Microbial Growth

1. Microbial Growth • Factors that influence growth: • Physical/Environmental • Chemical/Nutritional

2. Microbial adaptations are remarkable • Extremeophiles • May adapt to a point of no return

3. Temperature • Microbes within different ranges (-200C – 1200C) • Min and max typically ~ 30°C apart • Optimum temp closer to max than min

4. Psychrophiles: • Optimum temperature ~15°C • Psychrotrophs (moderate psychrophiles) • Optimum temperature ~25°C • Mesophiles: • Optimum temperature ~37°C

5. Thermophiles: • Optimum temperature ~60°C • Hyperthermophiles: • Optimum temperature above 70°C • Usually Archaea

7. pH • Most microbes are neutrophiles (6.5 – 7.5) • Optimum pH of most bacteria is 7 • Some bacteria are considered acid tolerant • Helicobacter pylori • Typically fungi grow over a wider pH range • Usually responsible for spoilage of acidic foods

8. Acidophiles optimum is lower (below 5.5) • Sulfolobus - Archaea from acidic hot springs • Lactobacillus – Bacteriaproduces lactic acid • Thiobacillus - Bacteria produces sulfuric acid • Alkalophiles have optimum above 8.0 • Bacillus alcalophilus ~ 10.5 • Vibriocholerae prefers pH of 9.0 outside host

9. Water Activity • Osmotic Pressure • Adaptations • inclusion bodies, compatible solutes, stretch receptors

10. Facultative halophiles • Osmotolerant/Halotolerant • Staphylococcus aureus • Fungi tend to be more tolerant than other microbes • Obligate halophiles • Most marine microbes • Extreme halophiles • Salt flats of Utah and Dead Sea

11. All organisms need: • Macroelements • CHNOPS • Microelements • K, Ca, Cl, Na • Trace elements • Growth factors

12. Carbon • One of the most important growth requirements • ½ the dry weight of a bacteria cell is carbon • Carbon skeleton base of organic compounds • Hydrocarbons

13. Metabolic Diversity • Organisms grouped by energy, carbon and electron source • Energy Source • Phototroph or Chemotroph • Carbon Source • Autotroph or Heterotroph • Electron Source • Lithotroph or Organotroph

14. Photolithoautotroph • CO2, Light and inorganic e- donor • Chemoorganoheterotroph • Organic carbon, organic chemicals and organic e- donor • Chemolithoautotroph • CO2, inorganic chemicals and inorganic e- donor • Photoorganoheterotroph • Organic carbon, light and organic e- donor • Chemolithoheterotroph • Organic carbon, inorganic chemicals and inorganic e- donor

15. Oxygen Requirements • Oxygen has many toxic forms • Organisms require enzyme systems to protect them • Superoxide (O2-) radical • Neutralized by superoxide dismutase (SOD) • 2 O2- + 2 H+ → H2O2 + O2

16. Peroxide • Detoxified by catalase or peroxidase • Catalase: H2O2 → H2O + O2 • Peroxidase: H2O2 + 2 H+ → 2 H2O

17. Obligate (Strict) Aerobes • only aerobic metabolism • Have SOD and catalase or peroxidase • Obligate (Strict) Anaerobes • Destroyed by oxygen • Do not have SOD, catalase or peroxidase • only anaerobic metabolism

18. Facultative Anaerobes • Have SOD and catalase or peroxidase • Grow with or without oxygen • aerobic or anaerobic metabolism • Grow faster in the presence of O2 • Aerotolerant Anaerobes • Have SOD • Grow with or without oxygen • only anaerobic metabolism • Grow faster in the absence of O2

19. Microaerophiles • Grow only in low levels O2 • small amounts of SOD and catalase • Produce toxic levels of superoxide free radicals and peroxide at high levels of O2 • only aerobic metabolism

20. Growth in liquid media Obligate Aerobes Facultative Anaerobes Obligate Anaerobes

21. Aerotolerant Anaerobes Microaerophiles

22. Nitrogen • Needed for amino acids, nucleic acids, and ATP • Amino acids from protein degradation • Nitrogen reduction • Reduce nitrate to ammonia then utilize the ammonia • Nitrogen fixation • assimilate gaseous nitrogen ( N2)

