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Laboratory culture: pure culture

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Laboratory culture: pure culture

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    1. Laboratory culture: pure culture

    2. Bacterial growth: basic concepts

    3. Bacterial growth: basic concepts autotroph: definition unglücklich: Energie aus Licht/ C-Quelle Co2 Energie au anorg. Molekülen/C-Quelle CO2 autotroph: definition unglücklich: Energie aus Licht/ C-Quelle Co2 Energie au anorg. Molekülen/C-Quelle CO2

    4. Microbial nutrition Fe: eigentilch Mikronutrient....muss aber v.a. bei Verwendung von Dest. H2O häufig extra zugesetzt werden Liste Interessant für Abwehr von Biowaffenprogrammen: Materialfluss weist auf Bakterienzucht im grossen Stil hinFe: eigentilch Mikronutrient....muss aber v.a. bei Verwendung von Dest. H2O häufig extra zugesetzt werden Liste Interessant für Abwehr von Biowaffenprogrammen: Materialfluss weist auf Bakterienzucht im grossen Stil hin

    5. Microbial nutrition: Growth factors

    6. Microbial growth media

    7. Microbial growth media

    8. Bacterial growth

    9. Bacterial growth: exponential growth 20 h: 5242 L Zellvolumen 80 h: 7 x 10 exp 36 Kubikmeter Zellmasse (Vielfaches des Erdvolumens)20 h: 5242 L Zellvolumen 80 h: 7 x 10 exp 36 Kubikmeter Zellmasse (Vielfaches des Erdvolumens)

    10. Bacterial growth: exponential growth

    11. Bacterial growth: calculate the generation time

    12. Bacterial growth: calculate the generation time

    13. Bacterial growth: calculate the generation time

    14. Bacterial growth: calculate the generation time

    15. Bacterial growth: calculate the generation time

    16. Bacterial growth: calculate the generation time Ergebnis: g = 30 minErgebnis: g = 30 min

    17. Bacterial growth: batch culture

    18. Batch culture: Lag phase

    19. Batch culture: exponential phase

    20. Batch culture: stationary phase 1 bacterium = 10-12 g g = 20 min ? after 48 h 4000 x weight of the earth1 bacterium = 10-12 g g = 20 min ? after 48 h 4000 x weight of the earth

    21. Batch culture: death phase

    22. Measurement of microbial growth

    23. total cell count

    24. viable cell count

    25. dilutions

    26. Turbidimetric measurements

    27. Turbidimetric measurements

    28. Continuous culture: the chemostat

    29. Continuous culture: the chemostat

    30. Continuous culture: the chemostat

    31. Factors affecting microbial growth

    32. Factors affecting microbial growth: Temperature Cardinal temperatures characteristic for an organism Can vary slightly dependiing on growth conditions etc. Pyrolobus fumarii (archaeon): optimum = 113 °C E. coli: Minimum 8°C, optimum 39°C, maximum 48°C Maximum: E. coli, for example, the Max. Temp. decrease 3 C (to 45 C) when it is grown in a medium lacking methionine. This effect is known to be caused by the thermolability of the biosynthetic enzyme homoserine trans-succinylase. Ron, E. Z. and Shani, M. (1971) Jour. Bact. 107, 397. Growth Rate of Escherichia coli at Elevated Temperatures: Reversible Inhibition of Homoserine Trans- Succinykinase. iii Ron, E. Z. and Davis, B. D. (1971) Jour. Bact. 107, 391. Growth Rate of Escherichia coli at Elevated Temperatures: Limitation by Methionine. iv Paek, K. and Walker, G. C. (1987) Jour. Bact. 169, 283. Escherichia coli dnaK Null Mutants are Inviable at High Temperature. v Kristjansson, J. K. (1991) Thermophilic Bacteria CRC Press, Inc., Boca Raton, Fla. vi Brock, T. D. (1985) Science 230, 132. Life at High Temperatures.Cardinal temperatures characteristic for an organism Can vary slightly dependiing on growth conditions etc. Pyrolobus fumarii (archaeon): optimum = 113 °C E. coli: Minimum 8°C, optimum 39°C, maximum 48°C Maximum: E. coli, for example, the Max. Temp. decrease 3 C (to 45 C) when it is grown in a medium lacking methionine. This effect is known to be caused by the thermolability of the biosynthetic enzyme homoserine trans-succinylase. Ron, E. Z. and Shani, M. (1971) Jour. Bact. 107, 397. Growth Rate of Escherichia coli at Elevated Temperatures: Reversible Inhibition of Homoserine Trans- Succinykinase. iii Ron, E. Z. and Davis, B. D. (1971) Jour. Bact. 107, 391. Growth Rate of Escherichia coli at Elevated Temperatures: Limitation by Methionine. iv Paek, K. and Walker, G. C. (1987) Jour. Bact. 169, 283. Escherichia coli dnaK Null Mutants are Inviable at High Temperature. v Kristjansson, J. K. (1991) Thermophilic Bacteria CRC Press, Inc., Boca Raton, Fla. vi Brock, T. D. (1985) Science 230, 132. Life at High Temperatures.

