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 95C
• Solidifies at 45C
• 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 differential media

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 (antimicrobialbile 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
Bacterial Growth in Labortory Conditions

• 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

19
Bacterial Growth in Laboratory Conditions

• 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

20
Bacterial Growth in Laboratory Conditions

• Continuous culture
• Bacterial culture can be maintained
• Continuous exponential growth can be sustained by
  use of chemostat

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 -5C to 15C
• Found in Arctic and Antarctic regions
• Psychrotroph
• 20C to 30C
• Important in food spoilage
• Mesophile
• 25C to 45C
• More common
• Disease causing
• Thermophiles
• 45C to 70C
• Common in hot springs
• Hyperthermophiles
• 70C to 110C
• 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.
• Hansens 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
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