BIO 245 Exam 2 Notes (2) PDF

Chapter 7 - Microbial Biochemistry Organic molecules- contain carbon ○ Atoms around carbon held together by covalent bonds Constitute 20-30% of cell’s mass Larger and more complex compounds ○ Carbohydrates, lipids, proteins Inorganic molecules- do not contain carbon...

Chapter 7 - Microbial Biochemistry Organic molecules- contain carbon ○ Atoms around carbon held together by covalent bonds Constitute 20-30% of cell’s mass Larger and more complex compounds ○ Carbohydrates, lipids, proteins Inorganic molecules- do not contain carbon ○ Ionic bonds ○ Exceptions: carbon oxides and carbonates (considered inorganic bc they don’t contain hydrogen) ○ Constitute 1% of cell’s mass ○ Small and simple compounds Water and salts Most carbon in organic molecules originates from inorganic carbon ○ Ex: CO2 captured via carbon fixation Biomolecules- organic molecules essential for biological and chemical processes Carbon- four valence electrons in outer orbitals = can form four covalent bonds ○ Methane (CH4) is simplest organic compound (also known as natural gas) ○ Carbon skeleton- carbon atoms bound together in straight, branched, or ring form Isomers- molecules with the same formula but hooked up differently ○ Impacts molecular function Structural isomers- glucose, galactose, glucose, differ in their structural formulas Stereoisomers- molecules that differ in how atoms are arranged in space Enantiomers Chiral - non superimposable mirror images These all have variable properties and functions Optical isomers- can rotate in plane of polarized light ○ Rotate light clockwise (+) as d form (dexter - on the right) ○ Rotate light counterclockwise (-) as l form (laevus - on the left) Functional groups- groups of atoms (in addition to C atoms) ○ Specific chemical reactions ○ Know hydroxyl, amino, carboxylic acid groups ○ remainder of molecule represented by R (side chain) Macromolecules- large biomolecules ○ Formed by linking together monomers (building blocks) to form polymers ○ Dehydration synthesis- monomers bind end to end; water is formed as a byproduct  Four main macromolecules ○ Carbohydrates ○ Proteins ○ Lipids ○ Nucleic acids Carbohydrates ○Most abundant biomolecules ○Composed of Carbon, Hydrogen, Oxygen ○Empirical formula (CH2O)n n = number of repeated units ○Ratio is 1:2:1 ○Can also contain N, P, S ○Diverse functions and uses: Part of ecosystems as food sources Components of DNA and RNA Building block of structural components (cellulose and chitin) Primary source of energy storage (starch and glycogen) Monosaccharides- simple sugars ○ Monomers for the synthesis of complex carbohydrates ○ Classified based on number of Carbon atoms Triose (3), tetrose (4), pentose (5), and hexose (6) Hexoses- glucose, galactose fructose Pentoses- ribose, deoxyribose Four or more carbon atoms more stable when they adopt a ring structure Disaccharides- composed of two monosaccharides, very sweet ○ Maltose - two glucose molecules - grain sugar ○ Lactose - galactose and glucose - milk sugar ○ Sucrose - glucose and fructose - table sugar ○ Glycosidic bond- covalent bond formed between hydroxyl groups of carbohydrates when dehydration synthesis occurs Polysaccharides- composed of hundreds of monosaccharides, not sweet ○ Glycans ○ Glycosidic bonds ○ Not soluble in water ○ Linear and branched configurations due to orientation of glycosidic linkages Cellulose- linear chains of glucose (cell walls in plants) Starch and glycogen- branched polymers of glucose ○ Energy storage Glycogen- in animals and bacteria  Starch- in plants ○ Modified polysaccharides NAG and NAM found in bacterial cell wall peptidoglycan Polymers of NAG form chitin (found in fungal cell walls) Lipids ○ Composed of C and H Can also contain N, O, P, S ○ Diverse structure and function Source of nutrients Energy storage Structural components for membranes and hormones ○ Chemically distinct Fatty acids and triglycerides Phospholipids and biological membranes Isoprenoids and sterols Fatty acids- long hydrocarbon chains with terminal carboxylic acid ○ Hydrophobic ○ Saturated fatty acids Contain only single bonds Straight, flexible carbon backbone Solid at room temperature ○ Unsaturated fatty acids