Lecture 4: Microbial Growth PDF

Lecture 4 Microbial Growth Dr Heather Nesbitt [email protected] Lecture 4: Microbial Growth Learning outcomes: A successful student will be able to show that he/she can: Define and describe the types of microbial growth Describe the techniques used to monitor microbial gr...

Lecture 4 Microbial Growth Dr Heather Nesbitt [email protected] Lecture 4: Microbial Growth Learning outcomes: A successful student will be able to show that he/she can: Define and describe the types of microbial growth Describe the techniques used to monitor microbial growth Describe the external factors that can affect microbial growth Describe the requirements for microbial growth Viruses  Growth Binary Fission When we speak of bacterial growth, we are talking about an increase in population size not an increase in the size of a single bacterium Bacterial cells replicate by a form of asexual reproduction called binary fission Budding in yeasts Growth of Microbial Populations  Generation Time Generation Time – time required to complete fission cycle from parent cell to two daughter cells. (Doubling time) In terms of a population it is the amount of time needed to double the population The length of the generation time is a measure of the Growth Rate of the microbe It varies depending on environmental conditions Different microbes have different generation times. Mycobacterium leprae  10-30 days Staphylococcus aureus  20-30 minutes Growth curve When the number of cells is plotted as the logarithm of the cell numbers versus time we obtain the sigmoid curve. What are the characteristics of each phase? Microbial Growth Cycle (batch culture) Batch culture: a closed-system microbial culture of fixed volume Typical growth curve for population of cells grown in a closed system is characterized by four phases Lag phase Log or Exponential phase Stationary phase Death phase  Microbial Growth Cycle (batch culture) Lag phase Interval of time between when a culture is inoculated and when growth begins Exponential phase Cells in this phase are typically in the healthiest state. Growth is at maximal rate. Stationary phase Growth rate of population is zero. Number new divisions=number of cells dying Either an essential nutrient is used up or waste product of the organism accumulates in the medium Death phase Lack of nutrients and increasing accumulation of wastes lead to… number of cell deaths > number of new divisions Under batch conditions, exponential growth can only be maintained for a few generations. Why? Since the nutrients are not renewed, exponential growth is limited to a few generations. Exponential Growth Number of cells doubles during each unit of time During exponential growth, the increase in cell number is initially slow but increases at a faster rate Cell population size can be represented by 2N (where n = the number of generations) Predicting the number of cells (N) Nfinal=(Ninitial)x2n Logarithmic versus arithmetic growth representation  Find out what Cryptic growth is Diauxic growth curve  Continuous Culture We may need to maintain cultures under constant environments for long periods How can we obtain continuous growth in the laboratory? Continuous culture: an open-system microbial culture of fixed volume Chemostat and turbidostat : most common types of continuous culture device  Chemostat Fresh medium continually supplied from a reservoir of sterile medium Volume maintained at constant level by overflow drain Bacteria grow at same rate as bacterial cells and spent medium are removed Rate of addition of fresh medium determines rate of growth  Turbidostat Turbidity of culture is held constant by manipulating rate at which medium is fed If turbidity increases, feed rate is increased to dilute turbidity back to set point If turbidity falls, feed rate is lowered so that growth can restore turbidity to its set point Turbidity is measured usually by spectrophotometer http://2012.igem.org/wiki/images/8/85/ Washington_Turbidostat.png  Important terminology Dilution rate, flow rate, volume; D= F/V Constant growth rate (µ) and (µmax.) Steady state condition Washout Why is it important to achieve a steady state before sampling for measurements ?  