Microbial Nutrition and Growth

Transcription

1 PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 6 Microbial Nutrition and Growth CSLO 5. Give examples of the range of metabolic diversity exhibited by microorganisms, impact of metabolic characteristics on growth, and control of growth. CHON 96% CaPPSS 4% TRACE ELEMENTS 1

2 Growth Requirements Nutrients: Chemical and Energy Requirements Sources of carbon, energy, and electrons Two groups of organisms based on source of carbon Autotrophs Heterotrophs Two groups of organisms based on source of energy Chemotrophs Phototrophs Two groups of organisms based on source of electrons Organotrophs Lithotrophs Figure 6.1 Four basic groups of organisms based on their carbon and energy sources. Growth Requirements Nutrients: Chemical and Energy Requirements Oxygen requirements Oxygen is essential for obligate aerobes Oxygen is deadly for obligate anaerobes How can this be true? Toxic forms of oxygen are highly reactive and excellent oxidizing agents Resulting oxidation causes irreparable damage to cells 2

3 Figure 6.3 Using a liquid thioglycollate growth medium to identify the oxygen requirements of organisms. Oxygen requirements Oxygen concentration High Loosefitting cap Low Obligate aerobes Obligate anaerobes Facultative anaerobes Aerotolerant anaerobes Growth Requirements Nutrients: Chemical and Energy Requirements Oxygen requirements Four toxic forms of oxygen Singlet oxygen Superoxide radicals (superoxide dismutase) Peroxide anion (catalase) Hydroxyl radical Anaerobes lack these enzymes Figure 6.2 Catalase test. Catalase destroys hydrogen peroxide to generate? 3

4 Growth Requirements Nutrients: Chemical and Energy Requirements Nitrogen requirements All cells recycle nitrogen from amino acids and nucleotides Nitrogen fixation by certain bacteria is essential to life on Earth N N Growth Requirements Nutrients: Chemical and Energy Requirements Other chemical requirements Phosphorus Sulfur Trace elements Only required in small amounts Growth factors (eg vitamins) Necessary organic chemicals that cannot be synthesized by certain organisms 4

5 Growth Requirements Physical Requirements Temperature Temperature affects three-dimensional structure of proteins Lipid-containing membranes of cells and organelles are temperature sensitive If too low, membranes become rigid and fragile If too high, membranes become too fluid Figure 6.4 The effects of temperature on microbial growth. Minimum Maximum Optimum 22ºC 30ºC 37ºC Figure 6.5 Four categories of microbes based on temperature ranges for growth. 5

6 Figure 6.6 An example of a psychrophile. Growth Requirements Physical Requirements ph Organisms sensitive to changes in acidity H + and OH interfere with H bonding Neutrophiles grow best in a narrow range around neutral ph Acidophiles grow best in acidic habitats Alkalinophiles live in alkaline soils and water Growth Requirements Physical Requirements Physical effects of water Microbes require water to dissolve enzymes and nutrients Water is important reactant in many metabolic reactions Most cells die in absence of water Two physical effects of water Osmotic pressure Hydrostatic pressure 6

7 Growth Requirements Physical Requirements Physical effects of water Osmotic pressure Pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane Hypotonic solutions have lower solute concentrations Cell placed in hypotonic solution swells Hypertonic solutions have greater solute concentrations Cell placed in hypertonic solution shrivels Restricts organisms to certain environments Obligate and facultative halophiles pg 322 Growth Requirements Physical Requirements Physical effects of water Hydrostatic pressure Water exerts pressure in proportion to its depth Barophiles live under extreme pressure Their membranes and enzymes depend on pressure to maintain their three-dimensional, functional shape Growth Requirements Associations and Biofilms Organisms live in association with different species Antagonistic relationships Synergistic relationships Symbiotic relationships 7

8 Growth Requirements Associations and Biofilms Biofilms Complex relationships among numerous microorganisms Form on surfaces, medical devices, mucous membranes of digestive system Form as a result of quorum sensing Many microorganisms more harmful as part of a biofilm Scientists seeking ways to prevent biofilm formation Figure 6.7 Biofilm development. 1 Free-swimming microbes are vulnerable to environmental stresses. Chemical structure of one type of quorum- sensing molecule Water flow Water channel Bacteria Escaping microbes Matrix 2 Some microbes 3 The cells begin producing 4 Quorum sensing 5 New cells arrive, 6 Some microbes escape land on a surface, an intracellular matrix and triggers cells to change possibly including new from the biofilm to resume such as a tooth, secrete quorum-sensing their biochemistry and species, and water a free-living existence and and attach. molecules. shape. channels form in the perhaps, form a new biofilm. biofilm on another surface. Culturing Microorganisms Culture Act of cultivating microorganisms or the microorganisms that are cultivated Inoculum introduced into medium Environmental specimens Clinical specimens Stored specimens 8

9 Nursing professionals Growth Requirements Microbial growth Increase in a population of microbes Due to reproduction of individual microbes Result of microbial growth is discrete colony An aggregation of cells arising from single parent cell Figure 6.8 Characteristics of bacterial colonies. Shape Circular Rhizoid Irregular Filamentous Spindle Margin Entire Undulate Lobate Curled Filiform Elevation Flat Raised Convex Pulvinate Umbonate Size Punctiform Small Moderate Large Colony Texture Smooth or rough Appearance Glistening (shiny) or dull Pigmentation Nonpigmented (e.g., cream, tan, white) Pigmented (e.g., purple, red, yellow) Optical property Opaque, translucent, transparent 9