Contain at least one double bond Have kinks in carbon skeleton → double bond causes a rigid bend Liquid at room temp Triglycerides- 3 fatty acids linked to a glycerol molecule (simple lipids) ○ Components of adipose tissue (body fat) ○ Energy storage molecules Complex lipids- composed of: ○ Glycerol molecule ○ Two fatty acids (saturated and/or unsaturated) ○ Additional component (phospholipid/glycolipid) ○ Amphipathic Hydrophilic heads Hydrophobic tails ○ Form uniquely functional structures in aqueous environments Micelles- spherical particle Interior is phospholipid tails Exterior is polar head Unit membranes- lipid bilayer sheets  vesicles/liposomes- lipid bilayer spheres Isoprenoids- branched lipids ○ Formed by chemical modification of isoprene ○ Technological uses Steroids- complex ringed structures ○ Found in cell membranes, some function as hormones ○ Most common are sterols Cholesterol- most common in animal tissues Strengthens cell membranes in eukaryotes and bacteria without cell walls (=mycoplasma) Prokaryotes- no cholesterol, similar compounds called hopanoids Strengthen bacterial membrane Fungi and some protozoa- similar compound called ergosterol Proteins ○ Made of CHNO, sometimes S ○ Essential in cell structure and function Enzymes that speed up chemical reactions Transporter proteins that move across chemical membranes Flagella that aid in movement Some bacterial toxins and cell structures ○ Composed of amino acids ○ Amino acids have alpha-carbon attached to: Hydrogen Carboxyl group Amino group Side chain Peptide bond- chemical bond formed between 2 amino acids ○ Formed between carboxylic acid group and amino group ○ Dehydration synthesis ○ Products: Peptides: 50 or fewer amino acids Oligopeptide- 20 amino acids Polypeptides- 50 amino acids Protein: very large number of amino acids or MULTIPLE polypeptides  Major determinants of protein structure/shape ○ Length of amino acids ○ Specific amino acid sequences ○ Protein shape is critical to its function ○ Levels of protein structure Primary structure- sequence of amino acids that make up the polypeptide chain ○ Flexible structure of peptide bonds Secondary structure- hydrogen bonding between amine and carboxyl functional groups within peptide backbone ○ 𝜶-helix: four amino acids apart (looks like a curl) ○ 𝝱-pleated sheets: amino acids further separated (pleated sheet) Tertiary structure- 3D shape of polypeptide chain, need this for protein to be functional (ALL PROTEINS HAVE TERTIARY) ○ Bonds between amino acid residues that are far apart in the chain Ex: disulfide bridges, hydrogen bonds, and ionic bonds ○ Protein folding- process where a polypeptide chain assumes tertiary structure ○ Native structure- folded proteins that are fully functional ○ Denatured proteins- 3D shape unfolded, protein no longer functional Loss of secondary, tertiary, and quaternary structure Typically irreversible Breakage of hydrogen and ionic bonds Heat denatures proteins Quaternary structure- exists ONLY in proteins consisting of several polypeptide chains  ○ All subunits must be present and configured to function Stabilized by weak interactions ○ Ex: hemoglobin has quaternary structure of four globular protein units 2 alpha and beta polypeptides, each one contains an iron based heme ○ Ex: cholera toxin Conjugated proteins composed of: ○ Protein portion ○ Non-protein portion Glycoprotein (carbohydrate) Lipoprotein (lipid) Nucleoprotein (RNA) ○ Important component of gram-neg cell membrane Chapter 8 - Microbial Metabolism Metabolism- buildup and breakdown of nutrients within a cell ○ Sum of all chemical reactions ○ Chemical reactions provide energy and create substances that sustain life ○ Many pathways of microbial metabolism are beneficial rather than pathogenic Catabolism- breaks down complex molecules into simpler ones ○ Exergonic- releases energy ○ Ex: breaks down glucose to CO2 and H20 Anabolism- builds complex molecules from simpler ones ○ Endergonic- uses energy ○ Ex: builds proteins from amino acids ATP- adenosine triphosphate ○ Links the reactions together ○ Stores energy released from catabolism ○ Releases energy to drive anabolic reactions Classification by carbon and energy source Source of