Chapter 7 Microbial Growth PDF

CHAPTER 7 MICROBIAL GROWTH Microbial Growth Phases of Growth Measurement of Growth Environmental Factors  Microbial Growth Microbial growth refers to the increase in: – cell size – cell number Microbes grow and divide by binary fission and budding ...

CHAPTER 7 MICROBIAL GROWTH Microbial Growth Phases of Growth Measurement of Growth Environmental Factors  Microbial Growth Microbial growth refers to the increase in: – cell size – cell number Microbes grow and divide by binary fission and budding  Cell Division Binary fission – equal cell division Budding – unequal cell division Binary Fission Budding in Yeast  Phases of Growth Four major phases of growth: Lag phase Log phase (exponential) Stationary phase Death phase Growth Curve  Lag Phase Little or no cell division Adapt/adjust to new conditions metabolically active - grow in size, synthesize enzymes, and incorporate molecules from medium produce large quantities of energy in the form of ATP  Exponential Phase also called logarithmic (log) phase Highest metabolic activity metabolically active most rapid growth population doubles every generation Number cell produced>number cells dying  Stationary Phase Number cell produced = number cells dying Cell division slows down total number of viable cells constant limited amount of nutrient limited oxygen supply metabolic waste accumulates critical population density  Death Phase also called decline phase Number cell produced < number cells dying(decreases at exponential rate) metabolic waste accumulates and becomes toxic to cells cell lose their ability to divide and thus die Bacterial Growth Phase  Generation (doubling) time – time required for a population to double in the number of cells – varies, depending on species of microorganism and environmental conditions – range from 20 minutes for most bacteria to several days for some eukaryotic microorganisms Can we maintain the growth of microorganisms in log phase?  Continuous Culture of Microorganisms achieved using continuous culture system growth in an open system – continual provision of nutrients – continual removal of wastes cells maintained in log phase at constant biomass concentration for extended periods e.g: bioreactor, chemostat, turbidostat Chemostat  Photobioreactor Uses light to cultivate phototrophic microorganisms Importance of Continuous Culture Methods constant supply of cells food and industrial microbiology study of microbial growth at very low nutrient concentrations (close to those present in environments) study of interactions of microbes under certain conditions Measurement of Growth Direct Methods of Measurement 1. Standard Plate Count 2. Membrane Filtration 3. Direct Microscopic Count Measurement of Growth Indirect Methods of Measurement 1. Turbidity - Spectrophotometric analysis 2. Metabolic activity (Most Probable Number (MPN) 3. Dry weight  Standard Plate Count Standard Plate Count – dilution of original bacterial culture with known volume – transfer each dilution onto agar plate (spread/pour plate) – measure viable cells only  Serial dilution Countable number of colonies (30 to 300 per plate) Countable number of colonies (30 to 300 per plate) Counting colonies using a digital bacterial colony counter  Membrane Filtration measure viable cells only useful in analyzing water purity  measure total coliform count  Direct Microscopic Count Petroff-Hausser counting chamber (hemocytometer) Electronic counter Petroff-Hausser counting chamber (hemocytometer) bacterial suspension introduced onto chamber with calibrated pipette microorganisms counted in specific calibrated areas number per unit volume is calculated using appropriate formula measure viable and non-viable cells Petroff-Hausser Counting Chamber  Direct Microscopic Count Electronic counter e.g. Coulter counter count larger microorganisms such as protozoa and yeast uses current flow that measures electrical resistance caused by microbial cells measure viable and non-viable cells  Direct Microscopic Count. Coulter counter suspension flows through capillary section between two electrodes resistance is measured when a particle passes through the current Most Probable Number (MPN) method to estimate number of cells used when samples contain too few organisms MPN test consists of five tubes each of three volumes (e.g. 10, 1, 0.1ml) principal: fermentation of carbohydrate fermentation products are acid (e.g. lactic acid, acetic acid) or  MPN Test (Carbohydrate Fermentation) +Gas/+Acid -Gas/ -Gas/-Acid +Acid lactose/ glucose nutrient broth pH indicator Durham tube +ve +ve -ve MPN test tubes with gas bubbles (labeled +) contain organisms Turbidity- Spectrophotometric Analysis cloudy appearance indicates bacterial growth Spectrophotometer measure turbidity determine degree of light transmission through bacterial culture measure viable and non-viable cells Turbidity As bacteria multiply, it becomes turbid Advantages No incubation time required Disadvantages Cannot distinguish live and dead High concentration bacteria required Metabolic activity As bacteria multiply, the accumulate certain products (CO2, acids) Measure metabolic products Disadvantages- Expensive Dry weight Microbes centrifuged Resulting cell pellet weighed Disadvantages- Cannot distinguish live and dead  