10 Culturing Microorganisms Obtaining Pure Cultures Cultures composed of cells arising from a single progenitor Progenitor is termed a colony-forming unit (CFU) Aseptic technique prevents contamination of sterile substances or objects Two common isolation techniques Streak plates Pour plates Figure 6.9 The streak-plate method of isolation. Can individually pick single cell of some large microorganisms and use to establish a culture Streak plate method 10

11 Figure 6.10 The pour-plate method of isolation. Sequential inoculations 1.0 ml 1.0 ml 1.0 ml Initial sample 9 ml broth 9 ml broth 9 ml broth 1.0 ml to each Petri dish, add 9 ml warm agar, swirl gently to mix Colonies Fewer colonies Culturing Microorganisms Culture Media Majority of prokaryotes have not been grown in culture medium Liquid or Solid Six types of general culture media Defined media Complex media Selective media Differential media Anaerobic media Transport media Figure 6.11 Slant tubes containing solid media. Agar is a common addition to many media Slant Butt 11

12 Defined media Figure 6.12 An example of the use of a selective medium. Bacterial colonies selective medium Fungal colonies ph 7.3 ph 5.6 Figure 6.13 The use of blood agar as a differential medium. Beta-hemolysis Alpha-hemolysis differential medium No hemolysis (gamma-hemolysis) 12

13 Figure 6.14 The use of carbohydrate utilization tubes as differential media. differential medium Durham tube (inverted tube to trap gas) No fermentation Acid fermentation with gas Figure 6.15 The use of MacConkey agar as a selective and differential medium. Selective & differential medium Escherichia coli Escherichia coli Escherichia coli Staphylococcus aureus Nutrient agar Staphylococcus aureus (no growth) MacConkey agar Salmonella enterica serotype Choleraesuis MacConkey agar Figure 6.16 An anaerobic culture system. Clamp anaerobic medium Airtight lid Chamber Palladium pellets to catalyze reaction removing O 2 Envelope containing chemicals to release CO 2 and H 2 Petri plates Methylene blue (anaerobic indicator) 13

14 Culturing Microorganisms Special Culture Techniques Techniques developed for culturing microorganisms Animals, bird eggs and cell culture E.g. Rabbits syphilis bacteria T. pallidum Low-oxygen culture Culturing Microorganisms Preserving Cultures Refrigeration Stores for short periods of time Deep-freezing Stores for years Lyophilization Stores for decades Figure 6.17 Binary fission. 2 1 Cytoplasmic membrane Chromosome Cell wall Replicated chromosome 30 minutes Generation Time 3 Septum 4 Completed septum 5 60 minutes Septum 90 minutes 120 minutes 14

15 Growth of Microbial Populations Generation Time Time required for a bacterial cell to grow and divide Dependent on chemical and physical conditions typical microbial growth curve. Growth of Microbial Populations Continuous Culture in a Chemostat Chemostat used to maintain a microbial population in a particular phase of growth Chemo stat is an Open system Requires addition of fresh medium and removal of old medium Used in several industrial settings Figure 6.21 Schematic of chemostat. Fresh medium with a limiting amount of a nutrient Flow-rate regulator Sterile air or other gas Log phase Limiting nutrient Culture vessel Culture Overflow tube 15

16 Growth of Microbial Populations Measuring Microbial Reproduction Direct or Indirect Requiring Incubation or Not Requiring Incubation Figure 6.22 The use of a cell counter for estimating microbial numbers. Cover slip Pipette Bacterial suspension Location of grid Overflow troughs Direct methods not requiring incubation Bacterial suspension Place under oil immersion Microscopic counts Growth of Microbial Populations Measuring Microbial Reproduction Direct methods not requiring incubation Electronic counters Coulter counters Flow cytometry Detector 16

17 Growth of Microbial Populations Measuring Microbial Reproduction Direct methods requiring incubation Serial dilution and viable plate counts Membrane filtration Most probable number Figure 6.23 A serial dilution and viable plate count for estimating microbial population size. 1 ml of original culture 1.0 ml 1.0 ml 1.0 ml 1.0 ml 9 ml of broth + 1 ml of original culture 1:10 dilution (10-1 ) 1:100 dilution (10-2 ) 1:1000 dilution (10-3 ) 1:10,000 dilution (10-4 ) 1:100,000 dilution (10-5 ) 0.1 ml of each transferred to a plate 0.1 ml 0.1 ml 0.1 ml 0.1 ml Incubation period Too numerous to count (TNTC) TNTC 65 colonies 6 colonies 0 colonies Serial dilution and viable plate counts Figure 6.24 The use of membrane filtration to estimate microbial population size. Sample to be filtered Membrane transferred to culture medium Membrane filter retains cells To vacuum Incubation Colonies 17

18 Figure 6.25 The most probable number (MPN) method for estimating microbial numbers. 1.0 ml 1.0 ml Undiluted 1:10 1:100 Inoculate 1.0 ml into each of 5 tubes Phenol red, ph color indicator, added Incubate Results 4 tubes positive 2 tubes positive 1 tube positive Growth of Microbial Populations Measuring Microbial Growth Indirect methods Turbidity Growth of Microbial Populations Measuring Microbial Growth Indirect methods Metabolic activity Dry weight Genetic methods Isolate DNA sequences of unculturable prokaryotes 18