Dynamics of Prokaryotic Growth

Transcription

1 Dynamics of Prokaryotic Growth Chapter 4 Principles of Bacterial Growth 1

2 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 health consequences Generation time Time it takes for population to double a.k.a. doubling time Varies among species Principles of Bacterial Growth Growth can be calculated N t = N 0 x 2 n (N t ) number of cells in population (N 0 ) original number of cells in the population (n) number of divisions Example N 0 = 10 cells in original population n = 12» 4 hours assuming 20 minute generation time N t = 10 x 2 12 N t = 10 x 4,096 N t = 40,960 Generation Times Vary for Each Organism E. coli Mycobacterium tuberculosis 2

3 Bacterial Growth in Laboratory Conditions Cells in laboratory grown in closed or batch system 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 Bacterial Growth in Laboratory Conditions Lag phase Number of cells does not increase Cells prepare for growth Tooling up Log gphase Period of exponential growth Doubling of population with each generation Stationary phase Overall population remains relatively stable Cells exhausted nutrients Cell growth = cell death 3

4 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 Environmental Factors on Growth Temperature Each species has welldefined temperature range Within range lies optimum growth temperature Prokaryotes divided id d into 5 categories Psychrophile Optimum temperature -5 C to 15 C 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 O 2 4

5 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 halophiles Nutritional Factors on Growth Growth of prokaryotes depends on nutritional factors as well as physical environment Main factors to be considered are: Required elements Growth factors Energy sources Nutritional diversity 5

6 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. H 2 S) Sunlight ( photo ) o Carbon sources: CO 2 ( auto ) (carbon fixation) Organic cmpds ( hetero ) troph = nourish Nutritional Factors on Growth Required Elements: Nutritional Factors on Growth Carbon Sources: CO 2 (Carbon fixation) Organic Compounds Nitrogen Sources: Organic Compounds Inorganic Compound N 2 (Nitrogen fixation) 6

7 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. Nutritional Factors on Growth Carbon Sources: CO 2 (Carbon fixation) Organic Compounds Nitrogen Sources: Organic Compounds Inorganic Compound N 2 (Nitrogen fixation) Limiting Nutrients Limiting Nutrients 7

8 Nutritional Factors on Growth 1 pkg chocolate chips 3 c sugar 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 growth in the lake by adding 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,.. 8

9 Bacterial Growth in Nature Biofilm layer 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 Bacterial Growth in Nature Interactions of mixed microbial communities 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 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 9

10 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 Chemically defined media Composed of precise amounts of pure chemical Generally not practical for routine laboratory use Special types of culture media Used to detect or isolate particular organisms Divided into selective and differential media 10

11 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 agar For isolation of Gram-negative bacteria 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 Providing appropriate atmospheric conditions Bacteria can be grouped by oxygen requirement Capnophile Microaerophile Anaerobe Aerobe 11

12 Obtaining a Pure Culture Streak Plate Detecting Bacterial Growth Variety of techniques to determine growth Numbers of cells Total mass Detection of cellular products 12

13 Detecting Bacterial Growth Direct cell count Useful in determining total number of cells Does not distinguish between living and dead d cells Methods include Direct microscopic count Use of cell counting instruments Direct Counts Counting chamber Counting Chamber Cell counting instruments 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 13

14 Viable Cell Counts Plate method Membrane filtration Viable Cell Counts: The Most Probable Number (MPN) Method Measure Biomass: Turbidity gives a rough estimate 14

15 Summary General principles binary fission, exponential growth, generation time Factors that influence growth environmental factors; temperature, O 2 availability, ph, a w, 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 15