CHAPTER 7 MICROBIAL GROWTH Microbial Growth Phases of Growth Measurement of Growth Environmental Factors  Environmental Factors most organisms grow in moderate environmental conditions extremophiles – grow under harsh conditions that would kill most other organisms  Environmental Factors Affecting Bacterial Growth Temperature pH Oxygen Hydrostatic pressure Water activity & solutes Radiation Temperature  Temperature Microorganisms can be classified based on their optimum growth temperature : Psychrophiles : 15 - 20oC Mesophiles : 25 - 40oC Thermophiles : 50 - 60oC Extreme thermophiles : Thermophilic sulfur bacteria can live and grow in runoff waters from such geysers despite the near-boiling temperatures Application of thermophiles Growth rates of psychrophilic, mesophilic, and thermophilic bacteria How microorganisms can survive in extreme temperatures?  Adaptation of Thermophiles stable protein structure – e.g. more H bonds, proline, chaperones histone-like proteins stabilize DNA stabilized membrane – e.g. more saturated, more branched and higher molecular weight lipids – e.g. ether linkages (archaeal membranes)  pH Acidophiles – optimum growth pH 0.1 - 5.5 Neutrophiles – optimum growth pH 5.5 and pH 7 Alkalophiles – optimum growth pH 8.5 and pH 11.5  pH Adaptation of acidophiles and alkalophiles: maintain internal pH near neutrality – plasma membrane is impermeable to protons synthesize proteins for protection – e.g. acid-shock proteins change pH of their habitat by producing acidic or basic waste products – most media contain buffers to prevent  Oxygen Obligate – organism needs strictly specific environmental condition Facultative – organism is able to adjust and tolerate to environmental condition  Oxygen Aerobes – require oxygen to grow Obligate aerobes – must have free oxygen for aerobic respiration (e.g. Pseudomonas sp.) Anaerobes – do not require oxygen to grow Obligate anaerobes – killed by free oxygen (e.g. Bacteroides sp.)  Oxygen Facultative anaerobes – perform aerobic metabolism when oxygen is present, but shift to anaerobic metabolism when oxygen is absent (e.g. E. coli, Staphylococcus sp.) Aerotolerant anaerobes – can survive in the presence of oxygen but do not use it in their metabolism (e.g. Lactobacillus sp.)  Oxygen Microaerophiles – grow best in presence of small amount of free oxygen (e.g. Campylobacter sp.) Capnophiles – thrive in the presence of high concentrations of carbon dioxide (require carbon dioxide to survive)  Oxygen need prefer ignore oxygen is < 2 – 10% oxyge oxygen oxygen toxic oxygen n  Basis of Different Oxygen Sensitivities oxygen easily reduced to toxic products – superoxide radical – hydrogen peroxide – hydroxyl radical aerobes produce protective enzymes – superoxide dismutase (SOD) – catalase Anaerobic jar used to grow obligate anaerobes sealed jar with GasPak GasPak contains chemical that generates H2 and CO2 H2 combines with oxygen and forms water CO2 causes rapid Anaerobi c chamber  Hydrostatic Pressure Water in oceans and lakes Pressure doubles with every 10m depth  Hydrostatic Pressure Barophiles – bacteria that live at high hydrostatic pressures, but die if left in laboratory at standard atmospheric pressure e.g. Halomonas salaria requires 100 MPa Barotolerant – grows best in standard atmospheric pressure, but can survive and grow in high pressure environments  Water Activity and Solutes water activity (aw) – amount of water available to organisms – reduced by interaction with solute molecules (osmotic effect) higher [solute]  lower aw Osmotolerant microorganisms grow over wide ranges of water activity many use compatible solutes to increase internal osmotic concentration – solutes that are compatible with metabolism and growth some have proteins and membranes that require high Effects of NaCl on Microbial Growth Halophiles – organisms that grow in environments with very high concentrations of salt – found in ocean (salt conc. 3.5%) – grow at >0.2 M of NaCl – e.g. marine organisms Cultivation of Marine Bacteria Can be cultured in:  marine agar  sea water agar (bacto agar + sea water) Marine agar is formulated to duplicate the major mineral concentration found in sea water Contains all necessary nutrients to cultivate most marine bacteria e.g: Gammaproteobacteria, Effects of NaCl on Microbial Growth Extreme halophiles – requires salt conc. 20-30% (e.g. Dead Sea) – require >2 M of NaCl – e.g. Halobacterium sp. Halotolerant – organism that does not require NaCl to grow but can tolerate its presence Effects of NaCl on Microbial Growth Dead Sea The Great Salt Lake in Utah Radiation  Radiation ionizing radiation – x rays and gamma rays – mutations death – disrupts chemical structure of many molecules, including DNA UV radiation – mutations death – causes formation of thymine dimers in DNA  Microbial Growth in Natural Environments microbial environments are complex, constantly changing often contain low nutrient concentrations (oligotrophic environment) may expose microorganism to overlapping gradients of nutrients  Responses to Oligotrophic Environments organisms become more competitive in nutrient capture and use of available resources morphological changes to increase surface area and ability to absorb nutrients mechanisms to sequester certain nutrients -- End of Chapter 7--