Dynamics of Prokaryotic Growth Chapter 4

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 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 Nt = N0 x 2n (Nt ) number of cells in population right now (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

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 Big question: how applicable is growing these things in lab if they do different things in nature.

Bacterial Growth in Nature Biofilm Formation begins when planktonic bacteria attach to surfaces Other bacteria attach and grow on initial layer Has characteristic architecture Contains open channels for movement of nutrients and waste Cells within biofilms can cause disease Treatment becomes difficult Architecture resists immune response and antimicrobials Bioremediation is beneficial use of biofilm Add nutrients - biostimulation Add other bacteria - bioaugmentation

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

Obtaining Pure Culture Pure culture defined as population of cells derived from single cell All cells genetically identical Cells grown in pure culture to study functions of specific species Pure culture obtained using special techniques Aseptic technique -minimizes potential contamination Cells grown on culture media Can be broth (liquid) or solid form

Obtaining Pure Culture Culture media can be liquid or solid Liquid is broth media Used for growing large numbers of bacteria Solid media is broth media with addition of agar Agar marine algae extract Liquefies at temperatures above 95°C Solidifies at 45°C Remains solid at room temperature and body temperature Bacteria grow in colonies on solid media surface All cells in colony descend from single cell Approximately 1 million cells produce 1 visible colony

Obtaining Pure Culture Streak-plate method Simplest and most commonly used in bacterial isolation Object is to reduce number of cells being spread Solid surface dilution Each successive spread decreases number of cells per streak Multiple techniques

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 especially in broths 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 phase Period of exponential growth Doubling of population with each generation Produce primary metabolites Compounds required for growth Late log phase Endospores Synthesize secondary metabolites Used to enhance survival Antibiotics

Bacterial Growth in Laboratory Conditions Stationary phase Overall population remains relatively stable Cells exhausted nutrients Cell growth = cell death Dying cell supply metabolites for replicating cells Death phase Total number of viable cells decreases Decrease at constant rate Death is exponential Much slower rate than growth

Bacterial Growth in Laboratory Conditions Phase of prolonged decline Once nearly 99% of all cells dead, remaining cells enter prolonged decline Marked by very gradual decrease in viable population Phase may last months or years Most fit cells survive Each new cell more fit than previous Evolution – change in genetics over time

World population

Bacterial Growth in Laboratory Conditions Colony growth on solid medium In colony, cells eventually compete for resources Position within colony determines resource availability Cells on edge of colony have little competition and significant oxygen stores Cells in the middle of colony have high cell density Leads to increased competition and decreased availability of oxygen

Chemostats allow for continuous growth

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 well defined temperature range Within range lies optimum growth temperature Prokaryotes divided 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 Psychro = “cold”

Environmental Factors on Growth Oxygen Prokaryotes divided based on oxygen requirements Obligate aerobes Absolute requirement for oxygen Use for energy production Facultative anaerobes Grow better with oxygen Use fermentation in absence of oxygen Microaerophiles Require oxygen in lower concentrations Higher concentration inhibitory Obligate anaerobes No multiplication in presence of oxygen May cause death Aerotolerant anaerobes Indifferent to oxygen, grow with or without Do not use oxygen to produce energy

Oxygen

Environmental Factors on Growth pH Bacteria survive within 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 Synthesize small organic molecules Osmotolerant bacteria tolerate high salt environments Halophiles - bacteria that require high salt for cell growth termed

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

Nutritional Factors on Growth Required elements Major elements Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium and iron Essential components for macromolecules Organisms classified based on carbon usage Heterotrophs Use organic carbon as nutrient source Autotrophs Use inorganic carbon (CO2) as carbon source Trace elements Cobalt, zinc, copper, molybdenum and manganese Required in minute amounts

Nutritional Factors on Growth Growth factors Some bacteria cannot synthesize some cell constituents These must be added to growth environment Organisms can display wide variety of factor requirements Some need very few while others require many These termed fastidious

Nutritional Factors on Growth Energy Sources Organisms derive energy from sunlight or chemical compounds Phototrophs Derive energy from sunlight Chemotrophs Derive energy from chemical compounds Organisms often grouped according to energy source

