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Information about growth

Published on September 5, 2010

Author: aditya0291


Growth & Culture of Bacteria : Growth & Culture of Bacteria Binary Division : Binary Division 1 to 2 to 4 to 8 to ? Phases of Growth : Phases of Growth Lag Adapt to nutrients Log Active growth Stationary Death = Growth rate Death Nutrients consumed pH too low (why?) Optimize curves in production Measuring Growth : Measuring Growth Direct Counts Petroff-Hauser Chamber Pro’s vs. Con’s Serial Dilution 10-fold serial dilutions Pro’s vs. Con’s MPN (Most Probable Number) Put 10, 1, and 0.1 ml into 10-mls broth Repeat 5 times per volume Statistical accurate sampling Public Health Standards are written for MPN Measuring Growth-cont’d : Measuring Growth-cont’d Turbidity Spectrophotometer Scale %Transmittance Optical Density or Absorbance Filtration 0.45 - 0.2 um sizes Grid Pattern on Filter Standards for Public Health 0 E.coli / 100 ml of water Also used for sterilization Microbial nutrition : Microbial nutrition • Cells mainly made up of macromolecules and water • Cells need nutrients – organisms differ in the types of nutrients they need; not all nutrients required in same amounts • Macronutrients – required in large amounts • Micronutrients – required in smaller, sometimes trace amounts Macronutrients : Macronutrients 1. Carbon Source Carbon sources • Most bacteria use organic compounds as a carbon source • Many use carbon-containing compounds (amino acids, fatty acids, organic acids, sugars, nitrogen bases, aromatic compounds …) as building blocks to synthesise cell components • Different bacteria utilise different compounds Macronutrients : Macronutrients • Chemoheterotrophic organisms obtain energy from glucose by glycolysis, fermentation and the Krebs cycle • Chemoheterotrophic bacteria synthesise some cell components from intermediates in the above pathways • On a dry weight basis a typical cell is about 50% carbon Macronutrients : Macronutrients 2. Nitrogen Source NO3- ==> NO2 ==> NH3 to Amino Acids ==> Proteins All organisms need N to synthesise enzymes, other proteins & nucleic acid • Some organisms obtain N from inorganic sources; a few obtain energy from metabolising inorganic N-containing substances • Many micro-organisms reduce nitrate (NO3 -) to amino groups (NH2) and use the amino groups to make amino acid • Many micro-organisms can utilise ammonia as the sole N source Slide 19: • Nitrogen fixing bacteria can take up nitrogen directly from the atmosphere Some can synthesise all 20 amino acids from amino groups; others must have some provided in their growth medium Some fastidious organisms require all 20 amino acids and other building blocks in their growth medium • Many disease causing organisms obtain amino acids from the cells of the humans or other 12 organisms they invade • A typical bacterial cell is 12% nitrogen by weight Slide 20: 3. Sulfur: usually from inorganic sources (sulphate or sulphide) required for Scontaining amino acids, some vitamins, coenzyme A 13 – Microbes involved in many chemical transformations of sulphur in the environment 4. Phosphorus: inorganic or organic – required for the synthesis of nucleic acids and phospholipids Slide 21: 5. Iron : key role in cellular respiration – key component in cytochromes and iron-sulphur proteins involved in electron transport • Under anoxic (anaerobic) conditions iron is in Fe2+ state & soluble; but under oxic (aerobic) conditions it is often Fe3+ & forms various insoluble minerals • Bacteria have developed iron-binding proteins (siderophores) that solubilise such iron & transport it into the cell eg hydroxamic acid derivatives Chelate Fe3+ very strongly – complex carried into cell – iron is split off and hydroxamate exits the cell and repeats the process Slide 22: –– examples of siderophores: Enterobactins – Escherichia coli and Salmonella Typhimurium Micronutrients: Trace Elements : Micronutrients: Trace Elements Critical to cell function even if only required in small amounts • Micronutrients are metals – structural role in many enzymes • Examples include cobalt, manganese, molybdenum nickel selenium zinc Growth factors : Growth factors Organic compounds – required in very small amounts and only by some cells • Include vitamins, amino acids, purines, Pyrimidines • Most micro-organisms can synthesise all of these compounds – some require one or more to be pre-formed in culture environment • Vitamins are the most commonly needed growth factors; most function as parts of co-enzymes • Most commonly required vitamins are thiamine (vitamin B1), biotin, pyridoxine (vitamin B6) and cobalamine (vitamin B12) Culture media : Culture media Different organisms have widely differing nutritional requirements Types of culture media 1. Synthetic (defined) • Need to ensure culture media used in the laboratory meets the needs of the organism you are trying to culture – Prepared by adding precise amounts of highly purified inorganic or organic chemicals to distilled water Culture media : Culture media – Exact chemical composition of medium is therefore known 2. Complex – Complex media – digests of casein ( milk protein), beef, soybeans, yeast cells ……other highly nutritious but chemically undefined 3. Nutrient – Simple complex media containing peptone and agar Categories of growth media : Categories of growth media Enriched – Nutrient medium containing enrichments such as blood or serum or yeast extract – Enrichment provides additional growth factors for more fastidious organisms (includes many pathogens – eg Blood agar Categories of growth media : Categories of growth media Enrichment – Contains special nutrients that allow the growth of a particular organism that may be present in low numbers and so masked by other organisms – Usually a broth culture medium – Eg Rappaport’s medium for salmonella, Enterococcosal Broth for enterococci Categories of growth media : Categories of growth media Selective – Encourages the growth of some organisms but suppresses the growth of others – Eg Mannitol salt agar for isolation of Staphylococcus aureus Differential – Contains a constituent that causes an observable change (change in colour or change in pH) Categories of growth media : Categories of growth media in the medium when a particular biochemical reaction occurs – eg fermentation of lactose in MacConkey medium causes a pH change – lactose fermenting colonies pink, non-lactose-fermenting colonies colourless Combined selective and differential media – Eg MacConkey Agar – contains crystal violet & bile salts which inhibit Gram-positive bacteria plus lactose and pH indicator Components of media : Components of media General nutrient sources – Peptones – water soluble protein hydrolysates – Infusions (extracts) • Specific growth factors (vitamins, amino acids) • Solidifying agent – agar • pH indicators • Reducing agents • Selective agents

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