Environmental Forcing Functions:

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1 Microbial Growth Environmental Forcing Functions: Temperature: Psychrophile, Mesophile, Thermophile & Hyperthermophile Cardinal Temps: Min*, Max, & Optimal* Q 10 Rule: 10 C rise will double the growth rate* Pressure: Barophiles (Most are also psychrophiles!) Found only in the deep ocean...so far

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3 Methanopyrus kandleri 116 Takai, et. al PNAS

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5 Growth temperature ranges for various life forms

6 Summer Surface Temperature max 7 o C

7 Morning Glory Pool 70 o C 20 o C

8 Average Ocean Depth

9 Microbial Growth Environmental Forcing Functions: ph: acidophiles & alkaliphiles Cytoplasm still near neutral Eh: available electron donors & terminal electron acceptors affects the chemistry of the environment

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12 Thermodynamics: The Chemical Fuels and Oxidants of Life e- source e- sink

13 Microbial Growth Environmental Forcing Functions: Salt: Halophiles Compatible solutes: amino acid derivatives (e.g., proline & glycine), sugars, & alcohols. Water Activity: Xerophiles (live in very dry habitats) Rem: All microbes are osmotrophs, must use organic material in solution! Oxygen Usage: aerobe, facultative (an)aerobe, microaerophile, obligate anaerobe DeTox enzymes: Catalase, Peroxidase, SOD

14 3%

15 Compatible solutes

16 Compatible solutes

17 Compatible solutes

18 Water activity is a measure of the energy status of the water in a system. It is defined as the vapor pressure of a liquid divided by that of pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one. a w p/p o p = vapor pressure of water in the substance p o = vapor pressure of pure water

19 Response of bacterial growth to oxygen availability * * Strict anaerobes will not grow if culture medium is prepared aerobically or if in plastic tubes

20 Change in Colony Phenotype due to Redox-Active compounds Phenazine production modulates colony morphology in P. aeruginosa PA14. Cultures were spotted onto agar plates containing Congo Red and Coomassie Blue, and incubated at 20 C for 6 days. The phenazine null strain (Δphz) started to wrinkle on day 2, the wild type (wt) wrinkled on day 3, whereas a pyocyanin overproducer (DKN370) remained smooth and white after 6 days. L.E.P. Dietrich et. al Science

21 Anaerobic Glove Bag N 2 /H 2 Atmosphere For Obligate Anaerobes A bi J Anaerobic Jar Atmosphere by GasPak

22 Reactive & Toxic forms of Oxygen Nasty!

23 4 electron reduction of O 2 to water

24 Bacterial Enzymes that Protect the Cell Against Toxic Forms of Oxygen Catalase Superoxide Dismutase Aerobe + + Faculatative anaerobe + + Microaerophile - + Obligate Anaerobe - - Absence of these enzymes leads to Oxygen sensitivity

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26 Catalase Test

27 Cytochrome Oxidase Test An important diagnostic indicator for the ID of Pseudomonas and Neisseria spp.

28 and now for something completely l different Microbial Growth

29 The Process of Growth Go Metabolism required for growth, both anabolic and catabolic. ~2000 reactions! Usual Definition: Increase in cell numbers Other definitions possible spores, UMC s s, respiration, viable but non-culturable, morphology changes (life cycle) Divide via Binary Fission: 3 mechanisms Cell Elongation cell wall DNA Replication rate limiting iti step Cell Division septum formation

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31

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33 FtsZ ring & cell division The divisome phase DNA stain FtsZ stain DNA & FtsZ

34 FtsZ similar to Tubulin MreB similar to Actin

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36 Bactoprenol, the lipid carrier of peptidoglycan building blocks.

37 Autolysins

38 Penicillin blocks this reaction

39 T d = 100 min T d = 20 min

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41 The Process of Growth Growth Rate: Time it takes to reproduce t ½ = ln2/μ = 0.693/μ Phases of Growth in Batch culture Lag, Log, Stationary, Death Measurement of Growth Total cell counts Viable cell counts Turbidity

42 The growth rate of a microbial culture

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44 Estimating the generation time of a microbial culture with semi-log plots in exponential phase t = time n = generations g = generation time

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47 Lag phase The Growth Cycle Cells synthesizing materials, not dividing Log gphase = exponential growth doublings increases density by ~1000 log 10 (N) increases linearly Stationary yphase Cells no longer growing Death phase

48 The Growth Cycle L l t Log scale necessary to show wide range of concentrations

49 Typical growth curve for an average bacterial population Cryptic Growth

50 Total Cell counts using the Petroff-Hausser Counter

51 Total Cell counts using the Petroff-Hausser Counter

52 Viable cell count methods on standard Petri Dish

53 Counting the number of viable cells by serial dilution and plate count Also known as dilution to extinction. Used to obtain a pure culture without using solid medium

54 (Part 1) Concentration of cells by membrane filtration

55 (Part 2) Concentration of cells by membrane filtration

56 Turbidity measurements of microbial growth

57 Turbidity measurements of microbial growth

58 The Process of Growth Continuous Culture: chemostat Steady State Reproducible Physiology Fine control Key parameters: Ks, μmax, Yield Closed systems vs. Open systems vs. Nature

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60 D = F/V = Where: D = dilution rate F = flow rate V = volume =growthrate rate Rem: At Steady State

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62 Starvation

63 Steady-state relationship between substrate concentration and output of bacterial mass D Ymax

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