Lecture 04.pdf

BIOL371: Microbiology Lecture 4 – Microbial growth Topics of today 1. Culturing microbes and measuring their growth 2. Dynamics of microbial growth Materials covered:  Chapter 4.1-4.10  Figures 4.1-4.8, 4.10, 4.11, 4.13, 4.16-4.19  Tables 4.1, 4.2 1. Culturing microbes and measuring their g...

BIOL371: Microbiology Lecture 4 – Microbial growth Topics of today 1. Culturing microbes and measuring their growth 2. Dynamics of microbial growth Materials covered:  Chapter 4.1-4.10  Figures 4.1-4.8, 4.10, 4.11, 4.13, 4.16-4.19  Tables 4.1, 4.2 1. Culturing microbes and measuring their growth 1. 2. 3. 4. 5. Cell nutrition Growth media and laboratory culture Microscopic counts of microbial cell numbers Viable counting and microbial cell numbers Turbidity as a measure of microbial cell numbers Nutritional requirements for microbial growth  Nutritional requirements vary among microbes, reflecting their diverse metabolism  Nutrients – supply of elements required by cells for growth  Macronutrients – nutrients required in large amounts  Micronutrients – nutrients required in minute amounts  Trace metals and growth factors Microbial periodic table of the elements Chemical makeup of a microbial cell  A single E. coli cell weighs ~1 pg (10–12 gram)  75% water  Dry weight of 184 fg (184 X 10–15 gram)  Carbon, oxygen, nitrogen, phosphorus, and sulfur account for 96% of dry weight Sources of macronutrients  Carbon  Heterotrophs: obtain carbon from breakdown of organic polymers and/or uptake of monomers (sugars, amino acids, fatty acids and other organics)  Autotrophs: synthesize organics from carbon dioxide  Nitrogen – from proteins, ammonia (NH3), nitrate (NO3–) or nitrogen gas (N2)  Oxygen and hydrogen from water  Phosphorus (P) – usually from inorganic phosphate (PO4–3); also from nucleic acids and phospholipids  Sulfur – from sulfate (SO4–2), sulfide (H2S) or organics (e.g., amino acids and vitamins) Trace elements (micronutrients) required by microbes Element Function Boron (B) Autoinducer for quorum sensing in bacteria; also found in some polyketide antibiotics Cobalt (Co) Vitamin B12; transcarboxylase (only in propionic acid bacteria) Copper (Cu) In respiration, cytochrome c oxidase; in photosynthesis, plastocyanin, some superoxide dismutases Iron (Fe)b Cytochromes; catalases; peroxidases; iron–sulfur proteins; oxygenases; all nitrogenases Manganese (Mn) Activator of many enzymes; component of certain superoxide dismutases and of the water-splitting enzyme in oxygenic phototrophs (photosystem Two ) Molybdenum (Mo) Certain flavin-containing enzymes; some nitrogenases, nitrate reductases, sulfite oxidases, DMSO–TMAO reductases; some formate dehydrogenases Nickel (Ni) Most hydrogenases; coenzyme F430; carbon monoxide dehydrogenase; urease Selenium (Se) Formate dehydrogenase; some hydrogenases; the amino acid selenocysteine Tungsten (W) Some formate dehydrogenases; oxotransferases of hyperthermophiles Vanadium (V) Vanadium nitrogenase; bromoperoxidase Zinc (Zn) Carbonic anhydrase; nucleic acid polymerases; many DNA-binding proteins Growth factors (micronutrients) required by microbes Growth factor Function PABA (p-aminobenzoic acid) Precursor of folic acid Folic acid One-carbon metabolism; methyl transfers Biotin Fatty acid biosynthesis; some CO2 fixation reactions B12 (Cobalamin) One-carbon metabolism; synthesis of deoxyribose B1 (Thiamine) Decarboxylation reactions B6 (Pyridoxine) Amino acid/keto acid transformations Nicotinic acid (Niacin) Precursor of NAD+ Riboflavin Precursor of FMN, FAD Pantothenic acid Precursor of coenzyme A Lipoic acid Decarboxylation of pyruvate and alpha-ketoglutarate Vitamin K Electron transport Coenzymes M and B Methanogenesis Classes of culture media for microbial growth  Nutrient solutions (culture media) used to grow microbes  Either liquid or solid  Sterilized typically in an autoclave; some supplements are heat labile and require filtration  Defined media: exact composition known  Complex media: composed of digests of microbial, animal, or plant products (e.g., yeast extract)  Selective media: contain compounds that selectively inhibit growth of