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1/21/2011 Dynamics of Prokaryotic Growth Chapter 4 1 1/21/2011 Principles of Bacterial Growth Principles of Bacterial Growth • Prokaryotic cells divide by binary fission – One cell divides into two • Two into four etc. – Cell growth is exponential • Doubling of population with each cell division • Exponential growth has important h lth consequences health – Generation time • Time it takes for population to double • a.k.a. doubling time • Varies among species 2 1/21/2011 Principles of Bacterial Growth • Growth can be calculated – Nt = N0 x 2n • • • • (Nt ) number of cells in population (N0 ) original number of cells in the population (n) number of divisions Example – N0 = 10 cells in original population – n = 12 » 4 hours assuming 20 minute generation time – Nt = 10 x 212 – Nt = 10 x 4,096 – Nt = 40,960 Generation Times Vary for Each Organism • E. coli • Mycobacterium tuberculosis 3 1/21/2011 Bacterial Growth in Laboratory Conditions • Cells in laboratory grown in closed or b t h system batch t – No new input of nutrient and no release of waste • Population of cells increase in predictable fashion – Follows a pattern called growth curve Bacterial Growth in Laboratory Conditions • The Growth Curve – Characterized by five distinct stages • Lag stage • Exponential or log stage • Stationary stage • Death stage • Phase of prolonged decline 4 1/21/2011 Bacterial Growth in Laboratory Conditions • Lag phase – Number of cells does not increase – Cells prepare for growth • “Tooling up” • Log phase – Period of exponential growth • Doubling of population with each generation • Stationary St ti phase h – Overall population remains relatively stable • Cells exhausted nutrients • Cell growth = cell death Environmental Factors on Growth • As group, prokaryotes inhabit nearly all environments – Some live in “comfortable” habitats – Some live in harsh environments • Most of these are termed extremophiles and belong to domain Archaea • Major conditions that influence growth – – – – Temperature Oxygen pH Water availability 5 1/21/2011 Environmental Factors on Growth • Temperature • – Optimum temperature -5°C to 15°C – Each species has wellwell defined temperature range • Within range lies optimum growth temperature – Prokaryotes divided into 5 categories Psychrophile • Found in Arctic and Antarctic regions • Psychrotroph 20°C to 30°C • Important in food spoilage • Mesophile 25°C to 45°C • More common • Disease causing • Thermophiles 45°C to 70°C • Common in hot springs • Hyperthermophiles 70°C to 110°C • Usually members of Archaea • Found in hydrothermal vents Environmental Factors on Growth Decreasing O2 6 1/21/2011 Environmental Factors on Growth • pH – Bacteria survive within various pH range – Neutrophiles • Multiply between pH of 5 to 8 – Maintain optimum near neutral – Acidophiles • Thrive at pH below 5.5 – Maintains neutral internal pH, pumping out protons (H+) – Alkalophiles • Grow at pH above 8.5 – Maintain neutral internal pH through sodium ion exchange » Exchange sodium ion for external protons Environmental Factors on Growth • Water availability – All microorganisms require water for growth – Water not available in all environments • In high salt environments – Bacteria increase internal solute concentration – Osmotolerant bacteria tolerate high salt environments – Bacteria that require high salt for cell growth termed h l hil halophiles 7 1/21/2011 Nutritional Factors on Growth • Growth of prokaryotes depends on nutritional t iti l ffactors t as wellll as physical h i l environment • Main factors to be considered are: – Required elements – Growth factors – Energy sources – Nutritional diversity Nutritional Factors on Growth To live/reproduce, all living things need: an energy source a source of carbon and other required elements Energy sources: Chemicals (“chemo”) Organic (“organo”) (ex. glucose) Inorganic (“litho”) (ex. H2S) Sunlight (“photo”) Carbon sources: CO2 (“auto”) (carbon fixation) Organic cmpds (“hetero”) troph = nourish 8 1/21/2011 Nutritional Factors on Growth Required Elements: Nutritional Factors on Growth Carbon Sources: •CO2 (Carbon fixation) •Organic Compounds Nitrogen Sources: •Organic Compounds •Inorganic Compound •N2 (Nitrogen fixation) 9 1/21/2011 Nutritional Factors on Growth Hartford Courant, March 6, 2009 Early Vegetables A Rewarding Way To Welcome Spring Peas are not fussy, but it does pay to soak the seed for about a half hour before planting, then cover it with an inoculant. You can buy an inexpensive inoculant at nurseries; it's a black powder that "inoculates" the soil with nitrogen-fixing bacteria, prompting the peas, which are legumes, to start