Microbial Growth

1. Microbial Growth 

2. I. The Growth Curve

3. Closed system = Batch Culture • Closed culture vessel • One batch of culture medium • Different from continuous culture (see below) • Nutrients used up, culture eventually dies • Four stages of bacteria growth in batch culture

4. Lag Phase • A period of apparent inactivity in which the cells are adapting to a new environment and preparing for reproductive growth, usually by synthesizing new cell components • ATP • Ribosomal proteins • rRNA • tRNA • Co-factors • Enzymes

5. Varies in length depending upon the condition of the microorganisms and the nature of the medium • Assessment of medium – Receptors • DNA synthesized – initiation of cell division

6. Exponential phase (Log Phase) • Optimal growth rate and cell division dependent on medium, O2, temperature, pH, genetic composition • Regular, constant cell division (logarithmically) • Smooth curve – division not synchronous • Most useful phase for biochemical, physiological and DNA replication studies • Biotechnology applications – competent cells – uptake of plasmid DNA • Late log = optimal plasmid concentration • The population is most uniform in terms of chemical and physical properties during this period

7. Stationary Phase • When the population reaches ~109/ml (106 for protozoan and algal cultures), cell division = cell death (stasis) • Nutrients become scarce • O2 is depleted • Toxic waste accumulates • The number of viable microorganisms remains constant either because metabolically active cells stop reproducing or because the reproductive rate is balanced by the rate of cell death

8. Death Phase • Viable cell mass decreases • Often logarithmic • Cells not viable when inoculated into fresh medium • Cells have reached the carrying capacity of their environment

9. The mathematics of growth-microbial growth can be described by certain mathematical terms: • Mean generation (doubling) time (g) is the time required for the population to double • Mean growth rate constant is the number of generations per unit time, often expressed as generations per hour

10. Generation times vary markedly with the species of microorganism and environmental conditions • they can range from 10 minutes for a few bacteria to several days with some eukaryotic microorganisms • Population size = 2n where n = the number of generations

11. II. Measurement of Microbial Growth

12. Counting cells directly (live and dead) • Petroff-Hausser Counting Chamber • Slide with depressed etched grids (25 squares) • Covered with a coverslip • 25 squares (area) = 1mm2 • Depth = 0.02mm • Volume = 2 x 10-5 ml in 25 squares • Determination of cell numbers: • 20 cells in one square x 25 squares/2 x 10-5 ml = 2.5 x 107 cells/ml

13. Electronic Counter • Coulter counter • Measures electrical resistance as cells pass single file through a thin stream • RBC and WBC are counted • Less accurate with small cells • High interference • Clumping

14. Counting only live cells • Plating techniques (spread plate, pour plate) using serial dilutions • Colony forming units (CFU) usually arise from one organism (but may be several if clumpy) • Membrane filtration assay • Membrane traps bacterial on the surface • Membrane transferred to an agar plate • Colonies grow  counted • Can use selective media (e.g. Endo agar for coliform counts in contaminated water supplies)

15. Measurement of cell mass • Cell mass increases as cell number increases • Dry weight measurements • Growth, concentration, wash, dried, weighed • Spectrophotometric determination • Light is scattered and is proportional to cell number • Linear relationship between absorbance and cell density • Often written as % transmittance (as absorbance increases, transmittance decreases) • Requires cultures to be ~107/ml and upwards (slight turbidity)

16. III. Continuous Culture Techniques

17. Used to maintain cells in the exponential growth phase at a constant biomass concentration for extended periods of time • Conditions are met by continual provision of nutrients and removal of wastes = OPEN SYSTEM • Constant conditions are maintained

18. Chemostat • A continuous culture device that maintains a constant growth rate by: • supplying a medium containing a limited amount of an essential nutrient at a fixed rate • removing medium that contains microorganisms at the same rate • As fresh media is added to the chamber, bacteria are removed • Limiting nutrients control growth rates • Cell density depends on nutrient concentration

19. Turbidostat • A continuous culture device that regulates the flow rate of media through the vessel in order to maintain a predetermined turbidity or cell density • There is no limiting nutrient • Absorbance is measured by a photocell (optical sensing device) • The number of cells in culture controls the flow rate and the rate of growth of culture adjusts to this flow rate

20. Balanced and Unbalanced Growth

21. Balanced (exponential) growth occurs when all cellular components are synthesized at constant rates relative to one another • Unbalanced growth occurs when the rates of synthesis of some components change relative to the rates of synthesis of other components. • This usually occurs when the environmental conditions change

22. IV. Environmental Factors Affecting Microbial Growth

23. Solutes and Water Activity • Osmotic concentrations affect microbes (e.g. plasmolysis in hypertonic solutions) • Water activity (aw) = measurement of availability of water in particular environments • Aw = Psolution/Pwater (P = vapor pressure) = inversely related to osmotic pressure • If the solution has a high osmotic pressure (high extracellular solute concentration), then its Aw = low

