Microbial Nutrition and Growth

1. 6 Microbial Nutrition and Growth

2. Metabolism Results in Reproduction • Result of microbial growth is discrete colony – an aggregation of cells arising from single parent cell • Reproduction results in growth

3. Growth Requirements • Organisms use a variety of nutrients for their energy needs and to build organic molecules and cellular structures • Most common nutrients – those containing necessary elements such as carbon, oxygen, nitrogen, and hydrogen • Microbes obtain nutrients from variety of sources

4. Nutrients: Chemical and Energy Requirements • Sources of carbon, energy, and electrons • Oxygen requirements • Nitrogen requirements • Other chemical requirements

5. Sources of Carbon, Energy, and Electrons • Organisms are categorized into two groups based on source of carbon • Those using an inorganic source of carbon (carbon dioxide) are autotrophs • Those catabolizing reduced organic molecules (proteins, carbohydrates, amino acids, and fatty acids) are heterotrophs

6. Sources of Carbon, Energy, and Electrons (continued) • Organisms categorized into two groups based on whether they use chemicals or light as source of energy • Those that acquire energy from redox reactions involving inorganic and organic chemicals are chemotrophs • Those that use light as their energy source are phototrophs

7. Four Basic Groups of Organisms Figure 6.1

8. Oxygen Requirements • Oxygen is essential for obligate aerobes (final electron acceptor in ETC) • Oxygen is deadly for obligate anaerobes • How can this be true? • Neither gaseous O2 nor oxygen covalently bound in compounds is poisonous • The forms of oxygen that are toxic are excellent oxidizing agents • Resulting chain of oxidations causes irreparable damage to cells by oxidizing compounds such as proteins and lipids

9. Four Toxic Forms of Oxygen • Singlet oxygen – molecular oxygen with electrons boosted to higher energy state • Occurs during photosynthesis so phototropic organisms have carotenoids that remove the excess energy of singlet oxygen • Superoxide radicals – some form during incomplete reduction of oxygen in aerobic and anaerobic respiration • So reactive that aerobes produce superoxide dismutases to detoxify them • Anaerobes lack superoxide dismutase and die as a result of oxidizing reactions of superoxide radicals formed in presence of oxygen

10. Four Toxic Forms of Oxygen (continued) • Peroxide anion – formed during reactions catalyzed by superoxide dismutase and other reactions • Aerobes contain either catalase or peroxidase to detoxify peroxide anion • Obligate anaerobes either lack both enzymes or have only a small amount of each

11. Four Toxic Forms of Oxygen (continued) • Hydroxyl radical – results from ionizing radiation and from incomplete reduction of hydrogen peroxide • The most reactive of the four toxic forms of oxygen • Not a threat to aerobes due to action of catalase and peroxidase • Aerobes also use antioxidants such as vitamins C and E to protect against toxic oxygen products

12. Classification of Organisms Based on Oxygen Requirements • Aerobes – undergo aerobic respiration • Anaerobes – do not use aerobic metabolism • Facultative anaerobes – can maintain life via fermentation or anaerobic respiration or by aerobic respiration • Aerotolerant anaerobes – do not use aerobic metabolism but have some enzymes that detoxify oxygen’s poisonous forms • Microaerophiles – aerobes that require oxygen levels from 2-10% and have a limited ability to detoxify hydrogen peroxide and superoxide radicals

13. Nitrogen Requirements • Anabolism often ceases due to insufficient nitrogen needed for proteins and nucleotides • Nitrogen acquired from organic and inorganic nutrients; also, all cells recycle nitrogen from amino acids and nucleotides • The reduction of nitrogen gas to ammonia (nitrogen fixation) by certain bacteria is essential to life on Earth because nitrogen is made available in a usable form

14. Other Chemical Requirements • Phosphorus required for phospholipid membranes, DNA, RNA, ATP, and some proteins • Sulfur is a component of sulfur-containing amino acids, disulfide bonds critical to tertiary structure of proteins, and in vitamins (thiamin and biotin) • Trace elements – usually found in sufficient quantities in tap water • Growth factors – organic chemicals that cannot be synthesized by certain organisms (vitamins, certain amino acids, purines, pyrimidines, cholesterol, NADH, and heme)

15. Physical Requirements for Growth • Temperature • pH • Osmolarity • Pressure

16. Temperature • Effect of temperature on proteins • Effect of temperature on lipid-containing membranes of cells and organelles • If too low, membranes become rigid and fragile • If too high, membranes become too fluid and cannot contain the cell or organelle

