently, bacteria were the only known type of procaryotic cell, and the discipline of biology related to their study is called bacteriology. Figure 1 (Right) The structure of a typical procaryotic cell, in this case, a Gram-negative bacterium, compared with (Left) a typical eucaryotic cell (plant cell). The procaryote is about 1 micrometer in diameter and about the size of the eucaryotic chloroplast or mitochondrion. Table 1. Phenotypic properties of Bacteria and Archaea compared with Property Eucarya. Biological Domain Eucarya Bacteria Archaea Cell configuration eucaryotic Prokaryotic Prokaryotic Nuclear membrane present Absent Absent Number of chromosomes >1 1 1 Chromosome topology linear Circular circular Murein in cell wall - + - ester-linked glycerides; ester-linked glycerides; Cell membrane lipids unbranched; saturated or ether-linked branched; saturated unbranched; polyunsaturated monounsaturated Cell membrane sterols present Absent Absent Organelles (mitochondria and present Absent Absent chloroplasts) Ribosome size 80S (cytoplasmic) 70S 70S Cytoplasmic streaming + - - Meiosis and mitosis present Absent Absent Transcription and translation - + + coupled Amino acid initiating protein methionine N-formyl methionine methionine synthesis Protein synthesis inhibited by - + - streptomycin and chloramphenicol  IDENTIFICATION OF BACTERIA  The criteria used for microscopic identification of procaryotes include cell shape and grouping, Gram-stain reaction, and motility. Bacterial cells almost invariably take one of three forms: rod (bacillus), sphere (coccus), or spiral (spirilla and spirochetes).  Rods that are curved are called vibrios. Fixed bacterial cells stain either Gram-positive (purple) or Gram-negative (pink); motility is easily determined by observing living specimens.  Bacilli may occur singly or form chains of cells; cocci may form chains (streptococci) or grape-like clusters (staphylococci); spiral shape cells are almost always motile; cocci are almost never motile.  This nomenclature ignores the actinomycetes, a prominent group of branched bacteria which occur in the soil. But they are easily recognized by their colonies and their microscopic appearance. Figure 2. Gram stain of Bacillus anthracis, the cause of anthrax. Such easily-made microscopic observations, combined with knowing the natural environment of the organism, are important aids to identify the group, if not the exact genus, of a bacterium - providing, of course, that one has an effective key. Such a key is Bergey's Manual of Determinative Bacteriology, the "field guide" to identification of the bacteria. Bergey's Manual describes affiliated groups of Bacteria and Archaea based on a few easily observed microscopic and physiologic characteristics. Further identification requires biochemical tests which will distinguish genera among families and species among genera. Figure 3. Size and fundamental shapes of procaryotes revealed by three genera of Bacteria (l to r): Staphylococcus (spheres), Lactobacillus (rods), and Aquaspirillum (spirals). Figure 4. Chains of dividing streptococci. Electron micrograph of Streptococcus pyogenes. Figure 5. Different shapes and arrangements of bacterial cells, with examples.  STRUCTURE AND FUNCTION OF PROCARYOTIC CELLS  A procaryotic cell has five essential structural components:  1- Genome (DNA),  2- Ribosomes,  3- Cell membrane,  4- Cell wall,  5- some sort of Surface layer.  At one time it was thought that bacteria were essentially "bags of enzymes".  The development of the electron microscope, in the 1950s, revealed the distinct anatomical features of bacteria and confirmed that they lacked a nuclear membrane.  Structurally, a procaryotic cell has three architectural regions: 1- Appendages (attachments to the cell surface) in the form of flagella and pili (or fimbriae); 2- Cell envelope consisting of a capsule, cell wall and plasma membrane; 3- Cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions.  Characteristics of typical bacterial cell structures. Structure Function(s) Predominant chemical composition Flagella Swimming movement Protein Pili Sex pilus Mediates DNA transfer during conjugation Protein Attachment to surfaces; protection against Common pili or fimbriae Protein phagotrophic engulfment Attachment to surfaces; protection against phagocytic Capsules (includes "slime layers" Usually polysaccharide; occasionally engulfment, occasionally killing or digestion; reserve and glycocalyx) polypeptide of nutrients or protection against desiccation Cell wall Prevents osmotic lysis of cell protoplast and confers Peptidoglycan (murein) complexed with Gram-positive bacteria rigidity and shape on cells teichoic acids Peptidoglycan prevents osmotic lysis and confers Peptidoglycan (murein) surrounded by rigidity and shape; outer membrane is permeability Gram-negative bacteria phospholipid protein-lipopolysaccharide barrier; associated LPS and proteins have various "outer membrane" functions Permeability barrier; transport of solutes; energy Plasma membrane Phospholipid and protein generation; location of numerous enzyme systems Ribosomes Sites of translation (protein synthesis) RNA and protein Often reserves of nutrients; additional specialized Highly variable; carbohydrate, lipid, protein Inclusions functions or inorganic Chromosome Genetic material of cell DNA Appendages Flagella  Are filamentous protein structures attached to the cell surface that provide the swimming movement for most motile procaryotes.  