MICROBIOLOGY BACTERIAL GROWTH

1
MICROBIOLOGYBACTERIAL GROWTH

• Instructor
• Terry Wiseth
• Northland Community Technical College

2
BACTERIAL GROWTH

• Culture
• Increase in the population of cells
• Generation time
• the time it takes to divide (double) is called

3
BINARY FISSION

• division exactly in half
• most common means of bacterial reproduction
• forming two equal size progeny
• genetically identical offspring
• cells divide in a geometric progression doubling
  cell number

4
BINARY FISSION

• Doubling time is the unit of measurement of
  microbial growth

5
CULTURE GROWTH

• Growth of culture goes through four phases with
  time
• 1) Lag phase
• 2) Log or Logarithmic
  phase
• 3) Stationary phase
• 4) Death or Decline
  phase

6
BACTERIAL GROWTH CURVE
7
LAG PHASE

• Organisms are adjusting to the environment
• little or no division
• synthesizing DNA, ribosomes and enzymes
• in order to
  breakdown
  nutrients, and to
  be used
  for growth

Mouse click for lag phase adjustment
8
LOGARITHMIC PHASE

• Division is at a constant rate (generation time)
• Cells are most susceptible to inhibitors

9
STATIONARY PHASE

• Dying and dividing organisms are at an
  equilibrium
• Death is due to reduced nutrients, pH changes,
  toxic waste and reduced oxygen
• Cells are smaller and have fewer ribosomes
• In some cases cells do not die but they are not
  multiplying

10
STATIONARY PHASE
11
DEATH PHASE

• The population is dying in a geometric fashion so
  there are more deaths than new cells
• Deaths are due to
• 1) factors in stationary phase
• 2) lytic enzymes that are released when bacteria
  lyse

12
DEATH PHASE
13
ENUMERATION OF BACTERIA

• 1) viable plate count
• 2) direct count
• 3) most probable number (MPN)

4
1
5
9
10
3
8
6
2
7
14
VIABLE PLATE COUNT

• Most common procedure for assessing bacterial
  numbers
• 1) serial dilutions of a suspension of bacteria
  are plated and incubated

15
VIABLE PLATE COUNT

• 2) the number of colonies developing are then
  counted
• it is assumed that each colony arises from an
  individual bacterial cell

16
VIABLE PLATE COUNT

• 3) by counting the colonies and taking into
  account the dilution factors the concentration of
  bacteria in original sample can be determined
• 4) only plates having between 30 and 300 colonies
  are used in the calculations

See next slide for bigger diagram
17
VIABLE PLATE COUNT
18
VIABLE PLATE COUNT

• 5) multiply the number of colonies times the
  dilution factor to find the number of bacteria in
  the sample
• Example
• Plate count 54
• Dilution factor 110,000 ml
• Calculation
• 54 X 10,000 540,000 bacteria/ml
  

19
VIABLE PLATE COUNT

• TNTC
• if the number of colonies is too great (over 300)
  the sample is labeled TNTC
• Too Numerous To Count
• limitation of viable plate count
• selective as to the bacterial types that will
  grow given the incubation temperature and
  nutrient type
  

20
VIABLE PLATE COUNT
Dilution factor of 1/1,000 (10 -3)
Click to incubate
417 colonies
TNTC
21
VIABLE PLATE COUNT
Dilution factor of 1/1,000,000 (10 -6)
Click to incubate
22 colonies
Too few the count is less than 30
22
VIABLE PLATE COUNT
Dilution factor of 1/100,000 (10 -5)
Click to incubate
42 colonies
Calculate the number of bacteria per ml
23
VIABLE PLATE COUNT

• Calculate
• 42 colonies
• dilution factor of 100,000
• 42 X 100,000 ???
• 4,200,000 bacteria/ml

24
DIRECT COUNT

• Dilutions of samples are observed under a
  microscope
• the number of bacterial cells from a given volume
  of sample are
  counted
• dead cells are also
  counted
• automated particle
  counters can be used

25
DIRECT COUNT
26
DIRECT COUNT
Cell density is determined by 1) Counting the
number of bacteria in one small square 2)
Multiplying by the dilution factor of the sample
Dilution factor of 107
Number of bacteria in square 5
5 X 107 50,000,000 bacteria per ml
27
MOST PROBABLE NUMBER

• Statistical method based on probability theory
• multiple serial dilutions are performed to reach
  a point of extinction
• dilution level at which no cells are deposited

28
MOST PROBABLE NUMBER

• Criteria have been developed for indicating
  whether a dilution tube has bacteria present
• the pattern of
  positive and
  negative results
  are compared
  
  with a table of
  statistical
  probabilities for
  obtaining those
  results

29
MOST PROBABLE NUMBER

• The pattern to tubes that show growth (brown) and
  those that do not (orange) are compared with a
  statistical table to calculate MPN