carbon ○ Autotrophs- convert inorganic CO2 into organic compounds ○ Heterotrophs- organic compounds as nutrients Source of energy (electrons) ○ Phototrophs- electrons from light ○ Chemotrophs- electrons from chemicals Organotrophs- electrons in organic compounds  Lithotrophs- electrons in inorganic compounds (unique to microbes) ○ Chemoheterotrophs- use organic molecules as both their energy and carbon sources (most organism) REDOX reactions- transfer of electrons between molecules is important because most of the energy is in the form of high energy electrons; oxidation reaction paired with a reduction reaction ○ Oxidation- loss of electrons (OIL) ○ Reduction- gain of electrons (RIG) Energy carriers ○ Energy released via catabolism can be stored via: Reduction of electron carriers In bonds of ATP ○ Electron carriers Bind and carry high energy electrons Easily reduced or oxidized B vitamin group origin and nucleotide derivatives NAD NADP FAD (NAD+/NADH); (FAD/FADH2); (NADP+/NADPH) Left = oxidized form, right = reduced form Recycled continuously ATP as an energy carrier ○ ATP is “energy currency” of the cell ○ Enables the cell to store energy safely and released it as needed ○ Adenine molecule bonded to ribose molecule and 3 phosphate groups AMP (one phosphate group); ADP (two phosphate groups) ○ Phosphorylation- addition of an inorganic phosphate group (Pi) to ADP with the input of energy High energy phosphate bonds (terminal bonds between phosphate groups) ○ Dephosphorylation- breakage of high energy bonds Energy is released One phosphate (inorganic phosphate Pi) Two phosphates (pyrophosphate PPi) ○ Energy released from dephosphorylation of ATP is used to drive cellular work ○ Enzymes  Catalysts- substances that speed up chemical reactions without being altered Enzymes are biological catalysts → they inc reaction rate without raising temperature Activation energy- the energy needed to form or break chemical bonds and convert reactants to products Enzymes lower the Ea by binding to reactant molecules and speeding up reactions Substrates- reactant to which an enzyme binds (think key) Fits like a key in 3D shape of specific amino acids on active site Active site- location on the enzyme where the substrate binds (think lock) Enzymes have specificity for particular substrates Same compound can be a substrate for many different enzymes ○ Some enzymes are made entirely of proteins Most consist of protein and non-protein component Apoenzyme- protein component of enzyme; inactive by itself non-protein component of enzyme: ○ Cofactor- inorganic ions such as Fe2+ and Mg2+ ○ Coenzyme- organic that assists enzymes to transfer electrons Derived from vitamins: CoA, NAD+, FMN Holoenzyme- apoenzyme + cofactor (whole, active enzyme) As temperature increases, rate of reactions increases ○ Lower temps- molecules move slowly ○ Higher temps- molecules move quickly, more collisions ○ Optimum temperature- maximum rate of reaction Beyond this, molecules slow down again ○ Denaturation- loss of tertiary structure (3D) Breakage of hydrogen bonds Extreme changes in pH Change in arrangement of amino acids in active site Loss of catalytic ability Optimum pH- when enzyme is most active ○ Above or below means reduced activity  High concentration of substrate- enzyme gets saturated ○ Active site always occupied by substrate ○ Catalyzing at its maximum rate Further increase of substrate does not affect rate Under normal conditions, enzymes are not saturated Competitive inhibitors- fill active site of an enzyme and compete with the substrate ○ Shape and structure is very similar to substrate ○ Once bound, does not form products Inhibitor concentration needs to be equal to substrate concentration Noncompetitive inhibitors- interact with allosteric site rather than active site ○ Changes shape of active site ○ Inhibitor concentration is much lower than substrate concentration ○ Allosteric inhibition Allosteric activators- bind to another part to increase affinity of enzyme Feedback inhibition- end product of a reaction allosterically (noncompetitively) inhibits enzymes from earlier in the pathway ○ Biochemical control ○ Stops cell from making more of a substance than it needs Carbohydrate