Procaryotic flagella are much thinner than eukaryotic flagella.  The diameter of a procaryotic flagellum is about 20 nanometers, well- below the resolving power of the light microscope.  The flagellar filament is rotated by a motor apparatus in the plasma membrane allowing the cell to swim in fluid environments.  Bacterial flagella are powered by proton motive force (chemiosmotic potential) established on the bacterial membrane, rather than ATP hydrolysis which powers eukaryotic flagella.  About half of the bacilli and all of the spiral and curved bacteria are motile by means of flagella. Very few cocci are motile, which reflects their adaptation to dry environments and their lack of hydrodynamic design.  About 50 genes are required for flagellar synthesis and function.  The flagellar apparatus consists of several distinct proteins: a system of rings embedded in the cell envelope (the basal body), a hook-like structure near the cell surface, and the flagellar filament.  The innermost rings, the M and S rings, located in the plasma membrane, comprise the motor apparatus.  The outermost rings, the P and L rings, located in the periplasm and the outer membrane respectively, function as bushings to support the rod where it is joined to the hook of the filament on the cell surface.  As the M ring turns, powered by an influx of protons, the rotary motion is transferred to the filament which turns to propel the bacterium.  Flagella may be variously distributed over the surface of bacterial cells in distinguishing patterns.  Basically, flagella are either polar (one or more flagella arising from one or both poles of the cell) or peritrichous (lateral flagella distributed over the entire cell surface).  Flagellar distribution is a genetically-distinct trait that is occasionally used to characterize or distinguish bacteria.  For example, among Gram-negative rods, pseudomonads have polar flagella to distinguish them from enteric bacteria, which have peritrichous flagella.  Procaryotes are known to exhibit a variety of types of tactic behavior, i.e., the ability to move (swim) in response to environmental stimuli. For example, during chemotaxis a bacterium can sense the quality and quantity of certain chemicals in its environment and swim towards them (if they are useful nutrients) or away from them (if they are harmful substances).  Other types of tactic response in procaryotes include phototaxis, aerotaxis and magnetotaxis. The occurrence of tactic behavior provides evidence for the ecological (survival) advantage of flagella in bacteria and other procaryotes.  Flagellar stains of three bacteria a. Bacillus cereus b. Vibrio cholerae c. Bacillus brevis. Since the bacterial flagellum is below the resolving power of the light microscope, although bacteria can be seen swimming in a microscope field, the organelles of movenent cannot be detected. Desulfovibrio species. TEM. About 15,000X. The bacterium is motile by means of a single polar flagellum.  Fimbriae  Fimbriae and pili are interchangeable terms used to designate short, hair-like structures on the surfaces of procaryotic cells.  Like flagella, they are composed of protein.  Fimbriae are shorter and stiffer than flagella, and slightly smaller in diameter.  Generally, fimbriae have nothing to do with bacterial movement (there are exceptions, e.g. twitching movement on Pseudomonas).  Fimbriae are very common in Gram-negative bacteria, but occur in some archaea and Gram-positive bacteria as well.  Fimbriae are most often involved in adherence of bacteria to surfaces, substrates and other cells or tissues in nature.  In E. coli, a specialized type of pilus, the F or sex pilus, mediates the transfer of DNA between mating bacteria during the process of conjugation, but the function of the smaller, more numerous common pili is quite different.  Common pili  (almost always called fimbriae) are usually involved in specific adherence (attachment) of procaryotes to surfaces in nature.  In medical situations, they are major determinants of bacterial virulence because they allow pathogens to attach to (colonize) tissues and/or to resist attack by phagocytic white blood cells.  