Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings PowerPoint ® Lecture Slide Presentation prepared by Christine L. Case Microbiology B.E Pruitt & Jane J. Stein AN INTRODUCTION EIGHTH EDITION TORTORA FUNKE CASE Microbial Growth and Nutrition

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Microbial Growth Microbial growth = increase in number of cells, not cell size

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Microbial Growth Microbes grow via binary fission, resulting in exponential increases in numbers The number of cells arising from a single cell is 2 n after n generations Generation time is the time it takes for a single cell to grow and divide

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Microbial Growth: Growth Curve lag phase: no growth and microorganisms are making macromolecules in preparation for growth log phase cultures are growing maximally Stationary phase occurs when nutrients are depleted and wastes accumulate (Growth rate = death rate) Death phase death rate is greater than growth rate

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Temperature pH Osmotic pressure Oxygen classes Environmental Factors Affecting Microbial Growth

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Environmental Factors : Temperature Temperature is a major environmental factor controlling microbial growth.. Minimum growth temperature: Temperature below which growth ceases, or lowest temperature at which microbes will grow.. Optimum growth temperature: Temperature at which the microbial growth rate is the fastest.. Maximum growth temperature: Temperature above which growth ceases, or highest temperature at which microbes will grow.

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Classification of Microorganisms by Temperature Requirements. Psychrophiles : Cold temperature optima (grow between o C).. Psychrotrophs: Cold temperature optima (grow between 0-30 o C).. Mesophiles ( 20 – 45 C) Midrange temperature optima (neither too hot nor too cold, typically between 20 and 45 °C). Found in warm-blooded animals. Thermophiles ( C) Growth temperature optima between 45ºC and 80ºC. Hyperthermophiles Optima greater than 80°C These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100ºC

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Environmental Factors : Temperature

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings pH Most bacteria grow between pH 6.5 and 7.5 (neutrophiles) Molds and yeasts grow between pH 5 and 6 Acidophiles grow in extreme acidic environments (less than 2) such as Helicobacter pylori Alkaliphiles grow best at high pH medium such as Vibrio cholera that can tolerate pH medium up to 9.5 Environmental Factors : pH

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Environmental Factors: Osmotic pressure organisms which thrive in high solute – osmophiles organisms which tolerate high solute – osmotolerant organisms which thrive in high salt – halophiles organisms which tolerate high salt – halotolerant. Xerophiles : are able to grow in very dry environments

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Environmental Factors: Osmotic pressure Osmotic Pressure Hypertonic environments, increase salt or sugar, cause plasmolysis Extreme or obligate halophiles require high osmotic pressure Facultative halophiles tolerate high osmotic pressure

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Environmental Factors: Oxygen. Obligate (strict) aerobes require O 2 in order to grow. Facultative anaerobes grow better in O 2. Obligate (strict) anaerobes cannot survive in O 2. Aerotolerant organisms don’t care about O 2. Microaerophiles require low levels of O 2 obligate aerobes Faultative anaerobes Obligate anaerobes Aerotolerant Microaerophiles Classification of organisms based on O 2 utilization

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Utilization of O 2 during metabolism yields toxic by- products including singlet oxygen:( 1 O 2 ), superoxide free radicals (O 2  ), hydrogen peroxide (H 2 O 2 ) and Hydroxyl radical(OH  ). Toxic O 2 products can be converted to harmless substances if the organism has catalase (or peroxidase) and superoxide dismutase (SOD) enzymes. Toxic Forms of Oxygen

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Toxic Forms of Oxygen. SOD converts O 2 - into H 2 O and O 2. Catalase breaks down the H 2 O 2. Peroxidase converts H 2 O 2 into H 2 O. In general any organism that can live or requires O 2 has SOD and catalase (or peroxidase).

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Reducing media Contain chemicals (thioglycollate or oxyrase) that combine O 2 Provides aerobic and anaerobic conditions Anaerobic Culture Methods

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Anaerobic jar Anaerobic Culture Methods

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Candle jar CO 2 -packet Capnophiles require high CO 2

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Microbial Nutrition The hundreds of chemical compounds present inside a living cell are formed from nutrients. Macronutrients : elements required in fairly large amounts Micronutrients : metals and organic compounds needed in very small amounts

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Main Macronutrients: Carbon Carbon (C), 50% of dry weight Structural organic molecules, energy source Autotrophs use CO2 Chemoheterotrophs use organic carbon sources

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Nitrogen, 12% of dry weight In amino acids, proteins Most bacteria decompose proteins Most bacteria can use Ammonia – NH 3 and many can use NO 3 - Nitrogen fixers can utilize atmospheric nitrogen (N 2 ) Main Macronutrients: Nitrogen

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Sulfur In amino acids, thiamine, biotin Most bacteria decompose proteins Some bacteria use SO 4 2  or H 2 S Phosphorus In DNA, RNA, ATP, and membranes PO 4 3  is a source of phosphorus Phosphate, potassium (K), magnesium(Mg), calcium (Ca), sodium (Na) and iron(Fe). Other Macronutrients

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings. Usually metals needed in very little amount but are critical to cell function. Usually as enzyme cofactors. e.g. Nickel(Ni), selenium (Se), Tungsten ( W), copper (Cu), Cobalt (Co). Micronutrients: Trace elements

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Organic Growth Factors Organic compounds obtained from the environment and needed in very small amount only by some cells Vitamins, amino acids, purines, pyrimidines The Requirements for Growth: Growth Factors

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Culture Medium: Medium containing nutrients that support microbial growth Sterile: No living microbes Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium Culture Media

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Types of Culture Media Media can be classified on three primary levels: Physical State Chemical Composition Functional type

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Types of Culture Media: Physical State Liquid Media: Fluid in nature, usually placed in test tubes, for example nutrient broth. Solid : Prepared by adding solidifying agents like gelatin and agar to the liquid medium, for example, nutrient agar. Solid :(can be converted into a liquid) Solid: (cannot be converted into a liquid) Semisolid

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Types of Culture Media: Chemical Composition Synthetic Media Chemically defined Contain pure organic and inorganic compounds Exact formula (little variation) Complex or Non-synthetic Media Contains at least one ingredient that is not chemically definable (extracts from plants and animals) No exact formula / tend to be general and grow a wide variety of organisms

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Culture Media: Chemical Composition Synthetic Medium Non-synthetic Medium

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Culture Media: Functional type Simple Medium: it contains only basic substance such as nitrogen, carbon and minerals that are essential for bacterial growth,.e.g. nutrient broth, nutrient agar, peptone water Enriched Medium: Some nutritionally enriched material like blood, serum or asctic fluid is added to the medium, required for proper growth of some bacteria,.e.g. blood agar Chocolate agar

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Culture Media: Functional type Differential Medium: it differentiates between two groups of bacteria, e.g., blood agar, MacConkey's Medium Selective Medium: In this media an inhibitory substance is added to the media which prevents growth of all organisms except the one for which it is designed. e.g. Lowenstein Jensen's medium

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings 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) Pure Culture

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Plate Counts: Perform serial dilutions of a sample Measurements of Microbial Growth: Plate count

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Measurements of Microbial Growth: Microscopic count. Need a microscope, special slides, high power objective lens.Typically only counting total microbe numbers

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Differences in electrical conductivity between species Fluorescence of some species Cells selectively stained with antibody + fluorescent dye Measurements of Microbial Growth: Flowcytometry

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Turbidity Estimating Bacterial Numbers by Indirect Methods: Turbidity