catabolism- the breakdown of carbohydrates to release energy ○ Involves enzymatic hydrolysis of glycosidic bonds in polysaccharides to form monomers Hydrolysis- splitting with addition of water (opposite of dehydration) Amylase- hydrolyzes glycogen or starch into glucose monomers Cellulase- hydrolysis into glucose monomers Most common carbohydrate is glucose Glucose is a highly reduced compound!!! ○ Contains lots of energy in the reduced bonds Processes of glucose catabolism- both start with Glycolysis ○ Cellular respiration ○ Fermentation Glycolysis- sugar lysis ○ Single 6 carbon glucose molecule split into 2 molecules of pyruvate (3 carbon sugar) ○ Most common pathway in many prokaryotes and eukaryotes for glucose catabolism ○ Occurs in the cytoplasm, 10 enzymatic steps ○ Anaerobic - does not require O2  ○ Types of glycolytic pathways Embden-Meyerhof- Parnas (EMP) pathway Found in animals and most common in microbes Entner-doudoroff (ED) pathway Pentose phosphate pathway (PPP) Glycolysis EMP pathway ○ Energy investment phase 2 ATP used 6 carbon sugar (Glucose) split into two phosphorylated 3 carbon molecules, (G3P) ○ Energy payoff phase The two G3P molecules are oxidized to 2 pyruvate molecules 4 ATP formed by SLP - substrate-level phosphorylation Net yield ATP = 4 - 2 = 2 ATP 2 NADH are produced Substrate level phosphorylation (SLP) - a phosphate group is removed from an organic molecule and is directly transferred to an available ADP molecule, producing ATP Transition (bridge) reaction ○ Pyruvate from glycolysis can be further oxidized in the Krebs cycle, producing more energy ○ Bridge step/transition reaction Decarboxylation (loss of CO2) occurs first Pyruvate (3 carbon) is oxidized to acetyl group (2 carbon) NAD+ is reduced to NADH Acetyl group attaches to coenzyme A (CoA) forming acetyl CoA Occurs in cytoplasm (prokaryotes) and mitochondrial matrix (eukaryotes) For every molecule of glucose, 2 acetyl CoA And 2 NADH are formed Then acetyl CoA enters Krebs cycle Krebs cycle (citric acid cycle) ○ Transfers remaining electrons present in acetyl group Occurs in cytoplasm (prokaryotes) and mitochondrial matrix (eukaryotes) Closed loop, 8 step cycle ○ Acetyl CoA loses the CoA, acetyl combines with oxaloacetate to form citric acid cycle ○ Oxidation of each acetyl group produces 3 NADH; 1 FADH2  1 GTP by substrate level phosphorylation (equivalent to 1 ATP) Releases 2 CO2 ○ Most important products of krebs cycle = NADH and FADH2 Contain most of the energy that was originally present in the glucose ○ Intermediates in krebs cycle- useful for many biosynthetic pathways (amino acids, fatty acids, nucleotides, etc) Cellular respiration ○ Begins when electrons are transferred from NADH and FADH2 Electrons produced in glycolysis, transition reaction, and Krebs cycle ○ To a final INORGANIC electron acceptor Oxygen- aerobic respiration Non-oxygen inorganic molecules- anaerobic respiration Occurs in inner part of cytoplasmic membrane (prokaryotes) and inner mitochondrial membrane (eukaryotes) Energy of electrons generates an electrochemical gradient which is used to make ATP via oxidative phosphorylation Electron transport chain (ETC) ○ Last component of cellular respiration ○ Comprised of membrane-associated protein complexes and mobile electron carriers (NADH, FADH2, etc) ○ Major membrane-associated electron carriers: Cytochromes Flavoproteins Iron-sulfur proteins Quinones Proton motive force (PMF) ○ As electrons move down ETC, protons (H+) are pumped to the outside of cytoplasmic membrane (bacteria) From the mitochondrial matrix across the inner mitochondrial space (eukaryotes) Buildup of protons ○ Establishes electrochemical gradient Higher concentration of protons on one side of the membrane Has potential energy called Proton Motive Force (PMF) Can be used to make ATP Can also be used for rotation of flagella or movement of ions  Chemiosmosis- ATP synthesis using energy from PMF ○ Proteins cannot diffuse back into the cytoplasm due to selectively permeable membrane Can move back through protein channels containing ATP synthase ATP synthase- catalyst for the conversion of PMF into ATP Releases energy as protons move through it Addition of inorganic