For example, pathogenic Neisseria gonorrhoeae adheres specifically to the human cervical or urethral epithelium by means of its fimbriae; enterotoxigenic strains of E. coli adhere to the mucosal epithelium of the intestine by means of specific fimbriae; the M-protein and associated fimbriae of Streptococcus pyogenes are involved in adherence and to resistance to engulfment by phagocytes. Fimbriae (common pili) and flagella on the surface of bacterial cells. Left: dividing Shigella enclosed in fimbriae. The structures are probably involved in the bacterium's ability to adhere to the intestinal surface. Right: dividing pair of Salmonella displaying both its peritrichous flagella and its fimbriae. The fimbriae are much shorter and slightly smaller in diameter than flagella. Both Shigella and Salmonella are enteric bacteria that cause different types of intestinal diarrheas. The bacteria can be differentiated by a motility test. Salmonella is motile; Shigella is nonmotile. Fimbriae of Neisseria gonorrhoeae allow the bacterium to adhere to tissues. Electron micrograph.  Some properties of pili and fimbriae. Bacterial species where Typical number on cell Distribution on cell surface Function observed Escherichia coli (F or sex mediates DNA transfer 1-4 uniform pilus) during conjugation Escherichia coli (common surface adherence to 100-200 uniform pili or Type 1 fimbriae) epithelial cells of the GI tract surface adherence to Neisseria gonorrhoeae 100-200 uniform epithelial cells of the urogenital tract Streptococcus pyogenes adherence, resistance to (fimbriae plus the M- ? uniform phagocytosis; antigenic protein) variability Pseudomonas aeruginosa 10-20 polar surface adherence  The Cell Envelope  The cell envelope is a descriptive term for the several layers of material that envelope or enclose the protoplasm of the cell.  The cell protoplasm (cytoplasm) is surrounded by the plasma membrane, a cell wall and a capsule.  The cell wall itself is a layered structure in Gram-negative bacteria.  All cells have a membrane, which is the essential and definitive characteristic of a "cell".  Almost all procaryotes have a cell wall to prevent damage to the underlying protoplast.  Outside the cell wall, foremost as a surface structure, may be a polysaccharide capsule, or at least a glycocalyx.  Profiles of the cell envelope the Gram-positive and Gram-negative bacteria. The Gram-positive wall is a uniformly thick layer external to the plasma membrane. It is composed mainly of peptidoglycan (murein). The Gram-negative wall appears thin and multilayered. It consists of a relatively thin peptidoglycan sheet between the plasma membrane and a phospholipid-lipopolysaccharide outer membrane. The space between the inner (plasma) and outer membranes (wherein the peptidoglycan resides) is called the periplasm.  Capsules  Most procaryotes contain some sort of a polysaccharide layer outside of the cell wall polymer.  In a general sense, this layer is called a capsule. A true capsule is a discrete detectable layer of polysaccharides deposited outside the cell wall.  A less discrete structure or matrix which embeds the cells is a called a slime layer or a biofilm. A type of capsule found in bacteria called a glycocalyx is a thin layer of tangled polysaccharide fibers which is almost always Bacterial capsules outlined by India ink viewed by light microscopy. This is a true capsule, a discrete layer of polysaccharide Negative stain of Streptococcus pyogenes viewed by surrounding the cells. transmission electron microscopy (28,000X). The halo around the chain of cells is the hyaluronic acid capsule that surrounds the exterior of the bacteria. The septa between dividing pairs of cells may also be seen.  Capsules have several functions and often have multiple functions in a particular organism.  Like fimbriae, capsules, slime layers, and glycocalyx often mediate adherence of cells to surfaces.  Capsules also protect bacterial cells from engulfment by predatory protozoa or white blood cells (phagocytes), or from attack by antimicrobial agents of plant or animal origin.  Capsules in certain soil bacteria protect cells from perennial effects of drying or desiccation.  Capsular materials (e.g. dextrans) may be overproduced when bacteria are fed sugars to become reserves of carbohydrate for subsequent metabolism.  Colonies of Bacillus anthracis.  The slimy or mucoid appearance of a bacterial colony is usually evidence of capsule production. In the case of B. anthracis, the capsule is composed of poly-D-glutamate.  The capsule is an essential determinant of virulence to the bacterium.  Bacteria may attach to surface, produce slime, divide and produce microcolonies within the slime layer, and construct a biofilm, which becomes an enriched and protected environment for themselves and other bacteria.  A classic example of biofilm construction in nature is the formation of dental plaque mediated by the oral bacterium, Streptococcus mutans.  The bacteria adhere specifically to the pellicle of the tooth by means of a protein on the cell surface. The bacteria grow and synthesize a dextran capsule which binds them to the enamel and forms a biofilm some 300-500 cells in thickness.  