1. Microbial Growth Environmental influences and adaptations Bio3124 Lecture #5

2. Growth Under favorable nutritional conditions Biosynthesis leads to increase in cellular constituents Cells divide and population increases Growth: increase in population size not individual cells size 2

3. Binary Fission and Exponential Growth 3 1 st division 2 nd division 3 rd division 4 th division N = N o. 2 n n = number of divisions (generations ) 2 0 =1 2 1 =2 2 2 =4 2 3 =8 2 4 =16

4. The Mathematics of Growth Two parameters are important 1. Generation time (g) time required for the population to double in size –Varies depending on species of microorganism and environmental conditions –range is from 10 minutes for some bacteria to several days for some eukaryotic microorganisms 4

5. The Mathematics of Growth 2. Mean growth rate constant (k) Mean number of generations (divisions) per hour –This shows how fast the cells are growing under culture conditions during log phase – it is the inverse of “g” value – ie. k=1/g (hr -1 ) How do you find the generation time? 5

6. 6 The Mathematics of Growth N 0 cells after “n” generations produce N cells over a given incubation time (t) Since population increases exponentially, then the final cell yield is an exponential function, N= N 0 2 n log N= log (N 0. 2 n ) log N= log N 0 + n.log2 n.log2=log N-log N 0

7. 7 Solving for n (number of generations) n = (log N - log N 0 )/log2 n = (log N - log N 0 )/0.301 Since “n” generations happened over “t” incubation time Then the mean generation time: g=t/n (min or hr) Graphically this corresponds to the time interval that cells doubled in the linear portion of log phase. The Mathematics of Growth

8. Problem A bacterial culture grows from 1 X 10 6 cells/ml to 6.4 X 10 7 cells/ml in 2 hours. What is the generation time, number of generations and the average growth rate constant of this bacterial culture? N 0 = 1 X 10 6, N= 6.4 X 10 7, t=120 min n = (log N - log N 0 )/0.301 n = (log 6.4 X 10 7 - log 1 X 10 6 )/0.301 n= 6 generations G=120/6 G=20 min or 0.33 hr K=1/g K=1/0.33 K=3 generations per hour 8

9. Experimental Growth Curve Plot of population vs time batch culture –culture incubated in a closed vessel with a single batch of medium usually plotted as logarithm of cell number versus time Has four distinct phases 9

10. Growth Curve 10 Has four distinct phases

11. Lag Phase cells synthesizing new components – reorganizing gene expression –adapt to new medium varies in length –in some cases can be very short or even absent 11

12. Exponential Phase also called log phase rate of growth is constant population is most uniform in terms of chemical and physical properties during this phase Balanced growth: cellular constituents manufactured at constant rates relative to each other 12

13. 13 Cellular Mass Time Total Growth Limiting nutrient concentration Yield: function of the availability of limiting nutrient

14. Stationary Phase Total number of viable cells remains constant –may occur because metabolically active cells stop reproducing –may occur because reproductive rate is balanced by death rate cells are smaller remodeling of gene expression secondary metabolites produced 14

15. Possible reasons for entry into stationary phase nutrient limitation limited oxygen availability toxic waste accumulation critical population density reached 15

16. Starvation responses decrease in size, protoplast shrinkage, and nucleoid condensation production of starvation proteins –Chaperones prevent protein denaturation –DBPs (DNA binding proteins) protect DNA –Increased PG cross-linking strengthens cell wall Activation of mechanisms for long-term survival –increased virulence –morphological changes e.g., endospore formation and differentiation Bacillus and ClostridiumSpore bearers: Genera Bacillus and Clostridium 16

17. Sporogenesis Also called endospore formation or sporulationAlso called endospore formation or sporulation normally commences when growth ceases because of lack of nutrientsnormally commences when growth ceases because of lack of nutrients Is a complex multistage processIs a complex multistage process 17

18. Sporulation 18

19. Death Phase (decline) Two alternative hypotheses –Cells are Viable But Not Culturable (VBNC) Cells alive, but dormant programmed cell death –Fraction of the population genetically programmed to die (commit suicide) 19

20. Prolonged Decline in Growth bacterial population continually evolves process marked by successive waves of genetically distinct variants natural selection occurs Secondary metabolites –Antibiotics –Modified amino acids 20

21. Measurement of Microbial Growth can measure changes in number of cells in a population can measure changes in mass of population 21

22. Measurement of Cell Number Direct cell counts –counting chambers –electronic counters –collecting on filter membranes and staining with fluorescent dyes Viable cell counts –plating methods –membrane filtration methods 22

