Lecture 9 - Microbial Growth and Metabolism PDF

BIOL1030 Biochemistry & Microbiology Microbial Growth & Metabolism  Bacteria replication- binary fission Binary fission is the type of cell division most frequently observed among procaryotes Cell reproduction leads to an increase in population size...

BIOL1030 Biochemistry & Microbiology Microbial Growth & Metabolism  Bacteria replication- binary fission Binary fission is the type of cell division most frequently observed among procaryotes Cell reproduction leads to an increase in population size Growth increases the size of the organism An increase in size enables the formation of multiple organisms Binary fission is a relatively simple type of cell division: the cell elongates replicates its chromosome separates the newly formed DNA molecules so there is one chromosome in each half of the cell a septum (or cross wall) is formed at midcell, dividing the parent cell into two progeny cells having its own chromosome and a complement of Two pathways function during the bacterial cell cycle: one pathway replicates and partitions the DNA into the progeny cells the other carries out cytokinesis—formation of the septum and progeny cells.  Growth curve Binary fission and other cell division processes bring about an increase in the number of cells in a population Population growth is studied by analyzing the growth curve of a microbial culture Population growth of microbes reproducing by binary fission in a batch culture can be plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has four distinct phases: Lag Exponential Stationary Death  Lag phase When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs This cycle is considered to be the standard bacterial growth curve Lag phase – Little or no cell division Post introduction of the species into the growth media Organisms are metabolically active Grow in size but not in numbers Cell numbers remain fairly constant  Exponential (log) phase Log phase – bacterial division is increased Cells divide and grow at a rapid rate Growth is constant The interval is called generation time This is the time required for the cell to divide: population doubles During log phase, several conditions emerge dividing at the maximal rate possible: Available nutrients decrease Wastes accumulate Living space becomes less The ability to produce energy and divide diminishes Growth rate slows  Stationary phase During stationary phase, the population stabilizes: The number of new cells produced equals the number of cells dying Growth curve becomes horizontal Lack of nutrients prevents positive population growth The number of living cells stays constant Final population size depends on nutrient availability and other factors, as well as the type of microorganism In stationary phase, the total number of viable microorganisms remains constant  Death phase Cells growing in batch culture cannot remain in stationary phase indefinitely. During this phase, the number of viable cells often declines exponentially, with cells dying at a constant rate. During death phase, the population begins to decline: The number of new cells produced is less than the number of cells dying Conditions become worse nutrition wise Cells cannot divide and begin to die Nutrition is gone  Media and Culturing Microorganisms A group of microorganisms that grow in a medium is culture A solid, liquid or semi-solid designed to support the growth of microorganisms is medium To be effective, the medium must contain all the nutrients the microorganism requires for growth Frequently a medium is used to select and grow specific microorganisms or to help identify a particular species Culture media can be classified based on several parameters: 1. the chemical constituents from which they are made 2. physical nature 3. function Culturing bacteria requires a pure sample Pure cultures are defined as: A culture that contains cells from a single species of an organism The medium selected to grow that culture must fit the organism enzyme and nutrition requirements Streak Plating is one method of collecting pure samples Pour Plates are another method  Streak Plating Pour Plate Method Isolate pure colonies Used primarily as a method for counting bacteria Effective for separating mixed cultures A serial dilution is performed from a liquid specimen https://www.youtube.com/watch?v=FutAgWDymAM  Culturing bacteria Culture Media-Media containing all nutrients needed for growth To grow we need to know the nutritional needs Have to have the ability to provide those needs in an artificial environment There is no single media that will permit the growth of all bacteria. Some bacteria require anaerobic conditions for incubation There are numerous types of media available for culturing Media can be classified in two ways: 1. Composition – the type of substances that make up the media a) Natural Media – naturally occurring substances: blood, soil, etc. b) Synthetic media – Media Prepared in a laboratory 1. Defined synthetic- you must know the exact nutritional requirements of the organism 2. Complex media – chemically non- defined; Contains familiar materials in media Chemical composition varies 2. Function – how the media affects the bacterial growth  Isolating and Identifying Bacteria As a microbiologist, much of what you do will be dedicated to identifying organisms based on specific criteria. These can include: The source of the