The bacteria are able to cleave sucrose (provided by the animal diet) into glucose plus fructose. The fructose is fermented as an energy source for bacterial growth.  The glucose is polymerized into an extracellular dextran polymer that cements the bacteria to tooth enamel and becomes the matrix of dental plaque.  The dextran slime can be depolymerized to glucose for use as a carbon source, resulting in production of lactic acid within the biofilm (plaque) that decalcifies the enamel and leads to dental caries or bacterial infection of the tooth. Cell Wall  Most procaryotes have a rigid cell wall. The cell wall is an essential structure that protects the cell protoplast from mechanical damage and from osmotic rupture or lysis.  Procaryotes usually live in relatively dilute environments such that the accumulation of solutes inside the procaryotic cell cytoplasm greatly exceeds the total solute concentration in the outside environment.  Thus, the osmotic pressure against the inside of the plasma membrane may be the equivalent of 10-25 atm. Since the membrane is a delicate, plastic structure, it must be restrained by an outside wall made of porous, rigid material that has high tensile strength. Such a material is murein, the ubiquitous component of bacterial cell walls. Electron micrograph of an ultra-thin section of a dividing pair of group A streptococci (20,000X). The cell surface fibrils, consisting primarily of M protein, are evident. The bacterial cell wall, to which the fibrils are attached, is also clearly seen as the light staining region between the fibrils and the dark staining cell interior. Cell division in progress is indicated by the new septum formed between the two cells and by the indentation of the cell wall near the cell equator. The streptococcal cell diameter is equal to approximately one micron. Electron micrograph of Streptococcus pyogenes.  The cell walls of bacteria deserve special attention for several reasons: 1. They are an essential structure for viability. 2. They are composed of unique components found nowhere else in nature. 3. They are one of the most important sites for attack by antibiotics. 4. They provide ligands for adherence and receptor sites for drugs or viruses. 5. They cause symptoms of disease in animals. 6. They provide for immunological distinction and immunological variation among strains of bacteria.  The cell walls of all Bacteria contain a unique type of peptidoglycan called murein.  Peptidoglycan is a polymer of disaccharides (a glycan) cross-linked by short chains of amino acids (peptides), and many types of peptidoglycan exist.  All Bacterial peptidoglycans contain N-acetylmuramic acid, which is the definitive component of murein. The cell walls of Archaea may be  In the Gram-positive Bacteria (those that retain the purple crystal violet dye when subjected to the Gram- staining procedure) the cell wall is thick (15-80 nanometers), consisting of several layers of peptidoglycan.  In the Gram-negative Bacteria (which do not retain the crystal violet) the cell wall is relatively thin (10 nanometers) and is composed of a single layer of peptidoglycan surrounded by a membranous structure called the outer membrane.  The outer membrane of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide (LPS or endotoxin), which is toxic to animals. In Gram-negative bacteria the outer membrane is usually thought of as part of the cell wall. Peptidoglycan structure  Peptidoglycan structure and arrangement in E. coli is representative of all Enterobacteriaceae, and many other Gram-negative bacteria, as well.  The glycan backbone is made up of alternating molecules of N-acetylglucosamine (G) and N-acetylmuramic acid (M) connected by a beta 1,4-glycoside bond.  The 3-carbon of N-acetylmuramic acid (M) is substituted with a lactyl ether group derived from pyruvate.  The lactyl ether connects the glycan backbone to a peptide side chain that contains L-alanine, (L-ala), D-glutamate (D- glu), Diaminopimelic acid (DAP), and D-alanine (D-ala). The structure of the muramic acid subunit of the peptidoglycan of Escherichia coli. This is the type of murein found in most Gram-negative bacteria. The glycan backbone is a repeat polymer of two amino sugars, N- acetylglucosamine (G) and N-acetylmuramic acid (M). Attached to the N-acetylmuramic acid is a tetrapeptide consisting of L-ala-D-glu-DAP-D-ala.b. Abbreviated structure of the muramic acid subunit. c. Nearby tetrapeptide side chains may be linked to one another by an interpeptide bond between DAP on one chain and D-ala on the other. d. The polymeric form of the molecule.  Strandsof murein are assembled in the periplasm from about 10 muramic acid subunits. Then the strands are connected to form a continuous glycan molecule that encompasses the cell.  The assembly of peptidoglycan on the outside of the plasma membrane is mediated by a group of periplasmic enzymes which are transglycosylases, transpeptidases and carboxypeptidases.  