Microbiology An Introduction, Chapter 6, Microbial Growth PDF

Microbiology an Introduction Twelfth Edition Chapter 6 Microbial Growth Copyright © 2016 Pearson Education, Inc. All Rights Reserved Microbial Growth Microbial growth = increase in number of cells, not cell...

Microbiology an Introduction Twelfth Edition Chapter 6 Microbial Growth Copyright © 2016 Pearson Education, Inc. All Rights Reserved Microbial Growth Microbial growth = increase in number of cells, not cell size Copyright © 2016 Pearson Education, Inc. All Rights Reserved The Requirements for Growth Physical requirements – Temperature – pH – Osmotic pressure Chemical requirements – Carbon – Nitrogen, sulfur, and phosphorous – Trace elements – Oxygen – Organic growth factors Copyright © 2016 Pearson Education, Inc. All Rights Reserved Physical Requirements Temperature – Minimum growth temperature – Optimum growth temperature – Maximum growth temperature Copyright © 2016 Pearson Education, Inc. All Rights Reserved Physical Requirements – Psychrophiles—cold- – Thermophiles loving (-15 to 200C)  Optimum growth temperature of 50 – Mesophiles—moderate- to 60°C temperature-loving (25-  Found in hot springs and organic 400C). compost – Psychrotrophs – Hyperthermophiles  Grow between 0°C  Optimum growth temperature > 80°C and 20-30°C  Optimum 4oC  Cause food refrigerator food spoilage Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.2 Food Preservation Temperatures (Psychrotrophs) The Effect of the Amount of Food on its Cooling Rate in a Refrigerator and its Chance of Spoilage Copyright © 2016 Pearson Education, Inc. All Rights Reserved pH Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Acidophiles grow in acidic environments Acidity inhibits most microbial growth and is used frequently for food preservation (e.g.: pickling). Alkalinity inhibits microbial growth, but not commonly used for food preservation. Copyright © 2016 Pearson Education, Inc. All Rights Reserved pH Acidophiles: “Acid loving”. – Grow at very low pH (0.1 to 5.4) – Lactobacillus produces lactic acid, tolerates mild acidity. Neutrophiles: – Grow at pH 5.4 to 8.5. – Includes most human pathogens. Alkaliphiles: “Alkali loving”. – Grow at alkaline or high pH (7 to 12 or higher) – Soil bacterium Agrobacterium grows at pH 12. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Osmotic Pressure Hypertonic environments (higher in solutes than inside the cell) cause plasmolysis due to high osmotic pressure Copyright © 2016 Pearson Education, Inc. All Rights Reserved Osmotic Pressure Cells are 80 to 90% water. Hypertonic solutions: High osmotic pressure removes water from cell, causing shrinkage of cell membrane (plasmolysis). Used to control spoilage and microbial growth. – Sugar in jelly. – Salt on meat. Hypotonic solutions: Low osmotic pressure causes water to enter the cell. In most cases cell wall prevents excessive entry of water. Microbe may lyse or burst if cell wall is weak. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Osmotic Pressure Halophiles: Require moderate to large salt concentrations. Ocean water contains 3.5% salt. – Most bacteria in oceans. Extreme or Obligate Halophiles: Require very high salt concentrations (20 to 30%). – Bacteria in Dead Sea, brine vats. Facultative Halophiles: Do not require high salt concentrations for growth, but tolerate 2% salt or more. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Chemical Requirements Carbon – Structural backbone of organic molecules – Chemoheterotrophs use organic molecules as carbon – Autotrophs use CO2 Nitrogen – Component of proteins, DNA, and ATP – Most bacteria decompose protein material for the nitrogen source – Some bacteria use NH4+ or NO3- from organic matter – A few bacteria use N2 in nitrogen fixation Copyright © 2016 Pearson Education, Inc. All Rights Reserved Chemical Requirements Sulfur – Used in amino acids, thiamine, and biotin – Most bacteria decompose protein for the sulfur source – Some bacteria use SO4 2 or H2S Phosphorus – Used in DNA, RNA, and ATP – Found in membranes ‒ PO43  is a source of phosphorus Copyright © 2016 Pearson Education, Inc. All Rights Reserved Chemical Requirements Oxygen: Organisms that use molecular oxygen (O2), produce more energy from nutrients than anaerobes. Obligate Aerobes: Require oxygen to live. – Disadvantage: Oxygen is not found in all environments and dissolves poorly in water. – Example: Pseudomonas, common nosocomial pathogen. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Chemical Requirements Facultative Anaerobes: Prefer to use oxygen, but can grow in its absence. Have complex set of enzymes – Examples: E. coli, Staphylococcus, yeasts, and many intestinal bacteria. Obligate Anaerobes: Cannot use oxygen and are harmed by the presence of toxic forms of oxygen. – Examples: Clostridium bacteria that cause tetanus and botulism. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Chemical Requirements Aerotolerant Anaerobes: Can’t use oxygen, but tolerate its presence. Can break down toxic forms of oxygen. Oxygen has no effect on growth. – Example: Lactobacillus carries out fermentation regardless of oxygen presence. Microaerophiles: Require oxygen, but at low concentrations. Sensitive/toxic to oxygen. – Example: Campylobacter. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Trace Elements & Organic Growth Factors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines Trace elements: Inorganic elements required in small amounts Usually as enzyme cofactors Include iron, copper, molybdenum, and zinc Copyright © 2016 Pearson Education, Inc. All Rights Reserved Biofilms Microbial communities Form slime or hydrogels that adhere to surfaces – Bacteria communicate cell-to- cell via quorum sensing Share nutrients Shelter bacteria from harmful environmental factors Copyright © 2016 Pearson Education, Inc. All Rights Reserved Biofilms Found in digestive system and sewage treatment systems; can clog pipes 1000x resistant to microbicides Involved in 70% of infections – Catheters, heart valves, contact lenses, dental caries Copyright © 2016 Pearson Education, Inc. All Rights Reserved Culture Media Culture medium: nutrients prepared for microbial growth Sterile: no living microbes Inoculum: introduction of microbes into a medium Culture: microbes growing in or on a culture medium Copyright © 2016 Pearson Education, Inc. All Rights Reserved Culture Media Agar – Complex polysaccharide – Used as a solidifying agent for culture media in Petri plates, slants, and deeps – Generally not metabolized by microbes – Liquefies at 100°C – Solidifies at ~40°C Copyright © 2016 Pearson Education, Inc. All Rights Reserved Culture Media Chemically defined media: exact chemical composition is known – Fastidious organisms are those that require many growth factors provided in chemically defined media Complex media: extracts and digests of yeasts, meat, or plants; chemical composition varies batch to batch Nutrient broth Nutrient agar Copyright © 2016 Pearson Education, Inc. All Rights Reserved Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia Coli Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli Constituent Amount Glucose 5.0 g Ammonium phosphate, monobasic (NH4H2PO4) 1.0 g Sodium chloride (NaCl) 5.0 g Magnesium sulfate (MgSO4·7H2O) 0.2 g Potassium phosphate, dibasic (K2HPO4) 1.0 g Water 1 liter Copyright © 2016 Pearson Education, Inc. All Rights Reserved Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria Constituent Amount Peptone (partially digested protein) 5.0 g Beef extract 3.0 g Sodium chloride 8.0 g Agar 15.0 g Water 1 liter Copyright © 2016 Pearson Education, Inc. All Rights Reserved Anaerobic Growth Media and Methods Reducing media – Used for the cultivation of anaerobic bacteria – Contain chemicals (sodium thioglycolate) that combine O2 to deplete it – Heated to drive off O2 Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.6 A Anaerobic Jar for Cultivating Anaerobic Bacteria on Petri Plates Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.7 An Anaerobic Chamber Copyright © 2016 Pearson Education, Inc. All Rights Reserved Special Culture Techniques Capnophiles Microbes that require high CO2 conditions – CO2 packet – Candle jar Copyright © 2016 Pearson Education, Inc. All Rights Reserved Special Culture Techniques Biosafety levels – BSL-1: no special precautions; basic teaching labs – BSL-2: lab coat, gloves, eye protection – BSL-3: biosafety cabinets to prevent airborne transmission – BSL-4: sealed, negative pressure; "hot zone"  Exhaust air is filtered twice through HEPA filters Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.8 Technicians in a Biosafety Level 4 (BSL-4) Laboratory. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Selective and Differential Media Selective media – Suppress unwanted microbes and encourage desired microbes – Contain inhibitors to suppress growth of unwanted – Saboraud’s Dextrose Agar  pH of 5.6 prevents bacterial growth and used to isolate fungi Copyright © 2016 Pearson Education, Inc. All Rights Reserved Selective and Differential Media Differential media – Allow distinguishing of colonies of different microbes on the same plate Some media have both selective and differential characteristics Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.10 Differential Medium Copyright © 2016 Pearson Education, Inc. All Rights Reserved Enrichment Culture Encourages the growth of a desired microbe by increasing very small numbers of a desired organism to detectable levels Unlike selective medium, does not necessarily suppress the growth of other microbes. – Used mainly for fecal and soil samples. After incubation in enrichment medium, greater numbers of the organisms, increase the likelihood of positive identification. Usually a liquid Copyright © 2016 Pearson Education, Inc. All Rights Reserved Table 6.5 Culture Media Table 6.5 Culture Media Type Purpose Chemically Defined Growth of chemoautotrophs and photoautotrophs; microbiological assays Complex Growth of most chemoheterotrophic organisms Reducing Growth of obligate anaerobes Selective Suppression of unwanted microbes; encouraging desired microbes Differential Differentiation of colonies of desired microbes from