Growth and Multiplication of Bacteria Hugh B. Fackrell Sept 1997 Filename: Growth.ppt

Requirements for Growth/Multiplication ALL required nutrients correct pH temperature salinity, moisture redox potential atmosphere

Growth Liquid vs Solid Media Liquid: clear >>turbid Solid: individual colonies each colony derived from a single cell

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)

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

Binary Fission Light micrograph

Binary Fission

Consequences of Binary Fission Very large number of cells very fast Mathematical 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 numbers Phases of growth demonstrated Generation time easily calculated

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

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

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

Growth curves for exponentially increasing population Number of cells Log number of cells Time (hours)

Bacterial Growth Curve Stationary phase Death phase Log phase Lag phase 1 5 10 Time (hours)

Measurement of Growth Constants G: Generation Time K: 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 Log Number of Bacteria Log phase Double # cells Generation time 1 5 10 Time (hours)

Slope of Log phase proportional to generation time Time (hours) Log Number of bacteria Fast Medium Doubling number Slow

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 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

Factors influencing lag phase Age 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

Growth Responses: Temperature Rate of Growth Thermophile Mesophile Extreme Thermophile Psychrotroph Pyschrophile -10 10 20 30 40 50 60 70 80 90 100 Temperature (o C)

Growth Responses: pH pH Rate of Growth Neutrophile Alkalophile Acidophile 1 2 3 4 5 6 7 8 9 10 11 12 pH

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

Diauxic Growth: 2 carbon sources [Sugar] Arabinose Glucose Time (hr)

Synchronous Growth Filtration Temperature shock Starvation 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 Number of Cells Asynchronous growth Synchronous growth Time (min)

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

Growth in Limited Nutrients Total Growth Growth Rate [Nutrient]

Applications of Limiting [Nutrient] Chemostat (continuous culture) Bio-Assay

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)

Bio-Assay Vitamin B-12 measurement in Green beans Lactobacillus leichmanni Microbial Growth Growth of known [nutrient] Growth of unknown [Nutrient] in unknown [Nutrient] mg/ml

Chemostat Description of Instrument Principle Steady State Sample Results Application

Chemostat: Description of Instrument

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 Steady State K = D Flow rate Vessel volume D =

Chemostat: Sample Results Measurement Value Cell density or biomass Nutrient conc Generation time Dilution Rate of Nutrient

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

Bacterial Growth in 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 Prostheses Tampons IUD Foley: Intravenous: latex silicone Intravenous: polyurethane S. epidermidis Prostheses Hip joints Dental implants voicebox Tampons IUD

Biofilms: Water Dental lines Spacecraft Drinking filters ALL surfacces Biofilm in gut of a mollusc

Biofilms: Disease Cystic fibrosis Ulcers Dental caries lung-alveolar surface Ulcers Helicobacter jejuni Dental caries Streptococccus spp