The mechanism of action of penicillin and related beta- lactam antibiotics is to block transpeptidase and carboxypeptidase enzymes during their assembly of the murein cell wall. Hence, the beta lactam antibiotic are said to "block cell wall synthesis" in the bacteria.  The glycan backbone of the peptidoglycan molecule can be cleaved by an enzyme called lysozyme that is present in animal serum, tissues and secretions, and in the phagocytic lysosome.  The function of lysozyme is to lyse bacterial cells as a constitutive defense against bacterial pathogens. Some Gram-positive bacteria are very sensitive to lysozyme and the enzyme is quite active at low concentrations.  Lachrymal secretions (tears) can be diluted 1:40,000 and retain the ability to lyse certain bacterial cells. Gram-negative bacteria are less vulnerable to attack by lysozyme because their peptidoglycan is shielded by the outer membrane.  The exact site of lysozymal cleavage is the beta 1,4 bond between N-acetylmuramic acid (M) and N-acetylglucosamine (G) , such that the muramic acid subunit is the result of the action of lysozyme on bacterial peptidoglycan.  In Gram-positive bacteria there are numerous different peptide arrangements among peptidoglycans. The best studied is the murein of Staphylococcus aureus. In place of DAP (in E. coli) is the diamino acid, L- lysine (L-lys), and in place of the interpeptide bond (in Gram-negatives) is an interpeptide bridge of amino acids that connects a free amino group on lysine to a free carboxy group on D-ala of a nearby tetrapeptide side chain.  This arrangement apparently allows for more frequent cross-bonding between nearby tetrapeptide side chains. In S. aureus, the interpeptide bridge is a peptide consisting of 5 glycine molecules (called a pentaglycine bridge).  Assembly of the interpeptide bridge in Gram-positive murein is inhibited by the beta lactam antibiotics in the same manner as the interpeptide bond in Gram-negative murein. Gram-positive bacteria are more sensitive to penicillin than Gram-negative bacteria because the peptidoglycan is not protected by an outer membrane and it is a more abundant molecule.  In Gram-positive bacteria, peptidoglycans may vary in the amino acid in place of DAP or L-lys in position 3 of the tetrapeptide, and in the exact Schematic diagram of the peptidoglycan sheet of Staphylococcus aureus. G = N-acetyl-glucosamine; M = N-acetyl- muramic acid; L-ala = L-alanine; D-ala = D-alanine; D-glu = D-glutamic acid; L-lys = L-lysine. This is one type of murein found in Gram-positive bacteria. Compared to the E. coli peptidoglycan there is L-lys in place of DAP (diaminopimelic acid) in the tetrapeptide. The free amino group of L-lys is substituted with a glycine pentapeptide (gly-gly-gly-gly-gly-) which then becomes an interpeptide bridge forming a link with a carboxy group from D-ala in an adjacent tetrapeptide side chain. Gram-positive peptidoglycans differ from species to species, mainly in regards to the amino acids in the third position of the tetrapeptide side chain and in the amino acid composition of the interpeptide bridge.  The Outer Membrane of Gram-negative Bacteria The outer membrane is a lipid bilayer intercalated with proteins, superficially resembling the plasma membrane. The inner face of the outer membrane is composed of phospholipids similar to the phosphoglycerides that compose the plasma membrane. The outer face of the outer membrane may contain some phospholipid, but mainly it is formed by a different type of amphiphilic molecule which is composed of lipopolysaccharide (LPS). Schematic illustration of the outer membrane, cell wall and plasma membrane of a Gram-negative bacterium. Note the structure and arrangement of molecules that constitute the outer membrane.  The LPS molecule that constitutes the outer face of the outer membrane is composed of a hydrophobic region, called Lipid A, that is attached to a hydrophilic linear polysaccharide region, consisting of the core polysaccharide and the O-specific polysaccharide. The Lipid A head of the molecule inserts into the interior of the membrane, and the polysaccharide tail of the molecule faces the aqueous environment. Where the tail of the molecule inserts into the head there is an accumulation of negative charges such that a magnesium cation is chelated between adjacent LPS molecules. This provides the lateral stability for the outer membrane, and explains why treatment of Gram- negative bacteria with a powerful chelating agent, such as EDTA, causes dispersion of LPS molecules.  Bacterial lipopolysaccharides are toxic to animals.  When injected in small amounts LPS or endotoxin activates macrophages to produce pyrogens, activates the complement cascade causing inflammation, and activates blood factors resulting in intravascular coagulation and hemorrhage.  Endotoxins may play a role in infection by any Gram-negative bacterium.  