some microbe but not others  Differential medium: contains an indicator, usually a dye, that detects particular metabolic reactions during growth  Enriched medium: contains the addition of specific compounds (like blood or extra vitamins) necessary to enhance the growth of fastidious (i.e. difficult to cultivate) bacteria Defined and complex culture media  Complex medium: can be readily used to grow a range of bacteria  Defined medium: require prior knowledge of nutrition requirement of microbes  Needed for genetic studies involving prototrophic mutants  Examples below for culturing Escherichia coli Defined culture medium Glucose K2HPO4 KH2PO4 (NH4)2SO4 MgSO4 CaCl2 Trace elements* Distilled water Adjust to pH 7.0 10g 7g 2g 1g 0.1g 0.02g 2-10 µg each 1 litre *Fe, Co, Mn, Zn, Cu, Ni, Mo Complex culture medium Glucose Yeast extract Peptone KH2PO4 Distilled water Adjust to pH 7.0 15g 5g 5g 2g 1 litre Uses of solid media  Solid media are prepared by addition of the gelling agent (agar) to liquid media.  When grown on solid media, cells form isolated masses (colonies).  A colony, when well isolated, is typically derived from a single bacterium  Colony morphology (visible characteristics)  Sometimes used to identify microorganisms  Routinely used to determine if a culture is pure (one microbe), contaminated, or mixed (a) Serratia marcescens on MacConkey agar. (b) Close-up of colonies outlined in part a. (c) Pseudomonas aeruginosa on trypticase soy agar. (d) Shigella flexneri on MacConkey agar. (e) Bacterial colonies that developed from plating a dilution of seawater. Aseptic technique: transfer without contamination  Airborne contaminants everywhere  Streak plate technique with inoculating loop to obtain pure cultures Counting microbial cell numbers with microscopes 1. Stain cells with a dye(s), dry a specified volume on a glass slide, and count under a bright-field microscope a. Vital dye can be used to distinguish live cells from dead cells 2. Put a coverslip on the counting chamber (thick glass slide etched with grids) and add liquid sample a. Count the number of cells inside the grid under a phasecontrast microscope b. Not very reproducible i. Prone to technical errors ii. Motile cells cannot be readily calculated unless they are Cell counting chamber fixed Viable cell counts  Microscopic cell counting does not distinguish reproducing cells from dormant cells  Growing cells on solid media provides information on viable cell counts  Generally one cell gives rise to one colony (not true for filamentous bacteria and biofilm-forming bacteria)  Numbers expressed as colony-forming units (CFUs) Serial dilutions for viable counts  Samples from many sources can contain too many bacteria to be counted accurately on agar plate (~58 cm2 in area) without dilutions  Make a series of successive (serial) of ten-fold dilutions  Plate defined volume of the dilutions on solid substrate and count colony-forming units (CFUs) Applications of viable counts  Plate count is quick, easy, and highly sensitive  Routinely used in food, dairy, medical, and aquatic microbiology  A combined used of differential, selective and complex media provides information on the presence of specific bacteria (e.g., pathogen) and total bacteria counts (estimate shelf life in food and dairy products)  Underestimate total bacterial counts in environmental samples because many bacteria require nutrients and conditions not found in complex media under laboratory conditions Listeria (food-borne pathogen) on chromogenic (differential) medium Group A Streptococcus (causative agent of strep throat) on blood-infused medium (lysis of blood cells) Turbidity as a measure of bacterial cell numbers  Cell suspensions are turbid (cloudy) because cells scatter light – more cells, more turbid  Turbidity measurements are fast, widely used methods  Measured with a spectrophotometer  Unit is determined as optical density (OD) at specified wavelength (visible light; e.g., green light 540 nm)  For unicellular organisms, OD is proportional to cell number within limits.  