1. © 2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Chapter 6 Microbial Nutrition and Growth

2. Growth Requirements Microbial growth –Increase in a population of microbes Result of microbial growth is discrete colony –An aggregation of cells arising from single parent cell Reproduction results in growth © 2012 Pearson Education Inc.

3. Colonies on a plate

4. Growth Requirements Organisms use a variety of nutrients for their energy needs and to build organic molecules and cellular structures Most common nutrients contain necessary elements such as carbon, oxygen, nitrogen, and hydrogen Microbes obtain nutrients from variety of sources © 2012 Pearson Education Inc.

5. Growth Requirements Nutrients: Chemical and Energy Requirements –Sources of carbon, energy, and electrons –Two groups of organisms based on source of carbon –Autotrophs –Heterotrophs –What is our source of carbon? –What is a plant’s source of carbon? –Two groups of organisms based on source of energy –Chemotrophs –Phototrophs © 2012 Pearson Education Inc.

6. Growth Requirements Nutrients: Chemical and Energy Requirements –Oxygen requirements –Obligate aerobes—organisms that cannot live without oxygen. –Obligate anaerobes—organisms that cannot live in the presence of oxygen. –Facultative anaerobes—can live with or without oxygen. –Aerotolerant anaerobes—do not use aerobic metabolism, but can tolerate oxygen. © 2012 Pearson Education Inc.

7. 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 © 2012 Pearson Education Inc.

8. Growth Requirements Nutrients: Chemical and Energy Requirements –Oxygen requirements –Two common toxic forms of oxygen –Superoxide radicals (O 2 - ) –Peroxide anion (O 2 2- ) –Organisms that grow in the presence of oxygen must have enzymes that can break down the toxic forms of oxygen. © 2012 Pearson Education Inc.

9. Growth Requirements Superoxide radicals (O 2 - ) Superoxide dismutase 2O 2 - + 2H +  H 2 O 2 + O 2 Peroxide anion (O 2 2- ) Catalase 2H 2 O 2  2H 2 O + O 2 Also peroxidase can break down H 2 O 2 Other than these enzymes, aerobes use antioxidants such as vitamins C and E in order to protect against toxic oxygen products.

10. Figure 6.2 Catalase test

11. Figure 6.3 Oxygen requirements of organisms-overview

12. Growth Requirements Nutrients: Chemical and Energy Requirements –Nitrogen requirements –Anabolism often ceases because of insufficient nitrogen –All cells recycle nitrogen from amino acids and nucleotides –Nitrogen fixation by certain bacteria is essential to life on Earth because most organisms cannot use nitrogen gas (N 2 ) even though it is the most makes up 79% of our atmosphere. © 2012 Pearson Education Inc.

13. Growth Requirements Nutrients: Chemical and Energy Requirements –Other chemical requirements –Phosphorus –Sulfur –Trace elements –Required only in small amounts © 2012 Pearson Education Inc.

14. Growth Requirements Physical Requirements –Temperature –Effect of temperature on proteins –If temperature is too high, proteins can denature. –If temperature is too low, proteins may not be active. –Effect of temperature on membranes of cells and organelles –If too low, membranes become rigid and fragile –If too high, membranes become too fluid © 2012 Pearson Education Inc.

15. Figure 6.4 Microbial growth-overview

16. Temperature Requirements Psychrophiles—grow best at temperatures below 15 o C. Mesophiles—grow best in temperatures ranging from 20 o C to 40 o C. Thermophiles—grow at temperatures above 45 o C. Hyperthemophiles—grow at temperatures above 80 o C.

17. Celsius to Fahrenheit o C x 9/5 + 32 37 o C = 98.6 o F ~30 o C = skin temperature 25 o C = room temperature 65 o C = will denature most enzymes

18. Figure 6.5 Four categories of microbes based on temperature ranges for growth Psychrophiles Mesophiles Thermophiles Hyperthermophiles Growth rate Temperature (°C)

19. Figure 6.6 An example of psychrophile-overview

20. Quick Lab—comparison of temperature requirements among microbes. Each group of three will receive a TSA plate and will divide it into four quadrants with a wax pencil. One quadrant will be spot-inoculated with S. epidermidis, one with S. marcescens, and one with Bacillus stearothermophilus. Some plates will be incubated overnight at 25 o C, some at 37 o C, and some at 65 o C. Make sure your inoculum is not too heavy; in other words, don’t put a massive hunk of bacteria onto the plate.