The toxic component of endotoxin (LPS) is Lipid A. The O-specific polysaccharide may provide ligands for bacterial attachment and confer some resistance to phagocytosis.  Variation in the exact sugar content of the O polysaccharide (also referred to as the O antigen) accounts for multiple antigenic types (serotypes) among Gram-negative bacterial pathogens. The Plasma Membrane  The plasma membrane, also called the cytoplasmic membrane, is the most dynamic structure of a procaryotic cell.  Its main function is a s a selective permeability barrier that regulates the passage of substances into and out of the cell. The plasma membrane is the definitive structure of a cell since it sequesters the molecules of life in a unit, separating it from the environment.  The bacterial membrane allows passage of water and uncharged molecules, but does not allow passage of larger molecules or any charged substances except by means special membrane transport processes and transport systems. Functions of the procaryotic plasma membrane.  1. Osmotic or permeability barrier  2. Location of transport systems for specific solutes (nutrients and ions)  3. Energy generating functions, involving respiratory and photosynthetic electron transport systems, establishment of proton motive force, and transmembranous, ATP-synthesizing ATPase  4. Synthesis of membrane lipids (including lipopolysaccharide in Gram- negative cells)  5. Synthesis of murein (cell wall peptidoglycan)  6. Assembly and secretion of extracytoplasmic proteins  7. Coordination of DNA replication and segregation with septum formation and cell division  8. Chemotaxis (both motility per se and sensing functions)  9. Location of specialized enzyme system Physical and Environmental Requirements for Microbial Growth  The procaryotes exist in nature under an enormous range of physical conditions such as O2 concentration, Hydrogen ion concentration (pH) and temperature.  Applied to all microorganisms is a vocabulary of terms used to describe their growth (ability to grow) within a range of physical conditions. A thermophile grows at high temperatures, an acidophile grows at low pH, an osmophile grows at high solute concentration, and so on.  This nomenclature will be employed in this section to describe the response of the procaryotes to a variety of physical conditions. The Effect of Oxygen  Oxygen is a universal component of cells and is always provided in large amounts by H2O. However, procaryotes display a wide range of responses to molecular oxygen O 2  Obligate aerobes require O2 for growth; they use O2 as a final electron acceptor in aerobic respiration.  Obligate anaerobes (occasionally called aerophobes) do not need or use O2 as a nutrient. In fact, O2 is a toxic substance, which either kills or inhibits their growth. Obligate anaerobic procaryotes may live by fermentation, anaerobic respiration, bacterial photosynthesis, or the novel process of methanogenesis.  Facultative anaerobes (or facultative aerobes) are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O2) they grow by fermentation or anaerobic respiration, but in the presence of O 2 they switch to aerobic respiration.  Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O 2. They live by fermentation alone whether or not O2 is present in their environment. The Effect of pH on Growth  The pH, or hydrogen ion concentration, [H+], of natural environments varies from about 0.5 in the most acidic soils to about 10.5 in the most alkaline lakes.  The range of pH over which an organism grows is defined by three cardinal points: the minimum pH, below which the organism cannot grow, the maximum pH, above which the organism cannot grow, and the optimum pH, at which the organism grows best.  For most bacteria there is an orderly increase in growth rate between the minimum and the optimum and a corresponding orderly decrease in growth rate between the optimum and the maximum pH, reflecting the general effect of changing [H+] on the rates of enzymatic reaction.  Microorganisms which grow at an optimum pH well below neutrality (7.0) are called acidophiles.  Those which grow best at neutral pH are called neutrophiles and those that grow best under alkaline conditions are called alkaliphiles.  Obligate acidophiles, such as some Thiobacillus species, actually require a low pH for growth since their membranes dissolve and the cells lyse at neutrality.  In the construction and use of culture media, one must always consider the optimum pH for growth of a desired organism and incorporate buffers in order to maintain the pH of the medium.  Many pathogenic bacteria exhibit a relatively narrow range of pH over which they will grow. Most diagnostic media for the growth and identification of human pathogens have a pH near 7.  Growth rate vs pH for three environmental classes of procaryotes. Most free- living bacteria grow over a pH range of about three units. Note the symmetry of the curves below and above the optimum pH for growth. The Effect of Temperature on Growth  A particular microorganism will exhibit a range of temperature over which it can grow, defined by three cardinal points in the same manner as pH.  