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Unformatted text preview: Important Point: Each Species is Unique Chapter 4: Dynamics of Prokaryotic Growth Pure-Culture Basics Sterile = completely free of microbes. Bacteria are incredibly diverse, but... Each bacterial species can grow in only a limited set of environments. Each bacterial species can grow only if presented with the right nutrients/conditions. In addition, bacteria produce characteristic byproducts (e.g., waste products). We can take advantage of these growth characteristics to identify bacteria phenotypically. To do these identifications we first have to get organisms in Pure Culture. Unfortunately, only about 1% of microorganisms currently can be grown in pure culture. (mostly) Isolated Colonies Aseptic Technique = procedures that minimize unintentional introduction of microorganisms to media (cultures) or from cultures to surrounding environment. Solid media is usually employed to obtain pure cultures. Agar is usually employed to make solid media. Agar melts at 95°C and solidifies below 45°C. Colony = pile of cells descended from single cell (or clump of cells). Petri Dish = container to which agar is added to obtain pure culture. Agar Plate (plate) = agar-containing petri dish. 1 Optimum Growth Temperature The interval, division to division, is called the Generation or Doubling Time. Note that not all daughter cells fully separate after division, e.g. streptococci, etc. Growth temperature optimum. Storing Pure Cultures Stored pure cultures are often called “Stock Cultures” Stock cultures often are stored as/using: Frozen in glycerol solution Lyophilized = freeze drying On agar slants As stabs Environmental Factors Binary Fission This is how most bacteria undergo cell division (how they replicate). Temperature Ranges Streak-Plate Method Max due to enzyme denaturation. Min due to enzyme & membrane fluidity problems. 2 Important for food spoilage. Oxygen Requirements: The Shake Tube Important source of heat-stable enzymes (e.g., Taq polymerase or laundry detergent enzymes). Oxygen Requirements: The Shake Tube Note maximum growth nearer to surface (where oxygen is plentiful; this is not shown well in image). I.e., organisms adapted to growth at body temperature. Mesophiles Psychotrophs Thermophiles Most human pathogens are mesophiles. Aerotolerant Anaerobe! Don’t worry about enzyme names. Just recall “O2 (product) detoxification”. Medically Important Examples Pseudomonas spp. are obligate aerobes. E.g., Clostridium spp. such as C. botulinum. Enterics such as Escherichia coli are facultative anaerobes 3 Plasmolysis Food preservation: jams, jellies, bacon, anchovies, etc. Energy & Carbon Source Types Terms for Nutrient Needs Water Availability Heterotrophs = require organic carbon (e.g., glucose). Autotrophs convert CO2 to organic carbon. Carbon fixation = conversion of CO2 to organic carbon. Nitrogen fixation = conversion of N2 to non-gaseous form (i.e., ammonia). Growth factors = small organic molecules (e.g., vitamins, amino acids) that must be provided for growth (some bacteria require no “exogenous” “growth factors”). E.g., Neisseria spp. can require 40 growth factors to grow. We would describe such a bacterium as fastidious. E. coli requires no growth factors. We would describe such a bacterium as non-fastidious. Culture Media Types Peptone = predigested protein “Chemoheterotroph” is good enough for now. “Chemoautotroph” is good enough for now. Chemoheterotrophs differ in the number (and types) of organic compounds they can use. Some Pseudomonas species can utilize 80 different compounds. Other bacteria are limited to as little as only a single organic compound type. Culture Media Types Enrichment Culture Means of isolating rare organisms with specific characteristics from heterogeneous populations. Note that, confusingly, many media are both selective and differential, e.g., MacConkey agar. 4 Direct Microscopic Count Direct Microscopic Count Requires relatively high bacterial densities. Viable Counts: Plate Counts Viable Counts: Plate Counts Usually can’t distinguish living cells from dead cells. Note the enumeration of colonyforming units (CFUs). Quantifies number of cells (CFUs) capable of replicating. Serial Dilutions Most Probable Number (MPN) Note Serial Dilution. These are gas-filled tubes, an indication of bacterial growth (fermentation). Looking for sufficient dilution that ~half of tubes show growth. Reciprocal of that dilution ≈ bacterial density. 5 Most Probable Number (MPN) Growth Curve Useful particularly when enumerating organisms that won’t grow on/in agar media. Durham tube. Growth Curve Death rate = Birth rate. Phase of Prolonged Decline Constant per-capita death rate (exponential). Division at constant rate (exponential). Biofilms are polysaccharide-encased bacterial communities attached to environmental surfaces. Chemostats are a means of keeping a culture in log phase indefinitely. Biofilms Continuous Culture, Chemostat Time of gearing up for division following change in culture conditions. Biofilms include slippery rocks (in aquatic environments), slime coating sink drains, yuck yucking up what was once your clean toilet bowl, tarter on your teeth, etc. “It is estimated that 65% of human bacterial infections involve biofilms.” “Biofilms are particularly troublesome because they protect organisms against harmful chemicals such as disinfectants” and antibiotics. They can accumulate on non-sterile medical devices kept in contact with patients over relatively long periods, e.g., on catheters. 6 Chapter 6 Notes Don’t worry too much about the details of Glycolysis, Cellular Respiration, or Photosynthesis onward. These topics are covered on pp. 144-151 and pp. 156-163. Note, however, that we will cover Glycolysis and Cellular Respiration in class at least from the perspectives of the importance of NAD+ regeneration. Link to Next Presentation 7 Oxygen Requirements: The Shake Tube 4 Culture Media Types Peptone = predigested protein 4 ...
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