Chapter 4 Lecture Outline Bacterial Culture, Growth, and Development.

Chapter 4Lecture Outline

Bacterial Culture, Growth, and Development

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 2

Microbial Nutrition


Microbial Nutrition

All life requires:Materials, to

make cell parts

Nutrients (C and others)

Energy, to move electrons

Electron flow, to drive all life processes Drives ions into, out of cells Used to create ATP


Microbial Nutrition: Nutrients Must be supplied from environment Macronutrients

Major elements in cell macromolecules C, O, H, N, P, S

Ions necessary for protein function Mg2+, Ca2+, Fe2+, K+

Micronutrients Trace elements necessary for enzyme function Cobalt, manganese, nickel, zinc, etc.

Additional complex growth factors for fastidious organisms Amino acids, haemin, NAD, etc

Some bacteria need nitrogen as N2 gas from air Some bacteria cannot be grown on artificial media


Obligate Intracellular Bacteria

Chicken fibroblast

Rickettsia

SEM Giemsa Stain


Microbial Nutrition: Source of Carbon Heterotrophy

Organic compounds Generates and

releases CO2

Autotrophy CO2 from air

CO2 fixation


Microbial Nutrition: Source of Energy Phototrophs

Light energy Light absorption excites electrons to high energy state Perform photosynthesis

ChemotrophsChemical oxidation –reduction reactionsTransfer electrons from high energy compounds

to make products of lower energy


Microbial Nutrition: Electron Source Lithotrophs

Inorganic molecules are electron donors Sulfur, iron, etc.

OrganotrophsOrganic molecules are electron donors

Glucose etc.


Microbial Nutrition: Summary for Prefixes for Term “-trophy” Carbon source for biomass

Auto- Hetero-

Energy source Photo- Chemo-

Electron source Litho- Organo-



Nutrient Up-Take


Nutrient Uptake: Diffusion Passive diffusion

Some gases pass freely through membranes O2, CO2

Follows gradient of material Facilitated diffusion

Transporters pass material into/out of cell

Follows gradient of material


Nutrient Uptake—Active Transport Symport and Antiport

Gradient of one molecule transports another Electron transport creates Proton-Motive Force PMF transports other molecules

Transports material against its gradient

Symport: Gradient of pumps in same direction

Antiport: Gradient of pumps in opposite direction


Nutrient Uptake—Active Transport

ABC Transporters ATP Binding CassetteUse ATP energy to pass

material into cellTransport material

against gradientUsed for uptake and

efflux

SBP only for up-take


Siderophores and Iron Up-Take System

Siderophores have high affinity for

soluble ferric ion

siderophore

Periplasmic solute-binding protein

ABC transporter

Outer membrane protein


Nutrient Uptake—Active Transport

Phosphotransferase System (PTS) Uses ATP energy to pass

material into cellModifies material as it

enters cell Gradient is maintained,

pushing material into cell

glucose enters cell and is phosphorylated. As a result, gradient of pushes more glucose inside. (glucose-6-phosphate) cannot pass out of cell.


Culturing Bacteria


Culturing Bacteria Culture media has all materials necessary for growth

Varies for different bacterial speciesElectron sourceEnergy source

If not phototrophic

Carbon source If not autotrophic

Nitrogen source If not N2-fixer



Growth Media

Complex media: Exact composition unknown Examples: Beef bouillon, yeast extract

Enriched media Contain in addition blood components

Defined synthetic media: Exact composition known Examples:


Selective and Differential Media

Reveal differences in metabolism

Selective Suppresses growth of unwanted

microbes

Differential Includes ingredients to detect

certain biochemical reactionsMacConkey

Selective and differential


Isolating Bacteria


Obtaining Pure Cultures Dilution streaking

Streak cells on plateAgar inhibits spread of

microbes on plateAll cells in colony derive

from single cell Genetically identical Clone of that original cell


Obtaining Pure Cultures Dilution in liquid culture

Reduces number of cells in each tubeSpread liquid on plate to see single colonies


Determining the Concentration of Bacteria Counting Petroff-Hauser chamber

Counts cells directlyGives accurate numberCan’t tell if cells are alive or dead

Use stain to distinguish living cells


Determining the Concentration of Bacteria Spectrophotometer

Measures optical density “Shadow” of bacteria

Gives rapid measurementCan’t tell if cells are alive or deadSolution must be at 107–1010 cells/ml

Drawing of light bulb Photodetector


Determining the Concentration of Bacteria Viable counts

Counts only cells able to reproduce Form colonies Colony forming units (CFU/ml) Assumes single cell suspension

Requires time to form colonies (overnight)


Bacterial Growth Curve


The Growth Cycle Lag phase

Cells synthesizing materials, not dividing Log phase = exponential growth

1 2 4 8 16 … 10 doublings increases density by ~1000 log10(N) increases linearly

Stationary phaseCells no longer growing

Death phase


Stationary Phase

Total number of viable bacteria does not change Changes in gene regulation Quorum sensing induced Biofilm formation Up regulation of virulence factors Spore formation Cell differentiation

Speciesspecific


Cell to Communication


Biofilms Cells secrete material to hold to a surface

Cells acting together Multiple species or a single species

Cells signal to each other Quorum sensing

Protects against dispersionPrevents antibiotics

from infiltrating


Biofilm Formation in Pseudomonas aeruginosa


Endospore Formation Cells respond to changing environment

Endospores Protect against bad conditions Disseminates cells

Forms inside (“endo”) mother cellBacillus and Clostridium species

Cortex (peptidoglycan)

Spore coat (resistant, calcium rich)Dipicolinic acid

Small acid soluble proteins (DNA stabilizing)

Exosporium


Cell DifferentiationAnimation: Endospore Formation

Click box to launch animation


Cell Differentiation Cells respond to changing environment

Heterocysts Different cells produce different

nutrients Vegetative cells—energy Heterocysts—fixed nitrogen

Myxospores Form inside fruiting body

Multicellular structure


Cell Differentiation Cells respond to changing environment

Actinomycetes form spores Bacteria produce aerial hyphae

Specialized structures containing spores

When nutrients become limited Protect genetic material Disseminate cells

Spores


Concept QuizAll bacterial cells need to be supplied with a source of

a. electrons, energy, and nutrients.

b. carbon, nitrogen, and light.

c. carbon, fixed nitrogen, and water.


Concept QuizThe fastest way to measure cell density is by using a

a. Petroff-Hauser counting chamber.

b. spectrophotometer.

c. petri plate.


Concept QuizWhen food supplies dwindle, cells protect themselves by forming

a. biofilms.

b. heterocysts.

c. spores.

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Chapter 4 Lecture Outline Bacterial Culture, Growth, and Development.

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