culture specimen (water source, gut, lungs, etc) The microscopic appearance of the organism (shape, chains, etc) Its pattern of growth on selective media The organism’s metabolic, hemolytic, and fermentative properties on media The results of specific biochemical tests Selective media – Promote the growth of some organisms and inhibit the growth of others Media Type Examples: They select out an organism based on their ability to survive in the media Differential Eosin Mehtylene Blue Agar MacConkey Agar Differential media – They allow the growth of Hektoen Enteric Agar organisms but determine whether an organism has specific enzyme capabilities to utilize the Mannitol Salt Agar media. These do not select. Selective Media Bismuth Sulfite Agar Can produce colored colonies Enrichment Media Tryptic Soy Agar/ Broth (TSA/B) Byproducts of metabolism can alter the media of indicators found in the media Chocolate Agar Blood Agar Characteristic Media Triple Sugar Iron Media Enrichment media – Promotes the growth of Other Media Cooked Meat Medium SPECIFIC organisms that may be present in Thioglycollate Medium small numbers and outcompeted by other organisms Sabouraud Medium Media enables growth  Differential and Selective Media McConkey Agar – Selective and Differential Eosin Methylene Blue Agar selects out gram negative bacteria Used to detect fecal coliform bacteria differentiates between those that can and cannot ferment Selective for gram-negative bacteria lactose Exp. Escherichia coli colonies grow with a metallic fermentation of lactose produces organic acids, sheen with a dark center, Aerobacter aerogenes particularly lactic acid, which decreases the pH colonies have a brown center and non-lactose- turns pink under acidic conditions fermenting, gram-negative bacteria appear pink. lactose-fermenting-gram-negatives (lactose-fermenters) will form pink colonies, while non-lactose fermenters will form off-white opaque colonies Mannitol Salt Agar – Selective and Differential Hektoen Enteric Agar – Selective and Differential Isolates Staphylococci (gm +ve) Selects and differentiates Salmonella and Shigella Pathogenic (medically significant) Staphylococci species from other Enterobacteriaceae in fecal samples ferment mannitol The plates contain various sugar sources Mannitol is the fermentable carbohydrate (lactose, sucrose, and salicin), none of which can be source. Staphylococcus aureus grows on this used by either Shigella or Salmonella medium and ferments mannitol to produce Most bacteria acidify the medium and turn a pH yellow colonies. indicator yellow or red. Peptone metabolism by Shigella and Salmonella alkalises the medium, turning a pH indicator blue. Bismuth Sulfite Agar - Selective and Differential Isolates a specific species of Salmonella (S. typhi - Typhoid Fever) It uses glucose as a primary source of carbon Bismuth and brilliant green (dye) both inhibit gram-positive growth  Antimicrobial Drug Susceptibility Testing (AST) Essential for identifying the most effective Methods of Antimicrobial Susceptibility Testing: antibiotics against bacterial pathogens and determining resistance levels. Disk Diffusion Method (Kirby-Bauer Test) Broth Dilution Method This process is crucial in clinical settings to guide Agar Dilution Method etc… treatment decisions and in epidemiological studies to monitor resistance patterns https://www.youtube.com/watch?v=BXr_kcki4Ag  Metabolism Metabolism can be defined as all of the chemical reactions that occur in any living organism 1.Catabolism – catabolic reactions – Breaking down 2.Anabolism – anabolic reactions – Building up Carbon Sources Microbial organisms can also be described by the source that the organism uses for Carbon Autotrophs Heterotrophs organisms that use organic compounds other than Use carbon dioxide as their sole source of carbon carbon dioxide as a source of carbon Ex: Plants, algae, cyanobacteria Ex: humans, fungi, animals and protozoans. Most microorganisms are chemoheterotrophs Use nutrients obtained from hosts, living or dead.  Chemotrophs Energy Sources Use organic or inorganic molecules as an energy source Two categories of chemotrophs: Phototrophs Chemolithotrophs – use inorganic molecules as an energy source Chemoorganotrophs – use organic molecules as Use light as an energy source an energy source. Energy produced through photosynthesis The terms relating to an energy source can be combined with the terms relating to carbon source: Photoautotrophs – organisms that use photons as an energy source and carbon dioxide as their carbon source - Plants Photoheterotrophs – organisms that use light as an energy source but other organic molecules for carbon – rare form of metabolism Chemoautotrophs – use chemicals as an energy source and carbon dioxide as their sole carbon source – rare form of metabolism Chemoheterotrophs – use chemicals as their energy source and molecules other than carbon dioxide as their source of carbon – most organisms (except plants) The nutrients needed by Microorganisms include: Carbon, Nitrogen, Sulfur, Phosphorus & Vitamins Additional elements required include Sodium, potassium, chlorine, magnesium, calcium, iron, and iodine Organisms also require a continuous intake of essential nutrients that cannot be synthesized: Fatty acids and amino acids These combine to make the essential macromolecules of life: Carbohydrates, amino acids, lipids, proteins, and DNA/RNA