others Enrichment Similar to selective media but designed to increase numbers of desired microbes to detectable levels Copyright © 2016 Pearson Education, Inc. All Rights Reserved Obtaining Pure Cultures A pure culture contains only one species or strain A colony is a population of cells arising from a single cell or spore or from a group of attached cells A colony is often called a colony-forming unit (CFU) The streak plate method is used to isolate pure cultures Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.11 The Streak Plate Method for Isolating Pure Bacterial Cultures Copyright © 2016 Pearson Education, Inc. All Rights Reserved Preserving Bacterial Cultures Deep-freezing: −50° to −95°C Lyophilization (freeze-drying): frozen (−54° to −72°C) and dehydrated in a vacuum Copyright © 2016 Pearson Education, Inc. All Rights Reserved Bacterial Division Increase in number of cells, not cell size Binary fission Budding (few bacterium) Conidiospores (actinomycetes) Fragmentation of filaments Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.12a Binary Fission in Bacteria Copyright © 2016 Pearson Education, Inc. All Rights Reserved Generation Time Time required for a cell to divide – 20 minutes to 24 hours Binary fission doubles the number of cells each generation Total number of cells = 2number of generations Growth curves are represented logarithmically Copyright © 2016 Pearson Education, Inc. All Rights Reserved Phases of Growth Lag phase Log phase Stationary phase Death phase Copyright © 2016 Pearson Education, Inc. All Rights Reserved Microbial Phases of Growth Bacterial Growth Curve: When bacteria are inoculated into a liquid growth medium, we can plot of the number of cells in the population over time. Four phases of Bacterial Growth: 1. Lag Phase: Period of adjustment to new conditions. Little or no cell division occurs, population size doesn’t increase. Phase of intense metabolic activity, in which individual organisms grow in size. May last from one hour to several days. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Microbial Phases of Growth 2. Log Phase: Cells begin to divide and generation time reaches a constant minimum. Period of most rapid growth. Number of cells produced > Number of cells dying Cells are at highest metabolic activity. Cells are most susceptible to adverse environmental factors at this stage. Radiation Antibiotics Copyright © 2016 Pearson Education, Inc. All Rights Reserved Microbial Phases of Growth 3. Stationary Phase: Population size begins to stabilize. Number of cells produced = Number of cells dying Overall cell number does not increase. Cell division begins to slow down. Factors that slow down microbial growth: Accumulation of toxic waste materials Acidic pH of media Limited nutrients Insufficient oxygen supply Copyright © 2016 Pearson Education, Inc. All Rights Reserved Microbial Phases of Growth 4. Death or Decline Phase: Population size begins to decrease. Number of cells dying > Number of cells produced Cell number decreases at a logarithmic rate. Cells lose their ability to divide. A few cells may remain alive for a long period of time. Copyright © 2016 Pearson Education, Inc. All Rights Reserved Direct Measurement of Microbial Growth Direct measurements–count microbial cells – Plate count – Filtration – Most probable number (MPN) method – Direct microscopic count Copyright © 2016 Pearson Education, Inc. All Rights Reserved Plate Counts Count colonies on plates that have 30 to 300 colonies (CFUs) To ensure the right number of colonies, the original inoculum must be diluted via serial dilution Counts are performed on bacteria mixed into a dish with agar (pour plate method) or spread on the surface of a plate (spread plate method) Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.16 Serial Dilutions and Plate Counts Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.17 Methods of Preparing Plates for Plate Counts Copyright © 2016 Pearson Education, Inc. All Rights Reserved Filtration Solution passed through a filter that collects bacteria Filter is transferred to a Petri dish and grows as colonies on the surface Copyright © 2016 Pearson Education, Inc. All Rights Reserved Direct Microscopic Count Volume of a bacterial suspension placed on a slide Average number of bacteria per viewing field is calculated Uses a special Petroff-Hausser cell counter Number of cells counted Number of bacteria/ml = Volume of area counted Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.20 Direct Microscopic Count of Bacteria with a Petroff-Hausser Cell Counter Copyright © 2016 Pearson Education, Inc. All Rights Reserved Estimating Bacterial Numbers by Indirect Methods Turbidity—measurement of cloudiness with a spectrophotometer Metabolic activity—amount of metabolic product is proportional to the number of bacteria (glucose consumption) Dry weight—bacteria are filtered, dried, and weighed; used for filamentous organisms Copyright © 2016 Pearson Education, Inc. All Rights Reserved Figure 6.21 Turbidity Estimation of Bacterial Numbers Copyright © 2016 Pearson Education, Inc. All Rights Reserved