21. Growth Requirements Physical Requirements –pH –Organisms are sensitive to changes in acidity –Neutrophiles grow best in a narrow range around neutral pH –Acidophiles grow best in acidic habitats –Alkalinophiles live in alkaline soils and water © 2012 Pearson Education Inc.

22. 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 –Some have cell walls that retain water –Endospores cease most metabolic activity –Two physical effects of water –Osmotic pressure –Hydrostatic pressure © 2012 Pearson Education Inc.

23. 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 –Hypertonic solutions have greater solute concentrations –Restricts organisms to certain environments –Obligate and facultative halophiles—salt-loving organisms. »Staphylococcus aureus can colonize the skin because it can tolerate higher salt concentrations. © 2012 Pearson Education Inc.

24. 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 shape © 2012 Pearson Education Inc.

25. Growth Requirements Associations and Biofilms –Organisms live in association with different species –Antagonistic relationships –Synergistic relationships—both organisms benefit –Symbiotic relationships—both organisms benefit to the extent that they do not normally live outside the relationship. –We are dependent on the bacteria colonizing our bodies! © 2012 Pearson Education Inc.

26. Growth Requirements Associations and Biofilms –Biofilms –Complex relationships among numerous microorganisms –Develop an extracellular matrix –Adheres cells to one another –Allows attachment to a substrate –Sequesters nutrients –May protect individuals in the biofilm –Form on surfaces often as a result of quorum sensing—microorganisms attracted to others already present –Many microorganisms more harmful as part of a biofilm © 2012 Pearson Education Inc.

27. Figure 6.7 Plaque (biofilm) on a human tooth

28. Culturing Microorganisms Inoculum introduced into medium –Environmental specimens –Clinical specimens –Stored specimens Culture –Act of cultivating microorganisms or the microorganisms that are cultivated © 2012 Pearson Education Inc.

29. Figure 6.8 Characteristics of bacterial colonies-overview

30. Culturing Microorganisms Obtaining Pure Cultures –Cultures composed of cells arising from a single progenitor –Progenitor is termed a CFU –Aseptic technique prevents contamination of sterile substances or objects –Two common isolation techniques –Streak plates –Pour plates © 2012 Pearson Education Inc.

31. Figure 6.9 Streak plate method of isolation-overview

32. Streak plate Good and bad

33. Figure 6.10 Pour plate method of isolation-overview

34. Culturing Microorganisms Culture Media –Majority of prokaryotes have not been grown in culture medium –Four types of general culture media –General purpose media –Enriched media –Selective media –Differential media © 2012 Pearson Education Inc.

35. Figure 6.12 An example of the use of a selective medium Fungal colonies Bacterial colonies pH 7.3pH 5.6

36. Figure 6.13 The use of blood agar as a differential medium Beta-hemolysis Alpha-hemolysis No hemolysis (gamma-hemolysis)

37. Figure 6.15 Use of MacConkey agar as a selective and differential medium-overview

38. Culturing Microorganisms Special Culture Techniques –Most microorganisms on earth cannot be grown in laboratories! (~99%) –Techniques developed for culturing microorganisms –Animal and cell culture—microorganisms grown on cell culture because artificial media is inadequate, e.g., leprosy, viruses © 2012 Pearson Education Inc.

39. Culturing Microorganisms Preserving Cultures –Refrigeration –Stores for short periods of time –Deep-freezing –Stores for years –Lyophilization (freeze drying) –Stores for decades © 2012 Pearson Education Inc.

40. Figure 6.17 Binary fission events-overview

41. Growth of Microbial Populations Generation Time –Time required for a bacterial cell to grow and divide –Dependent on chemical and physical conditions © 2012 Pearson Education Inc.

42. Four Phases of Microbial growth Lag phase—bacteria are adjusting to environment. Log phase—rapid growth phase. Bacteria are doubling. Stationary phase—number of dying cells equals number of cells being produced. Death phase—number of dying cells exceeds number of new cells produced.

43. Figure 6.20 Typical microbial growth curve Stationary phase Death (decline) phase Log (exponential) phase Lag phase Time Number of live cells (log)