24. Energy is required by microbes to tolerate low aw because in order to keep water, solute concentration inside of cells must be kept high = Osmotolerance • S. aureus can tolerate up to 3M NaCl • Archaebacteria halophiles tolerate 2.8-6.2M NaCl (Great Salt Lake, Dead Sea) • Avoidance of plasmolysis

25. pH (Log scale of 0 – 14; each pH unit = 10x change) • pH is the negative logarithm of the hydrogen ion concentration

26. Acidophiles grow best between pH 0 and 5.5 • Neutrophiles grow best between pH 5.5 and 8.0 • Alkalophiles grow best between pH 8.5 and 11.5

27. Extreme alkalophiles grow best at pH 10.0 or higher • Despite wide variations in habitat pH, the internal pH of most microorganisms is maintained near neutrality either by proton/ion exchange or by internal buffering • Sudden pH changes can inactivate enzymes and damage PMs • Reason for buffering culture medium, usually with a weak acid/conjugate base pair (e.g. KH2PO4/K2HPO4 – monobasic potassium/dibasic potassium)

28. Temperature • Microorganisms are sensitive to temperature changes • Usually unicellular and poikilothermic • Enzymes have temperature optima • If temperature is too high, proteins denature, including enzymes, carriers and structural components • Temperature ranges are enormous (-20 to 100oC)

29. Organisms exhibit distinct cardinal temperatures (minimal, maximal, and optimal growth temperatures) • If an organism has a limited growth temperature range = stenothermal (e.g. N. gonorrhoeae) • If an organism has a wide growth temperature range = eurythermal (E. faecalis)

30. Psychrophiles can grow well at 0C, have optimal growth at 15C or lower, and usually will not grow above 20C • Arctic/Antarctic ocean • Protein synthesis, enzymatic activity and transport systems have evolved to function at low temperatures • Cell walls contain high levels of unsaturated fatty acids (semi-fluid when cold)

31. Psychrotrophs (facultative psychrophiles) can also grow at 0C, but have growth optima between 20C and 30C, and growth maxima at about 35C • Many are responsible for food spoilage in refrigerators • Mesophiles have growth minima of 15 to 20C, optima of 20 to 45C, and maxima of about 45C or lower • Majority of human pathogens

32. Thermophiles have growth minima around 45C, and optima of 55 to 65C • Hot springs, hot water pipes, compost heaps • Lipids in PM more saturated than mesophiles (higher melting points) • Hyperthermophiles have growth minima around 55C and optima of 80 to 110C • Sea floor sulfur vents

33. Oxygen concentration • Obligate aerobes are completely dependent on atmospheric O2 for growth • Oxygen is used as the terminal electron acceptor for electron transport in aerobic respiration • Facultative anaerobes do not require O2 for growth, but do grow better in its presence • Aerotolerant anaerobes ignore O2 and grow equally well whether it is present or not

34. Obligate (strict) anaerobes do not tolerate O2 and die in its presence • Microaerophiles are damaged by the normal atmospheric level of O2 (20%) but require lower levels (2 to 10%) for growth

35. Oxygen tolerance is determined by an organism’s ability to destroy toxic oxidizing products of oxygen reduction • Remember, because oxygen has two unpaired outer orbital electrons, it accepts electrons readily

36. Toxic compounds • Superoxide radical: • O2 + e- O2•- • Hydrogen peroxide: • O2•- + e- + 2H+  H2O2 • Hydroxyl radical: • H2O2 + e- + H+ H2O + OH• • These compounds are used deliberately by phagocytic WBC to break down intracellular microbes (respiratory burst)

37. Solution used by obligate aerobes and facultative anaerobes: • Produce enzymes that convert these toxic oxidizing products to non-toxic compounds • Superoxide dismutase 2O2•- + 2H+  O2 + H2O2 • Catalase 2H2O2  2H2O + O2 • Aerotolerant microbes have SOD; Obligate anaerobes lack SOD and catalase or have low concentrations

38. Laboratory considerations • Aerobic cultures • Shaken or sterile air introduced to medium

39. Anaerobic cultures • Remove oxygen • Include reducing agents in medium (e.g. thioglycollate or cysteine • Dissolved oxygen is destroyed • Growth beneath surface • Replace oxygen with nitrogen gas and CO2 gas

40. Gas-Pak jar • H2 + palladium catalyst + O2 H2O • Bags or pouches  CaCO3  CO2 rich atmosphere

41. Pressure • Barotolerant organisms are adversely affected by increased pressure, but not as severely as are nontolerant organisms • Barophilic organisms require, or grow more rapidly in the presence of, increased pressure

42. Radiation • Ultraviolet radiation damages cells by causing the formation of thymine dimers in DNA • Photoreactivation repairs thymine dimers by direct splitting when the cells are exposed to blue light • Dark reactivation repairs thymine dimers by excision and replacement in the absence of light

43. Ionizing radiation such as X rays or gamma rays are even more harmful to microorganisms than ultraviolet radiation • Low levels produce mutations and may indirectly result in death • High levels are directly lethal by direct damage to cellular macromolecules or through the production of oxygen free radicals