producing nitrogen on their own. 10 1/21/2011 Nutritional Factors on Growth Carbon Sources: •CO2 (Carbon fixation) •Organic Compounds Nitrogen Sources: •Organic Compounds •Inorganic Compound •N2 (Nitrogen fixation) g Nutrients Limiting Limiting Nutrients Nutritional Factors on Growth 1 pkg chocolate chips 3 c sugar 11 1/21/2011 Nutritional Factors that Influence Microbial Growth 1 pkg chocolate chips 10 c sugar Seattle Times, Linda Mapes, 3/18/08 Algae-prone Green Lake is staying clean Good news for Green Lake: It's clean and staying that way. Efforts in 2004 to reduce algae g g growth in the lake by y adding g aluminum sulfate, or alum, are still paying dividends. The alum binds with phosphorus in the water, tying up a key nutrient algae need to thrive. ….the addition of nitrogen and phosphorus from fertilizers and other pollutants, combined with warm, sunny days, can spell excessive growth. And cyanobacteria, while quite beautiful, are toxic to small animals and dogs,….. dogs 12 1/21/2011 Bacterial Growth in Nature • Conditions in nature have profound effect on microbial growth – Cells sense changing environment • Synthesize compounds useful for growth • Cells produce multicellular associations to increase survivability – Example » Biofilms » Slime layers Biofilm layer Bacterial Growth in Nature • Interactions of mixed microbial communities iti – Prokaryotes live in mixed communities • Many interactions are cooperative – Waste of one organism nutrient for another • Some cells compete for nutrient – Synthesize toxic substance to inhibit growth of competitors 13 1/21/2011 Laboratory Cultivation • Knowing environmental and nutritional factors makes it possible to cultivate organisms in the laboratory • Organisms are grown on culture media – Media is classified as complex media or chemically defined media Laboratory Cultivation • Complex media – Contains a variety of ingredients – There is no exact chemical formula for ingredients • Can be highly variable – Examples include • Nutrient broth • Blood agar • Chocolate agar 14 1/21/2011 Laboratory Cultivation • Chemically defined media – Composed of precise amounts of pure chemical – Generally not practical for routine laboratory use Laboratory Cultivation • Special types of culture media – Used to detect or isolate particular organisms – Divided into selective and differential media 15 1/21/2011 Laboratory Cultivation • Selective media – Inhibit the growth of unwanted organisms • Allow only sought after organisms to grow – Examples • Mannitol salts agar – For isolation of salt tolerant bacteria • MacConkey M C k agar – For isolation of Gram-negative bacteria Laboratory Cultivation • Differential media – Contains substance that bacteria change in recognizable way – Example • Blood agar – Certain bacteria produce hemolysin to break down RBC • MacConkey agar – Contains pH indicator to identify bacteria that produce acid 16 1/21/2011 Laboratory Cultivation • Providing appropriate atmospheric conditions diti • Bacteria can be grouped by oxygen requirement – Capnophile – Microaerophile – Anaerobe – Aerobe Laboratory Cultivation 17 1/21/2011 Obtaining a Pure Culture Streak Plate Detecting Bacterial Growth • Variety of techniques to determine growth – Numbers of cells – Total mass – Detection of cellular products 18 1/21/2011 Detecting Bacterial Growth • Direct cell count – Useful in determining total number of cells – Does not distinguish between living and dead cells – Methods include • Direct microscopic count • Use of cell counting instruments Direct Counts Counting chamber • Counting Chamber Cell counting instruments 19 1/21/2011 Detecting Bacterial Growth • Viable cell count – Used to quantify living cells • Cells able to multiply – Valuable in monitoring bacterial growth • Often used when cell counts are too low for other methods – Methods include • Plate counts • Membrane filtration • Most probable numbers Viable Cell Counts • Plate method • Membrane filtration 20 1/21/2011 Viable Cell Counts: The Most Probable Number (MPN) Method Measure Biomass: Turbidity gives a rough estimate 21 1/21/2011 Summary • General principles – binary fission, exponential growth, generation time • Factors that influence growth – environmental factors; temperature, O2 availability, pH, aw, nutrients – nutritional factors; required elements, growth factors, energy source • Bacterial growth in laboratory conditions – closed system; the growth curve • Bacterial growth in nature – open system; dynamic conditions, mixed microbial communities, biofilms • Cultivating bacteria in the laboratory – characteristics of media • Methods to detect and measure bacterial growth – direct, viable, measuring biomass 22