Growth and Multiplication of Bacteria

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Growth and Multiplication of Bacteria

• Hugh B. Fackrell
• Sept 1997
• Filename Growth.ppt

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Requirements for Growth/Multiplication

• ALL required nutrients
• correct
• pH
• temperature
• salinity,
• moisture
• redox potential
• atmosphere

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GrowthLiquid vs Solid Media

• Liquid clear gtgtturbid
• Solid individual colonies
• each colony derived from a single cell

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Growth Event

• Absorption of water nutrients
• Catabolism of carbon source
• inorganic or organic
• Biosynthesis of new cellular components
• major energy consumption
• Cell enlargement
• Cell division ( Binary Fission)

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Binary Fission

• DNA replication
• Plasma membrane invaginate
• Cell wall deposited in invaginated space
• Cross wall completed
• Cells separate

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Binary Fission

• Light micrograph

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Binary Fission
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Consequences of Binary Fission

• Very large number of cells very fast
• Mathematical progressions
• arithmetic (1gt2gt4gt6gt8gt10gt12gt14gt16)
• geometric(1gt2gt4gt8gt16)
• exponential expression (20 gt 21 gt 22 gt23gt24)
• logarithmic expression(0 gtlog21gtlog22gtlog23gtlog24

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Logarithmic Plots

• Can plot very large Range of numbers
• Phases of growth demonstrated
• Generation time easily calculated

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Cell Multiplication

• 1 20 0 l
• 2 21 log21 ll
• 4 22 log22 llll
• 8 23 log23 lllllllll
• 16 24 log24 lllllllllllllllll

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Mathematics of bacterial growth
Cells

• Generation Log 2 Log10
• 0 1 0 0.000
• 1 2 1 0.301
• 2 4 2 0.602
• 3 8 3 0.903
• 4 16 4 1.204
• 5 32 5 1.505
  
• 6 64 6 1.806
• 7 128 7 2.107
• 8 256 8 2.408

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Growth Data

• Generation cells Log10
• 1 1 0
• 5 32 1.51
• 10 1,024 3.01
• 15 32,768 4.50
• 20 1,048,576 6.02

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Growth curves for exponentially increasing
population
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Bacterial Growth Curve
1
5
10
Time (hours)
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Measurement of Growth Constants

• G Generation Time
• K Mean Growth Rate Constant

G 1/K
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G Generation time

• Time in minutes or hours for a population of
  bacteria to double in number

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Calculation of Generation Time
Log Number of Bacteria
1
5
10
Time (hours)
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Slope of Log phase proportional to generation time
Slow
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K Mean Growth Rate Constant

• K n/t
• K (log10Nt - log10Nt0)/ 0.301t
• N number of cells
• n number of generations
• t time (hr or min)
• K 1/slope ( semi log growth plot)
• Therefore G 1/K

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Sample calculation for K G

• Population increase from 103 to 109 in 10hrs
• K (log 109 - log 103) / 0.301 x 10
• K 9-3/3.01 2 generations/hours
• G 1/K 1/2 0.5 hr/generation

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Factors influencing lag phase

• Age of culture inoculum
• old culture -gt long lag
• young culture-gt short lag
• Size of inoculum
• few cells -gt long lag
• many cells -gt short lag
• Environment
• pH, temp, gases,salinity
• sub optimum -gt long lag
• optimum-gt short lag

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Growth Responses Temperature
10
20
30
40
50
60
-10
0
70
80
90
100
Temperature (o C)
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Growth Responses pH
3
4
5
6
7
8
1
2
9
10
11
12
pH
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Diauxic Growth

• Growth on two carbon sources
• Mixed sugars
• Each sugar used separately
• Glucose ALWAYS used first
• Second sugar ONLY used when glucose GONE

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Diauxic Growth 2 carbon sources
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Synchronous Growth

• Filtration
• Smaller cells
• all same size
• Temperature shock
• Hot/cold brings cells to same metabolic state
• Starvation
• deplete medium of selected nutrient

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Synchronous vs Asynchronous growth
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Growth in Limited Nutrients

• Limiting concentration of Required nutrient
• YIELD
• number of cells
• Linear increase yield with nutrient conc

Yield Mass of organisms formed
Mass of nutrients used
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Growth in Limited Nutrients
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Applications of Limiting Nutrient

• Chemostat (continuous culture)
• Bio-Assay

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Bio-Assay Procedure

• Bacterium CANNOT synthesize nutrient
• Medium all growth requirements except nutrient
  to be assayed
• Add
• equal amounts of medium to each tube
• equal numbers of bacteria to each tube
• increasing amounts of the nutrient to be assayed
• Unknown
• Incubate
• Measure growth (turbidity or viable count)

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Bio-Assay

• Vitamin B-12 measurement in Green beans
• Lactobacillus leichmanni

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Chemostat

• Description of Instrument
• Principle
• Steady State
• Sample Results
• Application

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Chemostat Description of Instrument
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Chemostat Principle

• Essential nutrient is limited
• Growth rate(K) controlled by supply rate of
  nutrient
• Yield controlled by concentration of nutrient
• Dilution rate (D) speed of nutrient flow into
  the culture vessel

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Chemostat Sample Results
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Chemostat Applications

• Growing large amounts of cells
• Industrial production
• vaccines
• pharmaceuticals
• hormones
• Long term studies of specific growth phase
• Selecting for specific mutants
• Aquatic systems

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Bacterial Growth in Natural Environments

• Natural Environments
• Animal Tissues
• Soil
• Water- freshwater- marine
• Plants

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Bacterial Growth in Natural Environments

• Active
• Short bursts of growth metabolism
• usually low rates of growth
• Quiescent
• Viable cannot culture
• Stressed
• starvation semi viable

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Biofilms Body

• Catheter
• Foley
• latex silicone
• Intravenous
• polyurethane S. epidermidis
• Prostheses
• Hip joints
• Dental implants
• voicebox
• Tampons
• IUD

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Biofilms Water

• Dental lines
• Spacecraft
• Drinking filters
• ALL surfacces

Biofilm in gut of a mollusc
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Biofilms Disease

• Cystic fibrosis
• lung-alveolar surface
• Ulcers
• Helicobacter jejuni
• Dental caries
• Streptococccus spp