17. Effects of Temperature on Growth Figure 6.4a

18. Effects of Temperature on Growth Figure 6.4b

19. Catagories of Microbes Based on Temperature Range Figure 6.5

20. pH • Organisms sensitive to changes in acidity because H+ and OH- interfere with H bonding in proteins and nucleic acids • Most bacteria and protozoa grow best in a narrow range around neutral pH (6.5-7.5) – these organisms are called neutrophiles • Other bacteria and fungi are acidophiles – grow best in acidic habitats • Acidic waste products can help preserve foods by preventing further microbial growth • Alkalinophiles live in alkaline soils and water up to pH 11.5

21. Physical Effects of Water • Microbes require water to dissolve enzymes and nutrients required in metabolism • Water is important reactant in many metabolic reactions • Most cells die in absence of water • Some have cell walls that retain water • Endospores and cysts cease most metabolic activity in a dry environment for years • Two physical effects of water • Osmotic pressure • Hydrostatic pressure

22. Osmotic Pressure • Is the pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane; related to concentration of dissolved molecules and ions in a solution • Hypotonic solutions have lower solute concentrations; cells placed in these solutions will swell and burst

23. Osmotic Pressure • Hypertonic solutions have greater solute concentrations; cells placed in these solutions will undergo crenation (shriveling of cytoplasm) • This effect helps preserve some foods • Restricts organisms to certain environments • Obligate halophiles – grow in up to 30% salt • Facultative halophiles – can tolerate high salt concentrations

24. Hydrostatic Pressure • Water exerts pressure in proportion to its depth • For every addition of depth, water pressure increases 1 atm • Organisms that live under extreme pressure are barophiles • Their membranes and enzymes depend on this pressure to maintain their three-dimensional, functional shape

25. Ecological Associations • Organisms live in association with different species • Antagonistic relationships • Synergistic relationships • Symbiotic relationships • Biofilms • Complex relationships among numerous individuals • Form on surfaces often as a result of quorum sensing

26. Culturing Microorganisms • Inoculum introduced into medium (broth or solid) • Environmental specimens • Clinical specimens • Stored specimens • Culture – refers to act of cultivating microorganisms or the microorganisms that are cultivated

27. Clinical Sampling Table 6.3

28. Obtaining Pure Cultures • Cultures composed of cells arising from a single progenitor • Progenitor is termed a CFU • Aseptic technique is used to prevent contamination of sterile substances or objects • Two common isolation techniques • Streak plates • Pour plates

29. Streak Plate Method Figure 6.8a

30. Streak Plate Method Figure 6.8b

31. Pour Plate Method Figure 6.9a

32. Pour Plate Method Figure 6.9b

33. Culture Media • Majority of prokaryotes have never been grown in culture medium • Six types of general culture media • Defined media • Complex media • Selective media • Differential media • Anaerobic media • Transport media

34. Special Media Techniques • Techniques developed for culturing microorganisms • Animal and cell culture • Low-oxygen culture • Enrichment culture

35. Preserving Cultures • Refrigeration • Deep-freezing • Lyophilization

36. Growth of Microbial Populations Figure 6.17a

37. Growth of Microbial Populations Figure 6.17b

38. Arithmetic Versus Logarithmic Growth Figure 6.18a-b

39. Phases of Microbial Growth Figure 6.20

40. Measuring Microbial Growth • Direct methods • Viable plate counts • Membrane filtration • Microscopic counts • Electronic counters • Most probable number

41. Viable Plate Counts Figure 6.21

42. Membrane Filtration Figure 6.22a

43. Microscopic Counts Figure 6.23

44. Most Probable Number Figure 6.24

45. Measuring Microbial Growth • Indirect Methods • Metabolic activity • Dry weight • Turbidity

46. Turbidity and Spectrophotometric Measurement Figure 6.25a

47. Turbidity and Spectrophotometric Measurement Figure 6.25c

48. Staining • Increases contrast and resolution by coloring specimens with stains/dyes • Smear of microorganisms (thin film) air dried to slide and then fixed to surface by heat or chemical fixation • Microbiological stains are usually salts composed of cation and anion and one is colored (chromophore) • Acidic dyes stain alkaline structures; basic dyes stain acidic structures and are used more commonly

49. Staining • Simple stains • Differential stains • Gram stain • Acid-fast stain • Endospore stain • Special stains • Negative (capsule) stain • Flagellar stain • Fluorescent stains • Staining for electron microscopy

50. Simple Stains Figure 4.16b