    33. Factors affecting microbial growth: Temperature

    34. Maximum temperature In E. coli, within several minutes following a shift from 37 to 42 C, one observes induction of several dozen specific HS proteins and the repression of normal protein synthesis. This response is controlled by reprogramming transcription by alternate sigma factors.In E. coli, within several minutes following a shift from 37 to 42 C, one observes induction of several dozen specific HS proteins and the repression of normal protein synthesis. This response is controlled by reprogramming transcription by alternate sigma factors.

    35. Factors affecting microbial growth: Temperature

    36. Growth at low Temperatures: „Homoviscous adaptation“ The production of saturated vs unsaturated fatty acids is regulated by a thermolabile enzyme in the pathway of FA biosynthesis. Lipids of psychrophilic bacteria: fatty acids with up to 9 double bonds (C31:9)The production of saturated vs unsaturated fatty acids is regulated by a thermolabile enzyme in the pathway of FA biosynthesis. Lipids of psychrophilic bacteria: fatty acids with up to 9 double bonds (C31:9)

    37. „Temperature classes“ of organisms Cardinal temperatures characteristic for an organism Can vary slightly dependiing on growth conditions etc. Pyrolobus fumarii (archaeon): optimum = 113 °CCardinal temperatures characteristic for an organism Can vary slightly dependiing on growth conditions etc. Pyrolobus fumarii (archaeon): optimum = 113 °C

    38. Psychrophilic vs. Psychrotolerant

    39. Growth at high temperatures

    40. Growth at high temperatures Industrial application: Longer shelf life catalysis at high temperature: i.e. Taq DNA polymeraseIndustrial application: Longer shelf life catalysis at high temperature: i.e. Taq DNA polymerase

    41. Bacterial growth: pH Very few species grow <pH2 or >pH10 Intracellular pH 4.6: acidophile P. oshimae (grows at pH 0.7)Very few species grow <pH2 or >pH10 Intracellular pH 4.6: acidophile P. oshimae (grows at pH 0.7)

    42. Very few species grow <pH2 or >pH10 Intracellular pH 4.6: most extreme acidophile Picrophilus oshimae (grows at pH 0.7).....lyses when transferred to pH >4Very few species grow <pH2 or >pH10 Intracellular pH 4.6: most extreme acidophile Picrophilus oshimae (grows at pH 0.7).....lyses when transferred to pH >4

    43. Bacterial growth: high pH

    44. Buffers in bacterial culture media

    45. Bacterial growth: Osmosis Osmotic pressure in dilute solutions behaves according to the laws for ideal gasses (Van‘t Hoff)Osmotic pressure in dilute solutions behaves according to the laws for ideal gasses (Van‘t Hoff)

    46. Bacterial growth: Osmosis

    47. Bacterial growth: Halophiles

    48. Bacterial growth at low aw: compatible solutes Capacity to accumulate compatible sôlutes defines aw-range a microorganism can tolerate Staphylococcus: tolerates up to 7.5% NaCl (isolation!!): accumulates ProCapacity to accumulate compatible sôlutes defines aw-range a microorganism can tolerate Staphylococcus: tolerates up to 7.5% NaCl (isolation!!): accumulates Pro

    49. Bacterial growth: Oxygen Anaerobes: often rich in flavinproteins... Convert oxygen to H2O2, O2-, OH.Anaerobes: often rich in flavinproteins... Convert oxygen to H2O2, O2-, OH.

    50. Bacterial growth: toxic forms of Oxygen Superoxides produced during respiration (flavoproteins, quinones, thiols, iron-sufur cluster proteins) If H2O2 is removed by catalase, there is going to be no OH.Superoxides produced during respiration (flavoproteins, quinones, thiols, iron-sufur cluster proteins) If H2O2 is removed by catalase, there is going to be no OH.

    51. Bacterial growth: Oxygen detoxification Catalase present in all aerobes/absent in obligate anaerobesCatalase present in all aerobes/absent in obligate anaerobes

    52. Bacterial growth: Anaerobes Redox indicator resazurin: pink in presence of oxygen Methanogens often killed by brief exposure to oxygen (glove box)Redox indicator resazurin: pink in presence of oxygen Methanogens often killed by brief exposure to oxygen (glove box)

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