Dynamics of Prokaryotic Growth

1. Dynamics of Prokaryotic Growth Chapter 4

2. Preview • Principles of bacteria growth. • Bacteria growth in nature. • Bacteria growth in laboratory. • Factors affect bacteria growth. • Detecting bacteria growth

3. Principles of Bacterial Growth • Prokaryotic cells divide by binary fission • One cell divides into two • Cell growth is exponential • population double with each cell division. • Cell divide at constant pace • Generation time • Time it takes for population to double • A.k.a doubling time • Varies among species

4. Practical Problem • 100 E. coli in potato salad. • How many bacteria are there in salad 2 hrs later (if the double time is 20 min)? • Why do we need to store the salad in cooler?

5. Principles of Bacterial Growth • Growth can be calculated • Nt = N0 x 2n • (Nt ) number of cells in population • (N0 ) original number of cells in the population • (n) number of divisions • Example • N0 = 10 cells in original population • n = 12 • 4 hours assuming 20 minute generation time • Nt = 10 x 212 • Nt = 10 x 4,096 • Nt = 40,960

6. Bacterial Growth in Nature • Conditions in nature have profound effect on microbial growth • Synthesize compounds useful for growth • produce multicellular associations to increase survivability • Biofilms: a community formed by a group of bacteria and their secreted slimes. Biofilm layer

7. Biofilm • Can cause disease • Difficult to kill • Architecture resist immune response and antimicrobial drugs • Can be beneficial • biofilm

8. Bacterial Growth in Nature • Prokaryotes live in mixed communities • Many interactions are cooperative • Some cells compete for nutrient

9. Bacteria growth in Laboratory • Culture media • broth media • Solid media is broth media with addition of agar • Agar marine algae extract • Liquefies above 95°C • Solidifies at 45°C • Remains solid at room temperature and body temperature • Bacteria grow in colonies on solid media surface • Single colony

10. Laboratory Cultivation • Special types of culture media • These are used to detect or isolate particular organisms • Are divided into selective and differentialmedia

11. Laboratory Cultivation • Selective media • Inhibits the growth of unwanted organisms • Allows only sought after organism to grow • Example • Thayer-Martin agar (multiple antimicrobial) • For isolation of Neisseria gonorrhoeae • MacConkey agar (antimicrobial+bile salt) • For isolation of Gram-negative intestinal bacteria

12. Laboratory Cultivation • Differential media • Contains substance that bacteria change in recognizable way • Example • Blood agar • Test for hemolysis • MacConkey agar • pH indicator

13. Obtaining Pure Culture • Pure culture is defined as population of cells derived from single cell • All cells are genetically identical • to study functions of specific species • Obtain pure culture • Aseptic technique

14. Obtaining Pure Culture • Streak-plate method • Simplest and most commonly used in bacterial isolation • Object is to reduce number of cells being spread

15. Bacterial Growth in Laboratory Conditions Cells in laboratory grown in closed or batch system Population of cells increase in predictable fashion Follows a pattern called growth curve

16. Bacterial Growth in Laboratory Conditions The Growth Curve Characterized by five distinct stages Lag stage Exponential or log stage Stationary stage Death stage Phase of prolonged decline

17. Bacterial Growth in Laboratory Conditions Lag phase synthesis of cell components and prepare for division Log phase exponential growth Cell divide at constant rate Produce primary metabolites Compounds required for growth Cells enter late log phase Cell wall and cell membrane component changes Synthesize secondary metabolites Used to enhance survival Antibiotics

18. Stationary phase Overall population remains relatively stable Cells exhausted nutrients and build up toxic waste Cell growth = cell death Death phase Total number of viable cells decreases 99% of cells die at constant rate Death is exponential Much slower rate than growth Bacterial Growth in Labortory Conditions

19. Phase of prolonged decline Marked by very gradual decrease in viable population Phase may last months or years Most fit cells survive Each new cell more fit that previous Bacterial Growth in Laboratory Conditions

20. Continuous culture Bacterial culture can be maintained Continuous exponential growth can be sustained by use of chemostat Bacterial Growth in Laboratory Conditions

21. Bacterial Growth in Laboratory Conditions • Colony growth on solid medium • Position within colony determines resource availability • Cells on edge of colony have little competition and significant oxygen stores • Cells in the middle of colony have high cell density • Leads to increased competition and decreased availability of oxygen

22. Questions • What is biofilm? • What is pure culture? • What are the different stages of bacterial growth? • Selective and differential medium.

