Growth and Multiplication of Bacteria

Hugh B. Fackrell

Sept 1997

Filename: Growth.ppt

Requirements for Growth/Multiplication

ALL required nutrientscorrect

pH temperature salinity, moisture redox potential atmosphere

GrowthLiquid vs Solid Media

Liquid: clear >>turbidSolid: individual colonies

each colony derived from a single cell

Growth Event

Absorption of water & nutrientsCatabolism of carbon source

inorganic or organic

Biosynthesis of new cellular components major energy consumption

Cell enlargementCell division ( Binary Fission)

Binary Fission

DNA replicationPlasma membrane invaginateCell wall deposited in invaginated spaceCross wall completedCells separate

Binary Fission

Light micrograph

Binary Fission

Consequences of Binary Fission

Very large number of cells very fastMathematical progressions

arithmetic (1>2>4>6>8>10>12>14>16) geometric(1>2>4>8>16)

• exponential expression (20 > 21 > 22 >23>24)

• logarithmic expression(0 >log21>log22>log23>log24

Logarithmic Plots

Can plot very large Range of numbersPhases of growth demonstratedGeneration time easily calculated

Cell Multiplication

1 20 0 2 21 log21 4 22 log22 8 23 log23 16 24 log24

Mathematics of bacterial growth

Generation # Log 2 Log10

0 1 0 0.0001 2 1 0.3012 4 2 0.6023 8 3 0.9034 16 4 1.2045 32 5 1.505 6 64 6 1.8067 128 7 2.1078 256 8 2.408

Cells

Growth Data

#Generation #cells Log10

1 1 05 32 1.5110 1,024 3.0115 32,768 4.5020 1,048,576 6.02

Growth curves for exponentially increasing population

Time (hours)

Number of cells

Log number of cells

Bacterial Growth Curve

1 5 10Time (hours)

Lag phase

Log phase

Stationary phase

Death phase

Measurement of Growth Constants

G: Generation TimeK: Mean Growth Rate Constant

G := 1/K

G: Generation time

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

number

Calculation of Generation Time

1 5 10Time (hours)

Log phase

Double # cells

Generation time

Log Number of Bacteria

Slope of Log phase proportional to generation time

Time (hours)

Log Number of bacteria

SlowDoubling number

Medium

Fast

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

Sample calculation for K & G

Population increase from 103 to 109 in 10hrsK= (log 109 - log 103) / 0.301 x 10K= 9-3/3.01 = 2 generations/hours

G = 1/K = 1/2 = 0.5 hr/generation

Factors influencing lag phaseAge of culture inoculum

old culture -> long lag young culture-> short lag

Size of inoculum few cells -> long lag many cells -> short lag

Environment pH, temp, gases,salinity sub optimum -> long lag optimum-> short lag

10 20 30 40 50 60-10 0 70 80 90 100

Temperature (o C)

Rate of Growth

Mesophile

PyschrophilePsychrotroph

Thermophile

Extreme Thermophile

Growth Responses: Temperature

3 4 5 6 7 81 2 9 10 11 12

pH

Rate of Growth

Neutrophile

Acidophile

Alkalophile

Growth Responses: pH

Diauxic Growth

Growth on two carbon sourcesMixed sugarsEach sugar used separatelyGlucose ALWAYS used firstSecond sugar ONLY used when glucose

GONE

Diauxic Growth: 2 carbon sources

Time (hr)

Growth [Sugar]

GlucoseArabinose

Synchronous Growth

Filtration Smaller cells all same size

Temperature shock Hot/cold brings cells to same metabolic state

Starvation deplete medium of selected nutrient

Synchronous vs Asynchronous growth

Time (min)

Number of Cells

Asynchronous growth

Synchronous

growth

Growth in Limited Nutrients

Limiting concentration of Required nutrientYIELD number of cellsLinear increase yield with nutrient conc

Yield = Mass of organisms formed

Mass of nutrients used

Growth in Limited Nutrients

[Nutrient]

Total Growth

Growth Rate

Applications of Limiting [Nutrient]

Chemostat (continuous culture)Bio-Assay

Bio-Assay: ProcedureBacterium: CANNOT synthesize nutrientMedium: all growth requirements except nutrient to be

assayedAdd

equal amounts of medium to each tube equal numbers of bacteria to each tube increasing amounts of the nutrient to be assayed

[Unknown]IncubateMeasure growth (turbidity or viable count)

Bio-Assay

[Nutrient] g/ml

Microbial Growth 0

Growth of unknown

0

0

0

0

Growth of known [nutrient]

[Nutrient] in unknown

Vitamin B-12 measurement in Green beans Lactobacillus leichmanni

Chemostat

Description of InstrumentPrincipleSteady StateSample ResultsApplication

Chemostat: Description of Instrument

Chemostat: PrincipleEssential nutrient is limitedGrowth rate(K) controlled by supply rate of nutrientYield controlled by concentration of nutrientDilution rate (D): speed of nutrient flow into the

culture vessel

Flow rate

Vessel volume

D = Steady State

K = D

Chemostat: Sample Results

Dilution Rate of Nutrient

Measurement

Value Generation time

Nutrient conc

Cell density or biomass

Chemostat: ApplicationsGrowing large amounts of cellsIndustrial production

vaccines pharmaceuticals hormones

Long term studies of specific growth phaseSelecting for specific mutantsAquatic systems

Bacterial Growth in Natural Environments

Natural Environments Animal Tissues Soil Water- freshwater- marine Plants

Bacterial Growth in Natural Environments

Active Short bursts of growth & metabolism usually low rates of growth

Quiescent Viable cannot culture

Stressed starvation semi viable

Biofilms: Body Catheter

Foley: • latex silicone

Intravenous:• polyurethane S. epidermidis

Prostheses Hip joints Dental implants voicebox

TamponsIUD

Biofilms: Water

Dental linesSpacecraftDrinking filtersALL surfacces

Biofilm in gut of a mollusc

Biofilms: Disease

Cystic fibrosis lung-alveolar surface

Ulcers Helicobacter jejuni

Dental caries Streptococccus spp