For example, organisms with an optimum temperature near 37 degrees (the body temperature of warm-blooded animals) are called mesophiles.  Organisms with an optimum T between about 45 degrees and 70 degrees are thermophiles.  Some Archaea with an optimum T of 80 degrees or higher and a maximum T as high as 115 degrees, are now referred to as extreme thermophiles or hyperthermophiles.  The cold-loving organisms are psychrophiles defined by their ability to grow at 0 degrees.  A variant of a psychrophile (which usually has an optimum T of 10-15 degrees) is a psychrotroph, which grows at 0 degrees but displays an optimum T in the mesophile range, nearer room temperature.  Psychrotrophs are the scourge of food storage in refrigerators since they are invariably brought in from their mesophilic habitats and continue to grow in the refrigerated environment where they spoil the food. Of course, they grow slower at 2 degrees than at 25 degrees. Growth rate vs temperature for five environmental classes of procaryotes. Most procaryotes will grow over a temperature range of about 30 degrees. The curves exhibit three cardinal points: minimum, optimum and maximum temperatures for growth. There is a steady increase in growth rate between the minimum and optimum temperatures, but slightly past the optimum a critical thermolabile cellular event occurs, and the growth rates plunge rapidly as the maximum T is approached. As expected and as predicted by T.D. Brock, life on earth, with regard to temperature, exists wherever water remains in a liquid state. Thus, psychrophiles grow in solution wherever water is supercooled below 0 degrees; and extreme thermophilic archaea (hyperthermophiles) have been identified growing near deep-sea thermal vents at temperatures up to 120 degrees. Theoretically, the bar can be pushed to even higher temperatures. GROWTH OF BACTERIAL POPULATIONS Measurement of Bacterial Growth  Growth is an orderly increase in the quantity of cellular constituents.  It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment.  In most bacteria, growth involves increase in cell mass and number of ribosomes, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division.  This asexual process of reproduction is called binary fission.  Electron micrograph of Streptococcus pyogenes. Bacterial growth by binary fission. Most bacteria reproduce by a relatively simple asexual process called binary fission: each cell increases in size and divides into two cells. During this process there is an orderly increase in cellular structures and components, replication and segregation of the bacterial DNA, and formation of a septum or cross wall which divides the cell into two progeny cells.  The process is coordinated by the bacterial membrane perhaps by means of mesosomes.  The DNA molecule is believed to be attached to a point on the membrane where it is replicated.  The two DNA molecules remain attached at points side-by-side on the membrane while new membrane material is synthesized between the two points.  This draws the DNA molecules in opposite directions while new cell wall and membrane are laid down as a septum between the two chromosomal compartments.  When septum formation is complete the cell splits into two progeny cells. The time interval required for a bacterial cell to divide or for a population of bacterial cells to double is called the generation time. Generation times for bacterial species growing in nature may be as short as 15 minutes or as long as several days. The Bacterial Growth Curve  In the laboratory, under favorable conditions, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n (where n = the number of generations).  This is called exponential growth. In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature.  When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve The typical bacterial growth curve. When bacteria are grown in a closed system (also called a batch culture), like a test tube, the population of cells almost always exhibits these growth dynamics: cells initially adjust to the new medium (lag phase) until they can start dividing regularly by the process of binary fission (exponential phase). When their growth becomes limited, the cells stop dividing (stationary phase), until eventually they show loss of viability (death phase). Note the parameters of the x and y axes. Growth is expressed as change in the number viable cells vs time. Generation times are calculated during the exponential phase of growth. Time measurements are in hours for bacteria with short generation times. 1. Lag Phase.  Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.  The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium. 2. Exponential (log) Phase.  The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression.  