PO4 to ADP (oxidative phosphorylation) Forms ATP - more readily usable form of energy ○ Total ATP yield: 4 from Substrate level phosphorylation, 34 from Oxidative phosphorylation NADH makes 3 ATP, FADH makes 2 ATP Aerobic respiration- final electron acceptor is Oxygen ○ Reduced to water by final ETC carrier cytochrome oxidase ○ Sometimes aerobic respiration is not possible due to missing cytochrome oxidase, other enzymes, or low amounts of oxygen available Anaerobic respiration- final electron acceptor is NOT oxygen ○ Nitrate reduced to nitrite or nitrogen gas ○ Sulfate reduced to hydrogen sulfide ○ Carbonate reduced to methane ○ Essential for nitrogen and sulfur cycles ○ Yields less ATP than in aerobic respiration!!!!!!! Only part of krebs cycle operates under anaerobic conditions Only some ETC carriers participate ○ Organisms using anaerobic respiration grow slowly compared to aerobes Fermentation- does not use krebs cycle or ETC, produces small amounts of ATP (only 2 ATP) ○ Many cells unable to carry out respiration because: Lack the inorganic final electron acceptor Lack genes for complexes and electron carriers in ETC Lack genes to make one or more enzymes in krebs cycle ○ NADH must be re-oxidized to NAD+ for reuse as an electron carrier for glycolysis to continue ○ Some use organic molecule (pyruvate) as a final electron acceptor Lactic acid fermentation- produces lactic acid from glucose ○ Seen in some bacteria and by animals in muscles during oxygen depletion Pyruvate + NADH → lactic acid + NAD+ Only 2 ATP produced (low energy process) ○ Homolactic fermentation: produces lactic acid only Lactic acid bacteria example: Streptococcus and Lactobacillus  ○ Heterolactic fermentation: produces lactic acid and other compounds Lactic acid fermentation can lead to food spoilage Can also produce yogurt, sauerkraut, pickles, etc ○ Whole process can not proceed if no NAD+ to generate electrons Alcohol fermentation- produces ethanol and CO2 from glucose ○ Glucose is oxidized to 2 pyruvic acid ○ Pyruvic acid is converted to acetaldehyde and CO2 ○ NADH reduces acetaldehyde to ethanol ○ Only 2 ATP produced (low energy process) ○ This process is carried out by many bacteria and yeasts Yeast (Saccharomyces cerevisiae) Baker’s yeast or brewer’s yeast Beer, wine (ethanol) Breads (CO2 makes bread dough rise) Lipid catabolism- in addition to glucose, lipids and proteins can also be broken down to supply energy ○ Main lipids- triglycerides and phospholipids ○ Broken down by hydrolytic enzymes Lipases break down triglycerides (3 fatty acids and glycerol) Phospholipases break down phospholipids ( 2 fatty acids attached to glycerol) ○ Products generated = glycerol and fatty acids Glycerol is phosphorylated and converted to G3P → continues through glycolysis and krebs cycle Fatty acids undergo 𝝱-oxidation to form acetyl CoA and enter krebs cycle Protein catabolism- proteins broken down by microbial proteases ○ The last thing the cell wants to do is break down proteins, but if it can't use other energy sources shown above it will! ○ Extracellular proteases cut proteins internally at specific amino acid sequences into smaller peptides that can be taken up by cells ○ Intracellular proteases (=peptidases) break down peptides further into individual amino acids after removal of functional groups, amino acids can enter krebs cycle Deamination- removal of amino group Decarboxylation- removal of carboxyl group Photosynthesis- conversion of light energy from sun into chemical energy ○ Location: Chloroplasts within thylakoids - eukaryotes  Thylakoids with photosynthetic membranes - prokaryotes ○ Light reactions: Conversion of light energy into chemical energy (ATP) by photosynthetic pigments NADPH or NADH (energy rich electron carriers) are produced ○ Dark reactions: ATP and NADPH/NADH reduce CO2 to sugar (=carbon fixation) Photosynthetic pigments- molecules used to absorb solar energy ○ other things besides chlorophyll can absorb energy beyond sunlight ○ Organized into Photosystems used to generate ATP by chemiosmosis Photosystem I (PSI) and Photosystem II (PSII) Cyanobacteria and chloroplasts have both Anoxygenic bacteria use only one Photophosphorylation- type of oxidative