Nutritional Factors on Growth Nutritional Diversity Organisms thrive due to their ability to use diverse sources of carbon and energy Photoautotrophs Use sunlight and atmospheric carbon (CO2) as carbon source Called primary producers (Plants) Chemoautotrophs a.k.a chemolithoautotrophs or chemolitotrophs Use inorganic compounds for energy and use CO2 as carbon source Photoheterotrophs Energy from sunlight, carbon from organic compounds Chemoheterotrophs a.k.a chemoorganoheterotrophs or chemoorganotrophs Use organic compounds for energy and carbon source

Laboratory Cultivation Knowing environmental and nutritional factors makes it possible to cultivate organisms in the laboratory Organisms are grown on culture media complex media 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

Laboratory Cultivation Chemically defined media Composed of precise amounts of pure chemical Generally not practical for routine laboratory use Invaluable in research Each batch is chemically identical Does not introduce experimental variable

Laboratory Cultivation Special types of culture media Used to detect or isolate particular organisms Are divided into selective and differential media

Laboratory Cultivation Selective media Inhibit the growth of unwanted organisms Allow only sought after organisms to grow Example Thayer-Martin agar - antibiotics For isolation of Neisseria gonorrhoeae MacConkey agar – crystal violet and bile salts For isolation of Gram negative bacteria of intestine

Laboratory Cultivation Differential media Contains substance that bacteria change in recognizable way Example Blood agar Certain bacteria produce hemolysin to break down RBC Hemolysis MacConkey agar Contains pH indicator to identify bacteria that produce acid

Laboratory Cultivation Providing appropriate atmospheric conditions Bacteria can be grouped by oxygen requirement Capnophile Microaerophile Anaerobe

Laboratory Cultivation Capnophile Require increased CO2 Achieve higher CO2 concentrations via Candle jar CO2 incubator Microaerophile Require higher CO2 than capnophile Incubated in gastight jar Chemical packet generates hydrogen and CO2

Laboratory Cultivation Anaerobe Die in the presence of oxygen Even if exposed for short period of time Incubated in anaerobe jar Chemical reaction converts atmospheric oxygen to water Organisms must be able to tolerate oxygen for brief period Reducing agents in media achieve anaerobic environment Agents react with oxygen to eliminate it Sodium thyoglycolate Anaerobic chamber also used for cultivation

Detecting Bacterial Growth Variety of techniques to determine growth Numbers of cells Total mass Detection of cellular products

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

Detecting Bacterial Growth Direct microscopic count One of the most rapid methods Number is measured in a known volume Liquid dispensed in specialized slide Counting chamber Viewed under microscope Cells counted Limitation Must have at least 10 million cells per mL to gain accurate estimate

Detecting Bacterial Growth Cell counting instruments Count cells in suspension Cells pass counter in single file Measure changes in environment Coulter counter Detects changes in electrical resistance Flow cytometer Measures laser light

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

Detecting Bacterial Growth Plate counts Measures viable cells growing on solid culture media Count based on assumption that one cell gives rise to one colony Number of colonies = number of cells in sample Ideal number to count between 30 and 300 colonies Sample normally diluted in 10-fold increments Plate count methods pour-plates spread-plates methods

Detecting Bacterial Growth Membrane filtration Used with relatively low numbers Known volume of liquid passed through membrane filter Filter pore size retains organism Filter is placed on appropriate growth medium and incubated Cells are counted

Detecting Bacterial Growth Most probable numbers (MPN) Statistical assay Series of dilution sets created Each set inoculated with 10-fold less sample than previous set Sets incubated and results noted Results compared to MPN table Table gives statistical estimation of cell concentration

Detecting Bacterial Growth Biomass measurement Cell mass can be determined via Turbidity Total weight Amounts of cellular chemical constituents

Detecting Bacterial Growth Turbidity Measures with spectrophotometer Measures light transmitted through sample Measurement is inversely proportional to cell concentration Must be used in conjunction with other test once to determine cell numbers Limitation Must have high number of cells

Detecting Bacterial Growth Total Weight Tedious and time consuming Not routinely used Useful in measuring filamentous organisms Wet weight Cells centrifuged down and liquid growth medium removed Packed cells weighed Dry weight Packed cells allowed to dry at 100°C 8 to 12 hours Cells weighed

Detecting Bacterial Growth Detecting cell products Acid production pH indicator detects acids that result from sugar breakdown Gas production Gas production monitored using Durham tube Tube traps gas produced by bacteria ATP Presence of ATP detected by use of luciferase Enzyme catalyzes ATP dependent reaction If reaction occurs ATP present  bacteria present