Measurement of growth Measurements of cell numbers Direct counting (Petroff-Hausseer counting chamber or Hemocytometers or Electronic Coulter Counters) Plating techniques (Colony Forming Units - CFUs) Pore plate Spread plate Membrane filter Measurements of cell mass Dry weight Scattering of light (spectrophotometrically) Measurements of cell constituents Total protein or nitrogen Chlorophyll ATP Nucleic acids  Measuring Growth (Direct Measurement) Total Cell Count  Direct Microscopic examination using special slides  Automated counters (flow cytometry)  Measuring Growth (Direct Measurement) Total Cell Count Advantages No incubation time required Disadvantages Cannot always distinguish between live and dead bacteria. Motile bacteria are difficult to count Requires a high concentration of bacteria (10 million/ml)  Viable Count (Direct Measurement) Measurement of living, reproducing population Two main ways to perform plate counts Spread-plate method Pour-plate method To obtain the appropriate colony number, the sample to be counted may need to be diluted (serial dilutions) Spread-Plate Method for the Viable Count Pour-Plate Method for the Viable Count Serial dilutions Procedure for Viable Counting Using Serial Dilutions  Viable Count (Direct Measurement) General Advantages Measures viable cells Disadvantages Takes 24 hours or more for visible colonies to appear Only counts between 25 and 250 colonies are accurate Must perform serial dilutions to get appropriate numbers/plate  Viable Count (Direct Measurement) Specific Spread plate Advantages Colonies on surface and not exposed to melted agar Pour plate Disadvantages Not useful for heat sensitive organisms Colonies appear under agar surface  Measuring Growth (Direct Measurement) Filtration Most Probable Number A statistical estimating technique http://classes.midlandstech.edu/carterp/courses/bio225/chap06/Microbial%20Growth%20ss5.htm  Measuring Growth (Indirect Measurement) Metabolic activity Oxygen consumption Carbon dioxide production Acid production Expensive Dry weight Bacteria or fungi in liquid media are centrifuged. Resulting cell pellet is weighed Does not distinguish live and dead cells.  Measuring Growth (Indirect Measurement) Turbidity As bacteria multiply in media, it becomes turbid Use a spectrophotometer to determine % transmission or absorbance Multiply by a factor to determine concentration Absorbance is related to the number of bacteria Advantages No incubation time required. Disadvantages Cannot distinguish between live and dead bacteria. Requires a high concentration of bacteria (10 to 100 million cells/ml) Turbidity  Factors affecting Growth Physical requirements for growth Temperature Psychrophiles, Psychrotorphs, Mesophiles, Thermophiles and Extreme Thermophiles pH and use of Buffers Most bacteria are neutrophiles (pH 6.5-7.5) Yeast and molds (pH 4.0-6.0) Some are acidophiles pH < 4 Extremes (e.g. Sulfur oxidizers) can grow at pH 1-2. Alkalophiles pH > 9.0 Osmotic Pressure Hypertonic solutions/environment leads to plasmolysis Hypotonic solutions/environment leads to plasmoptisis Halophilic & Saccharophilic microorganisms (osmotolerant)  Temperature and Microbial Growth Temperature is a major environmental factor controlling microbial growth Cardinal temperatures: the minimum optimum maximum temperatures at which an organism grows Effects of Temperature on Microbial Growth  Microbial Growth at Hot Temperatures Extremophiles Organisms that have evolved to grow optimally under very hot or very cold conditions Thermophiles love heat Hyperthermophiles produce enzymes widely used in industrial microbiology e.g., Taq polymerase; used to automate the repetitive steps in the polymerase chain reaction (PCR) technique Effects of Temperature on Microbial Growth Psychrophiles Organisms with cold temperature optima The most extreme representatives inhabit permanently cold environments Psychrotolerant Organisms that can grow at 0ºC but have optima of 20ºC to 40ºC More widely distributed in nature than psychrophiles Mesophiles Organisms that have midrange temperature optima Found in warm-blooded animals, terrestrial and aquatic environments, temperate and tropical latitudes pH relation to growth Organisms sensitive to changes in acidity because H+ and OH- interfere with H bonding in proteins and nucleic acids Most bacteria and protozoa grow best in a narrow range around neutral pH (6.5-7.5) – these organisms are called neutrophiles Other bacteria and fungi are acidophiles – grow best in acidic habitats Acidic waste products can help preserve foods by preventing further microbial growth Alkalinophiles live in alkaline soils and water up to pH 11.5 Physical Effects of Water Microbes require water to dissolve enzymes and nutrients required in metabolism Water is important reactant in many metabolic reactions Most cells die in absence of water Some have cell walls that retain water Endospores and cysts cease most metabolic activity in a dry environment for years Two physical effects of water or salt Osmotic pressure Hydrostatic pressure Halophiles (salt lovers) Isotonic Versus Hypertonic Solution - Plasmolysis Osmotic Pressure Is the pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane; related to concentration of dissolved molecules and ions in a solution Hypotonic solutions have lower solute concentrations; cells placed in these solutions will swell and burst Hypertonic solutions have greater solute concentrations; cells placed in these solutions will undergo crenation (shriveling of cytoplasm) This effect helps preserve some foods Restricts organisms to certain environments Obligate halophiles – grow in up to 30% salt Facultative halophiles – can tolerate high salt concentrations  Hydrostatic Pressure Water exerts pressure in proportion to its depth For every addition of depth, water pressure increases 1 atm Organisms that live under extreme pressure are barophiles Their membranes and enzymes depend on this pressure to maintain their three-dimensional, functional shape  Oxygen and Microbial Growth Aerobes require oxygen to live Pseudomonas Anaerobes do not require oxygen and may even be killed by exposure Clostridium Facultative organisms can live with or without oxygen E. coli, Staphylococcus Aerotolerant anaerobes can tolerate oxygen and grow in its presence even though they cannot use it Lactobacillus Microaerophiles can use oxygen only when it is present at levels reduced from that in air Campylobacter Growth Versus Oxygen Concentration Figure 6.27  Necessary enzymes for life with oxygen Superoxide dismutase (SOD) First defense against toxic intermediates of oxygen Accelerates spontaneous supeoxide dismutation reaction: O2- + O2- + 2H+ O 2 + H 2 O2 Catalase Second defense against toxic intermediates of oxygen Catalyses inactivation of peroxide: H 2 O2 + H 2 O2 O2 + 2 H 2 O Peroxidase Further defense against toxic intermediates of oxygen Also catalyses inactivation of peroxide: Remember these enzymes H2O2 + 2H+ 2 H 2O are absent from anaerobic bacteria  Chemical requirements for growth Carbon & energy sources Chemoautotrophes Chemohetrotrophes Photoautotrophes Photohetrotrphes Nitrogen: Makes up 14% of dry cell weight. Used to form amino acids, DNA, and RNA. Sources of nitrogen Protein: Most bacteria Ammonium: Found in organic matter Nitrogen gas (N2): Obtain N directly from atmosphere. Important nitrogen fixing bacteria, live free in soil or associated with legumes (peas, beans, alfalfa, clover, etc.). Legume cultivation is used to fertilize soil naturally. Nitrates: Salts that dissociate to give NO3-  Chemical requirements for growth Sulphur for Sulphur-containing amino acids and some vitamins (thiamin and biotin) Sources of sulphur Protein: Most bacteria Hydrogen sulphide Sulphates: Salts that dissociate to give SO 4 Phosphorus: Used to form DNA, RNA, ATP, and phospholipids Sources of phosphorus Mainly inorganic phosphate salts and buffers. Others are K, Mg, Ca, trace elements, and organic growth factors Nutritional categories in microorganisms  Viruses Obligate intracellular parasites of cells (mammalian, plant and microbial) Can exist in an extracellular form and can be transmitted from one cell (host) to another (i.e cause disease) Enveloped or non-enveloped (naked) viruses Types of virus: DNA or RNA Some important DNA viruses  Some important RNA viruses Polio HIV Influenza Measles Lecture 4: Microbial Growth Learning outcomes: A successful student will be able to show that he/she can: Define and describe the types of microbial growth Describe the techniques used to monitor microbial growth Describe the external factors that can affect microbial growth Describe the requirements for microbial growth Viruses