phosphorylation ○ Cyclic Photophosphorylation Uses Photosystem I ONLY! Electrons released from photosystem RETURN back! Preferred for higher production of ATP ○ Noncyclic Photophosphorylation Uses BOTH Photosystems I & II! Electrons released from photosystems DO NOT return! Instead gets incorporated into NADPH, replenished by breakdown of water ATP is produced Oxygenic photosynthesis- produces O2 ○ Electron donor is H2O ○ Ex: plants, algae, cyanobacteria Anoxygenic photosynthesis- does NOT produce O2, produces Sulfur and Sulfate (SO4) ○ Electron donor is H2S or S2O3 ○ Ex: bacterial phototrophs (purple and green bacteria) Dark reactions of photosynthesis ○ Calvin cycle- biochemical pathway for CO2 fixation Cytoplasm - in photosynthetic bacteria Stroma - in eukaryotic chloroplast ○ 3 stages: Fixation: RuBisCo catalyzes the addition of a CO2 to RuBP produces 3-PGA Reduction: ATP and NADPH used to convert 3-PGA into G3P Some G3P used to build glucose  Regeneration: remaining G3P used to regenerate RuBP to continue the cycle Biogeochemical cycle- recycling of inorganic matter between living organisms and nonliving environment ○ Involves the cycling of the most common elements in organic molecules (CHNOPS) Carbon cycle- exchange of carbon between heterotrophs and autotrophs ○ Primarily the exchange of atmospheric CO2 ○ Produced by heterotrophs via respiration or fermentation ○ Fixed by autotrophs Methanotrophs- bacteria and archaea ○ Use methane as carbon source ○ Help reduce atmospheric methane levels Methanogens- archaea ○ Produce methane (= methanogenesis) ○ In anaerobic environments when CO2 is used as terminal electron acceptor Significant environmental impact- powerful greenhouse gas, contributes to climate change Nitrogen cycle- transforming nitrogen between various forms Nitrogen fixation- conversion of nitrogen into organic form by free living and symbiotic bacteria ○ Cyanobacteria fix inorganic nitrogen into ammonia (NH3) ○ Rhizobium fix nitrogen and live symbiotically in the root nodules of legumes Conversion of nitrogen into nitrogen gas ○ Ammonification- nitrogenous waste converted into ammonia ○ Nitrification- ammonia oxidized to nitrite, then to nitrate ○ Denitrification- nitrate converted into nitrogen gas Artificial fertilizer ○ salt/freshwater eutrophication = overgrowth and subsequent death of aquatic algae Chapter 9 - Microbial Growth Bacterial division- increase in number of cells, not cell size ○ Binary fission (asexual reproduction); most common mechanism of cell replication in bacteria ○ Formation of new cells Replication of DNA  Partitioning of cellular components into two daughter cells Each offspring receives a complete copy of the parental genome Cytokinesis- division of the cytoplasm Binary fission ○ cytokinesis/cell division- directed by protein FtsZ FtsZ assembles into a Z ring on cytoplasmic membrane Anchored by FtsZ binding proteins and defines the division plane Additional proteins required are added to the Z ring to form divisome Divisome- synthesizes peptidoglycan cell wall, forms a division septum that divides the two daughter cells Generation time- formation of a new generation ○ Doubling time- time it takes for the population to double through one round of binary fission Ranges from 20 min to over 24 hours E. coli- 20 min M. tuberculosis- 15-20 hrs M. leprae- 14 days Dependent upon environmental conditions ○ Exponential growth- binary fission doubles the number of cells each generation Total number of cells = 2n Generation time (g) = t/n n = number of generations t = time Growth curve- graphical representation of microbial growth in a closed culture (aka a batch culture) ○ Growth in tube or flask- no nutrients are added and most waste is not removed ○ Reproducible growth pattern ○ Culture density- number of cells per unit volume Measure of the number of cells in the population ○ When the number of live cells is plotted against time, 4 distinct phases can be observed in the culture Lag phase Log (exponential) phase Stationary phase Death phase Lag phase- interval between when a culture is inoculated and when growth begins  ○ Little to no increase in cell numbers Adjustment to new environment ○ Intense metabolic