23. Environmental Factors on Growth • Major conditions that influence growth • Temperature • Oxygen • pH • Water availability

24. Environmental Factors on Growth • Psychrophile • Optimum temperature -5°C to 15°C • Found in Arctic and Antarctic regions • Psychrotroph • 20°C to 30°C • Important in food spoilage • Mesophile • 25°C to 45°C • More common • Disease causing • Thermophiles • 45°C to 70°C • Common in hot springs • Hyperthermophiles • 70°C to 110°C • Usually members of Archaea • Found in hydrothermal vents

25. Environmental Factors on Growth • Temperature and food storage • 4C can slow down bacteria growth • Freezing can stop bacteria growth • Temperature and disease • Different pathogen can only grow in different part of body. • Hansen’s disease • Syphilis disease

26. Environmental Factors on Growth • Oxygen • Prokaryotes divided based on oxygen requirements • Obligate aerobes • Absolute requirement for oxygen • Use for energy production • Micrococcus • Obligate anaerobes • No multiplication in presence of oxygen • May cause death • Clostridium

27. Environmental Factors on Growth • Facultative anaerobes • Grow better with oxygen • Use fermentation in absence of oxygen • E coil • Microaerophiles • Require oxygen in lower concentrations • Higher concentration inhibitory • Helicobacter pylori • Aerotolerant anaerobes • Indifferent to oxygen, grow with or without • Does not use oxygen to produce energy • Streptococcus

28. Decreasing O2 Environmental Factors - O2 availability

29. Environmental Factors - O2 availability

30. Environmental Factors on Growth • pH • Bacteria survive within pH range • Neutrophiles • Multiply between pH of 5 to 8 • Maintain optimum near neutral • Acidophiles • Thrive at pH below 5.5 • Maintains neutral internal pH pumping out protons (H+) • Alkalophiles • Grow at pH above 8.5 • Maintain neutral internal pH through sodium ion exchange • Exchange sodium ion for external H+

31. Environmental Factors on Growth • Water availability • All microorganisms require water for growth • Water not available in all environments • In high salt environments • Bacteria increase internal solute concentration • Synthesize small organic molecules • Osmotolerant bacteria tolerate high salt environments • Bacteria that require high salt for cell growth termed halophiles

32. Nutritional Factors on Growth • Growth of prokaryotes depends on nutritional factors as well as physical environment • Main factors to be considered are: • Required elements • Energy sources • Growth factors

33. Nutritional Factors on Growth • Required elements • Major elements • Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium and iron • Essential components for macromolecules • Organisms classified based on carbon usage • Heterotrophs • Use organic carbon as carbon source • Autotrophs • Use inorganic carbon (CO2) as carbon source • Trace elements • Cobalt, zinc, copper, molybdenum and manganese • Required in minute amounts

34. Nutritional Factors on Growth • Energy Sources • Organisms derive energy from sunlight or chemical compounds • Phototrophs • Derive energy from sunlight • Chemotrophs • Derive energy from chemical compounds • Organisms often grouped according to energy source

35. Nutritional Factors on Growth • Nutritional Diversity • Organisms thrive due to their ability to use diverse sources of carbon and energy • Photoautotrouphs • Use sunlight and atmospheric carbon (CO2) as carbon source • Called primary producers (Plants) • Chemolithoautotrophs • A.k.a chemoautotrophs or chemolitotrophs • Use inorganic carbon for energy and use CO2 as carbon source • Photoheterotrophs • Energy from sunlight, carbon from organic compounds • Chemoorganoheterotrophs • a.k.a chemoheterotrophs or chemoorganotrophs • Use organic compounds for energy and carbon source • Most common among humans and other animals

36. Nutritional Factors on Growth • Growth factors • Some bacteria cannot synthesize some cell constituents • These must be added to growth environment • Referred to as growth factors • Organisms can display wide variety of factor requirements • Some need very few while others require many • These termed fastidious

37. Questions • Major factors that affect bacteria growth • Growth factor • Carbon source and energy source of chemoheterotroph

38. Detecting Bacterial Growth • Variety of techniques to determine growth • Number of cells • Total mass • Detection of cellular products

39. Detecting Bacterial Growth • Direct cell count • Plate count

40. Detecting Bacterial Growth • Direct microscopic count • Number is measured in a know volume • Liquid dispensed in specialized slide • Counting chamber • Viewed under microscope • Cells counted • Limitation • Must have at least 10 million cells per ml to gain accurate estimate

41. Detecting Bacterial Growth • Plate counts • Measures viable cells growing on solid culture media • Count based on assumption the one cell gives rise to one colony • Number of colonies = number of cells in sample • Ideal number to count • Between 30 and 300 colonies • Sample normally diluted in 10-fold increments • Plate count methods • pour-plates • Spread-plates methods