To relate a direct cell count to a turbidity value, a standard curve must first be established because different shapes and sizes have different light scattering properties  Samples can be checked repeatedly  Ideal for measuring microbial growth Spectrophotometric measurement of microbial growth  Optical density (OD) is defined as the negative of the logarithm (base 10) of the transmission 2. Dynamics of microbial growth  Binary fission and the microbial growth cycle  Quantitative aspects of microbial growth  Continuous culture  Biofilm growth  Alternatives to binary fission Terms used in describing microbial growth  Growth: increase in the number of cells  Binary fission: cell division following enlargement of a cell to twice original size  Septum: partition between dividing cells, pinches off between two daughter cells  Generation (doubling) time: time required for microbial cells to double in number  Differs for different microbes and varies depending on conditions  example: Escherichia coli = 20 minutes  During cell division, each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell. Growth curve  Typical growth curve in a culture of fixed volume (closed system) has four phases: 1) lag phase, 2) exponential phase, 3) stationary phase, and 4) decline (death) phase Phases of bacterial growth in liquid culture  Lag phase: interval between inoculation of a culture and beginning of growth  new conditions require altering metabolic state  time needed for biosynthesis of new enzymes and to produce required metabolites before growth can begin  Exponential phase: doubling at regular intervals  Balanced growth – cells are metabolically identical  Rates vary with media, conditions, organism itself  continues until conditions can no longer sustain growth  Stationary phase: growth rate of population is zero  Metabolism continues at greatly reduced rate  Decline phase: total number decreases due to cell death  Cryptic growth: subpopulations adapt Generation time  Exponential growth: cell numbers double at regular intervals  Semi-logarithmic relationship; use semi-log plot for presentation of data  Generation time of an exponentially growing population: g = t/n; where, g is generation time, t is the duration of exponential growth, n is the number of generation during the period Chemostat culture  Chemostat: continuous culture where the volume of culture medium is added at the same rate as spent medium is removed  Steady state: when cell number nutrient, waste remain constant  Studying microbial physiology  Enrichment of environmental cultures Biofilm growth  Planktonic growth: growth of free-floating/free-swimming cells; e.g., growth in liquid suspension  Sessile growth: attached to surface  Biofilm: cells are enmeshed in polysaccharide matrix attached to surface  Biofilm is formed in flour stages     Planktonic cells attach (flagella, fimbriae, pili) Colonization: growth and extracellular polysaccharide production Development: metabolic changes Dispersal: colonize new sites Biofilms and human society      Microbial mats: multilayered sheets with different organisms in each layer Implicated in joint infections, implanted medical devices Responsible for cavities and cause gum disease Foul, plug, and corrode pipes Form in fuel tanks and on ship hulls Microbial mat of the purple phototrophic bacterium Thermochromatium tepidum Budding cell growth  Growth dynamics cannot be readily explained by standard growth equations  Budding cell division: unequal cell growth forming different daughter cells  Some budding bacteria form cytoplasmic extensions such as stalks (e.g., Caulobacter) and appendages (e.g., Ancalomicrobium) Filamentous bacteria  Hyphae growth: long filaments of the Gram-positive bacteria actinomycetes (e.g., Streptomyces)  Growth occurs only at hyphal tip  Cell growth not directly linked to division – no septa  Forms cross-walls that do not define cells but allow transport between compartments  Hyphae overlap to form mycelia  Arthrospores: survival structures, unlike endospores, they are not resistant to harsh environment  Developed by multiple fissions and the simultaneous formation of septa along a filament  Streptomyces species are major producers of antibiotics