activity- synthesis of proteins ○ Damaged or shocked cells- repair takes place Log (exponential phase)- maximum growth rate ○ Cells start dividing actively, short generation time! ○ Number increases exponentially Straight line graph ○ Constant growth rate and uniform metabolic activity Preferred for industrial applications and research work Most susceptible to the action of disinfectants and common antibiotics Stationary phase- number of live cells reaches a plateau ○ Due to nutrient depletion, toxic waste accumulation ○ Growth rate slows → # of deaths = # of new cells - equilibrium ○ Cells switch to a survival mode of metabolism Less susceptible to antibiotics Many cells form spores Antibiotics are synthesized Death phase- number of dying cells exceed number of dividing cells ○ Exponential decrease, shown as straight line graph ○ Most cells lyse and release nutrients ○ Allows surviving cells (=persisters) to maintain viability Persisters- associated with certain chronic infections that do not respond to antibiotic treatment Ex: tuberculosis Logarithmic representation of bacterial populations ○ Solid line - does not clearly show population changes ○ Dashed line- logarithmic, straight ○ Constant slope ○ Semi logarithmic graphs ○ Convenient and easy to estimate generation times Measurement of microbial growth ○ Direct measurements- count microbial cells Direct cell count- using a microscope or culture counter Plate count Filtration Most probable number (MPN) ○ Indirect measurements Turbidity  Dry weight Metabolic activity Direct cell count ○ Uses Petroff-Hauser chamber Often used to count number of bacteria in milk Sample of culture suspension is added to the chamber under a coverslip Observed under microscope avg number of bacteria per viewing field is calculated ○ Advantages: easy, inexpensive, quick ○ Disadvantages: Motile cells must be immobilized Need a high cell count Can't differentiate between live and dead cells Live = green, dead = red Plate counts- counts the number of living cells capable of forming colonies on plates ○ Colonies not always form from single cells CFU- colony forming unit, recorded as CFU/mL More than one cell may have landed on same spot to give rise to a single colony Bacteria that grow in clusters/chains are difficult to disperse and a single colony may represent several cells Count plates with 30-300 CFUs Serial dilutions- several dilutions in multiples of 10 relative to culture density ○ Goal is to obtain plates with 30-300 CFUs ○ Experimentally conducted in duplicate or triplicate for reliability Plate counts ○ Pour plate method Sample mixed with warm agar poured onto sterile plate, allowed to solidify and incubated Colonies form on surface and within agar Brief exposure to heat can kill cells ○ Spread plate method 0.1mL added to surface of pre-poured solidified agar Spread with hockey stick and incubated Colonies form on surface More than 0.1mL- does not soak in and causes colonies to spread  Membrane filtration method- used when numbers are expected to be very small ○ Drinking water ○ Sample must be concentrated rather than diluted before planting Known volumes (1ml, 10ml, 100ml) passed through a filter that collects bacteria Filter is transferred to an agar plate, count the colonies on surface, find OCD ○ Commonly used to detect coliforms (= gram negative rods that ferment lactose) Indicators of fecal contamination and of food and water Indirect cell counts ○ Turbidity- cloudiness due to light scatter More bacteria, more turbid ○ Using a spectrophotometer- beam of light passed through a sample to a light sensitive detector More cells, more light scattered ○ % transmission or absorbance (optical density/OD) Inverse relationship between both factors Biofilms- complex and dynamic ecosystems ○ Common ebay microorganisms grow in nature ○ Ex: dental plaque ○ Embedded in extracellular polymeric substances (EPS) Made of polysaccharides and other macromolecules Maintains integrity and function of biofilm ○ Channels in the EPS allow movement of nutrients, waste, and glasses throughout the biofilm Keeps cells hydrated, preventing desiccation Shelter bacteria from harmful environmental factors Ex: antibiotics or immune cells