30
MOST PROBABLE NUMBER

• MPN of the original sample is 170 per 100 mL

31
FACTORS INFLUENCING BACTERIAL GROWTH

• Rates of growth and death are greatly influenced
  by environmental parameters
• each bacterial species has a specific tolerance
  range for specific environmental factors
• outside this range of environmental conditions
  bacteria lose their viability
• ability to reproduce

32
FACTORS INFLUENCING BACTERIAL GROWTH

• 1) Nutrition
• 2) Temperature
• 3) Oxygen
• 4) Salinity
• 5) pH
• 6) Pressure
• 7) Radiation

33
NUTRITION

• Basic bacterial requirements
• water
• carbon
• nitrogen
• other

34
WATER

• Used to dissolve materials to be transported
  across the cytoplasmic membrane

35
CARBON

• required for the construction of all organic
  molecules
• autotrophs use inorganic carbon (CO2) as their
  carbon source
• heterotrophs use organic carbon

36
NITROGEN

• Obtained from
• inorganic source
• e.g. Nitrogen gas (N2), Nitrate (NO3),
  Nitrite(NO2), and Ammonia (NH3)
• organic source
• e.g. Proteins, broken down to amino acids
• Many organisms use nitrogen gas by nitrogen
  fixation to produce ammonia

37
OTHER NUTRIENTS

• Required in small amounts are
• Iron
• Sulfur
• Phosphorus

38
TEMPERATURE

• One of the most important factors
• optimal growth temperature
• temperature range at which the highest rate of
  reproduction occurs
• optimal growth temperature for human pathogens
  ????

39
TEMPERATURE

• Microorganisms can be categorized based on their
  optimal temperature requirements
• Psychrophiles
• 0 - 20 ºC
• Mesophiles
• 20 - 40 ºC
• Thermophiles
• 40 - 90 ºC
• Most bacteria are mesophiles especially pathogens
  that require 37 ºC

40
BACTERIAL TEMPERATURE REQUIREMENTS
100
Psychrophile
Thermophile
Max Growth
50
Mesophile
0
0 0C
37 0C
90 0C
Variable
41
BACTERIAL TEMPERATURE REQUIREMENTS
42
TEMPERATURE

• Psychrophiles
• some will exist below 0 oC if liquid water is
  available
• oceans
• refrigerators
• freezers

Pigmented bacteria in Antarctic ice
43
TEMPERATURE

• Mesophiles
• most human flora and pathogens

44
TEMPERATURE

• Thermophiles
• hot springs
• effluents from laundromat
• deep ocean thermal vents

45
OXYGEN

• Required for aerobic respiration and energy
  production
• Organisms are classified according to their
  gaseous requirements
• Obligate aerobes
• Facultative anaerobes
• Obligate anaerobes

46
OXYGEN

• Obligate aerobic
• grow only when oxygen is available
• Obligate anaerobic
• grow in the absence of oxygen
• Facultative anaerobe
• require oxygen but exhibit maximal growth rates
  at reduced oxygen concentrations

47
OXYGEN
Obligate Aerobe
Facultative Anaerobe
Obligate Anaerobe
48
SALINITY

• Halophiles
• bacteria that specifically require NaCl for
  growth
• Moderates
• grow best at 3 NaCl solution
• many ocean dwelling bacteria
• Extreme
• grow well at NaCl concentrations of greater than
  15
• salt lakes, pickle barrels

49
SALINITY

• Halophiles growing within salt lakes often turn
  the water pink
• this sometimes occurs in Great Salt Lake, Utah

50
SALINITY

• Staphylococcus are salt tolerant up to
  concentrations of 10 NaCl
• grow on surface of skin

51
BACTERIAL pH REQUIREMENTS

• microbes have different optimum pH requirements
• ACIDOPHILES
• some bacteria can grow in acid substrates
• NEUTROPHILES
• most microbes prefer a pH near neutrality
• ALKALINOPHILES
• microbes which can grow in very alkaline
  substrates

52
BACTERIAL pH REQUIREMENTS
53
BACTERIAL pH REQUIREMENTS
54
BACTERIAL pH REQUIREMENTS
55
PRESSURE

• Osmotic Pressure
• exerted on the plasma membranes due to solute
  concentrations of a solution
• osmotolerant
• bacteria able to grow in solutions with a high
  solute concentration

56
PRESSURE

• Hydrostatic Pressure
• exerted by the weight of water
• barotolerant
• bacteria able to grow at deep ocean depths

57
RADIATION

• Photosynthetic microorganisms
• require light at minimum levels of intensity and
  proper wavelengths
• exposure to light can cause the death of some
  microorganisms
• some bacteria will produce pigments that protect
  them from exposure to lethal effects of light

58
RADIATION

• Ultraviolet radiation is harmful to the DNA of
  bacteria
• causes abnormalities in cell growth and division

59
ENDBACTERIAL GROWTH