The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation.  The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population.  Generation time (G) is defined as the time (t) per generation (n = number of generations). Hence, G=t/n is the equation from which calculations of generation time derive. 3. Stationary Phase.  Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space".  During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing.  The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It is during the stationary phase that spore- forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process. 4. Death Phase.  If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines.  (Note, if counting by turbidimetric measurements or microoscopic counts, the death phase cannot be observed).  During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.  What Do Fungi Look Like? Unicellular form o it is consists of a single, minute, oval or spherical cell. o The cell wall is mainly made up only from glucan and chitin. o It contain nucleus and nucleolus and mitocondria, and the main reserve food products are: glycogen, oil, volutin and protein bodies.  Mycelial (filamentous): Aseptate hyphae Protoplasm may form a continuous, uninterrupted mass The hyphae in this case is coenocyte Septate hyphae Unicellular and primitively branched Chytrids (Chytridiomycota): Dimorphism (i.e. existing in two forms):  Introduction to the Structure of Hyphae Each Hypha is: essentially a tube - consisting of a rigid wall and containing protoplasm tapered at its tip - this is the region of active growth (i.e. the extension zone).  Some fungi possess septa.  Some septa possess one of more pores.  Plasma membrane is closely associated with the hyphal wall.  Each hyphal cell or compartment contains one or more nuclei.  The growing tip is structurally and functionally  Cytoplasm contains (endoplasmic reticulum, many or one vacuole)  Mitochondria and many food particles made up glycogen, oil droplets and lipids, other cell organelles include ribosomes, microbodies and crystals are also common.  There are no major organelles at the extreme tip  At the extreme tip there is an accumulation of membrane-bound vesicles -the apical vesicular cluster (complex) (AVC).  Vacuoles ……….. store and recycle cellular metabolites, e.g. enzymes and nutrients.  In the oldest parts of the hypha cytoplasm breakdown due to autolysis (self-digestion) heterolysis.  Hyphal aggregation (plectenchyma) 1. Prosenchyma 2. Pseudoparenchyma Haustoria: Ectoparasites- Endoparasites  The Fungal Wall  Protect the underlying protoplasm;  Determines and maintains the shape  Acts as an interface between the fungus and its environment.  Acts as a binding site for some enzymes.  Possesses antigenic properties - which allow interactions with other organisms. Chemical composition of the wall: Polymeric fibrils Chitin Cellulose (in the Oomycota) Amorphous matrix components Glucans- proteins – lipids- Heteropolymers (mixed polymers) of mannose, galactose, fructose and xylose  In the oldest parts of the hyphae (and in many fungal spores) lipids and pigments may be deposited in the wall:  Lipids serve as a nutrient reserve and help prevent desiccation  Pigments, such as melanin, help protect the protoplast against the damaging effects of UV radiation.  Hyphal Growth The growth of a hypha is closely linked to the presence of vesicles which form the apical vesicular cluster (AVC). The position of the vesicles is linked to the direction of growth of a hypha: - o when a hypha is growing straight ahead, the vesicles are positioned in the center of the hyphal tip. o movement of the vesicles to the left or right side of the hyphal tip is accompanied by a change in direction of growth of the hypha. Vesicles of the AVC contain:  Wall precursors - the sub-units or buildng blocks of the wall polymers - e.g. uridine diphosphate N-acetylglucosamine, the sub-unit of chitin.  Wall lytic enzymes - which help breakdown and separate wall components - e.g. chitinase, glucanase.  Wall synthase enzymes - which help assemble new wall components and so increase the size of the wall - e.g. chitin synthase, glucan synthase.  Importance of Fungi Fungi are important because they are:- o Agents of biodegradation and biodeterioration o Responsible for the majority of plant diseases and several diseases of animals (including humans) o Used in industrial fermentation processes o Used in the commercial production of many biochemicals o Cultured commercially to provide us with a direct source of food o Used in bioremediation o Beneficial in agriculture, horticulture and forestry  Growth of Fungi  Restricted growth  Unrestricted growth Phases of growth Unicellular fungi 1. Lag phase, 2. Phase of accelerated growth 3. Log phase 4. Phase of decline acceleration 5. Stationary phase 6. Death phase Filamentous Fungi 1. Lag phase 2. Exponential or log phase 3. Stationary phase 4. The death phase