23. Counting chambers Petroff-Hausser chamber or Hemocytomer easy, inexpensive and quick useful for counting both eukaryotes and prokaryotes cannot distinguish living from dead cells 23

24. Electronic counters: Coulter counter useful for large microorganisms Less sensitive for bacteria microbial suspension forced through small orifice movement of microbe through orifice impacts electric current that flows through orifice instances of disruption of current are counted Can’t tell the dead and live apart 24

25. Direct counts on membrane filters cells are stained with fluorescent dyes cells filtered through special membrane that provides dark background for observing cells useful for counting bacteria with certain dyes, can distinguish live from dead cells Propidium iodide (dead cells, red) Syto-9 (live cells, green) 25

26. Enumeration: Viable Counting Methods spread and pour plate techniques –diluted sample of bacteria is spread over solid agar surface or mixed with agar and poured into Petri plate –after incubation the numbers of organisms are determined by counting the number of colonies multiplied by the dilution factor –results expressed as colony forming units per volume (CFU/ml) 26

27. Plating methods… Pour-plate or Spread-plate simple and sensitive widely used for viable counts of microorganisms in food, water, and soil inaccurate results obtained if cells clump together 27

28. Another Viable Count Method – growing on Membrane filters 28 Especially useful for analyzing aquatic samples

29. Measurement of Cell Mass Dry weight –time consuming and not very sensitive Quantity of a particular cell constituent –e.g., protein, DNA, ATP, or chlorophyll –useful if amount of substance in each cell is constant Turbidometric measures (light scattering) –quick, easy, and sensitive 29

30. 30 more cells  more light scattered  less light detected Spectrophotometry: Cannot distinguish between dead and live cells

31. The Influence of Environmental Factors on Growth most organisms grow in fairly moderate environmental conditions extremophilesextremophiles –grow under harsh conditions that would kill most other organisms 31

32. Temperature organisms exhibit distinct cardinal growth temperatures –minimal –maximal –optimal 32

33. 33 Temperature ranges for microbial growth (Pathogens)

34. Adaptations of thermophiles protein structure stabilized by a variety of means –e.g., more H-bonds, hydrophobic core –e.g., more proline= less flexibility –e.g., chaperones histone-like proteins stabilize DNA membrane stabilized by variety of means –e.g., more saturated, more branched and higher molecular weight lipids –e.g., ether linkages (archaeal membranes) 34

35. Classification of bacteria on the basis of their Oxygen need 35 need oxygen prefer oxygen ignore oxygen oxygen is toxic < 2 – 10% oxygen

36. Oxygen is toxic oxygen easily reduced to toxic products that oxidize cellular componentsoxygen easily reduced to toxic products that oxidize cellular components –superoxide radical (O 2 +e - → O 2 - ) –hydrogen peroxide (O 2 - +e - +2H + → H 2 O 2 ) –hydroxyl radical (H 2 O 2 + e - +H + → H 2 O+OH · ) aerobes produce protective enzymesaerobes produce protective enzymes –superoxide dismutase (SOD) 2O 2 - +2 H + → O 2 +H 2 O –Catalase 2H 2 O 2 → O 2 + 2H 2 O 36

37. Solutes and Water Activity water activity (a w )water activity (a w ) –amount of water available to cell –Inversely related to osmotic pressure –higher [solute]  lower a w 37

38. Adaptations: effect of NaCl on microbial growth Nonhalophiles grow 0.1-1 M Halophiles grow optimally at >0.2 M Moderate halophiles –Optimal growth at ~2M extreme halophiles –require >2 M Adaptation: Know how to control the water activity 38

39. Control of water activity compatible solutesIn hypertonic environments many use compatible solutes to increase their internal osmotic concentration ie. reduce a w (eg. amino acids, choline, K + ) In hypotonic environment, release solute from internal environment by opening channels through signaling by a mechanoreceptor sensor protein Water can cross the cell membrane either by diffusion or more quickly using aquaporins 39

40. pH Measure of H + concentration Measure of H + concentration – pH range : 0.0 – 14 Optimal pH for growth:Optimal pH for growth: –Acidophiles : pH 0.0 – 5.5 –Neutralophiles : pH 5.5 – 8.0 –Alkaliphiles : pH 8.5 – 11.5 40

41. Effects of pH Effects of pH Extreme pH –Loss of enzymatic activity; denaturation and degradation of proteins. –Hydrolysis of DNA and RNA –Loss of membrane integrity 41

42. pH Homeostasis Strategy: maintain an internal pH near neutrality Synthesize proteins, provide protection –e.g., acid-shock proteins to maintain the normal protein folding 42 pH,9 pH,2 pH,5 Transporters balance internal H+