23. Sulfur • Needed for building some amino acids (cysteine and methionine), vitamins (thiamine and biotin) and some carbohydrates • Sulfur containing amino acids from protein degradation • Reduce sulfates (SO42-) or sulfides (H2S)

24. Phosphorus • Tends to be a limiting growth requirement • Phosphorus is needed for building nucleic acids, phospholipids, and ATP • Phosphate ion (PO43-)is an important source

25. Microelements • Several are essential for proper cell function • Signal molecules, membrane potential, enzyme cofactors • Trace Elements • Minerals required in very small amounts • Iron, copper and zinc

26. Growth Factors • Organic compounds essential growth • Cannot be synthesized by microorganisms • Vitamins • Amino acids • Nucleic acid bases • Fastidious • Neisseria

27. Cultivation Of Microorganisms • culture medium • nutrient material prepared for growing microorganisms • inoculation • introduction of a microorganism into medium • culture • growth of a microorganism observed on/in a medium

28. Types Of Culture Media • Chemically defined media: • Exact composition known • Complex media: • Exact composition varies

29. Selective media: • Favors the growth of desired microorganisms • Inhibits the growth of unwanted ones • Differential media: • Distinguishes between groups of microorganisms

30. MacConkey’s Agar • Selective medium: • Inhibits Gram-positive bacteria growth • Encourages Gram-negative bacteria growth • Differential medium: • Lactose fermenters produce acid and form pink colonies • Non-lactose fermenters form colorless colonies

31. MacConkey’s Agar Escherichia Salmonella

32. Differential media • Blood agar • Alpha Hemolysis • Beta Hemolysis • Gamma Hemolysis

33. Microbial Growth • Refers to increase in number of cells not size of individual cells • Bacteria typically reproduce by binary fission • Generation time • time required for a bacterial population to double • Typically 1-3 hours

35. Generation number is expressed as a power of 2 • Original cell is 20, 2nd generation (after one cell division) would be 21 • 20= 1 cell • 21= 2 cells • 22= 4 cells • 23= 8 cells

37. Phases of Bacterial Growth Curve • In closed system or batch culture • Lag phase • Log phase • Stationary phase • Decline phase

38. Phases of the growth curve can be observed in liquid media • Solid media, different colonies in different phases • Continuous cultures • Open system

39. Measuring Bacterial Growth • Direct Methods • Viable plate count, Membrane filtration, Microscopic count, Most Probable Number (MPN), Electronic Counters • Indirect Methods • Turbidity, Metabolic activity, Weight

40. Direct Methods • Viable Plate Count • Important to limit colonies to a countable number • 30-300 colonies (CFUs) • Serial dilutions ensure colony counts within range • Advantage: only living cells • Disadvantage: incubation time, growth requirements, may underestimate count

42. Plate count methods • pour-plates • Spread-plates methods

43. 2. Membrane Filtration • Sample (liquid) passed through filter • Filter placed on surface of solid medium • Organisms retained on filter will grow • Advantage: only living cells, can be used to count low cell concentrations • Disadvantage: must have at least 100 ml of media, requires incubation time, may underestimate count

45. 3. Microscopic Count • Known volume of sample placed in counting chamber • Viewed under microscope • Cells counted • Advantage: no incubation time is required • Disadvantage: dead cells may be counted, tedious, requires a high concentration of cell (10 million per ml)

47. 4. Most probable numbers (MPN) • Multi-tube statistical assay • Advantage: measures only living cells, useful for culturing cells that wont grow on solid media • Disadvantage: incubation time, expensive & time consuming

48. Series of dilution sets • Each set inoculated with 10X less sample than previous set • Incubated and results compared to MPN table • gives statistical estimation of cell concentration

49. 5. Electronic Counter • Coulter Counter – electrical current • Flow Cytometry – light transmission • Advantage: No incubation time • Disadvantage: dead cells may be counted, not very sensitive due to clumping and debris in media

50. Indirect methods 1.Turbidity • Uses spectrophotometer • Advantage: no incubation time • Disadvantage: must have high concentration of cells, may count dead cells