bacterial growth

Laboratory culture: pure culture- Contaminants = other microorganisms present in the sample- history of the pure culture:

- Koch employed gelatin as solidifying agent- Walter Hesse (+ wife) adopted agar- Petri (1887) invented Petri-dish

- culture medium: - rich/selective- growth inhibitors- liquid/solid - temperature- minimum: - source of energy

- sources of carbon, nitrogen, ...-Aseptic technique:

- sterilization of medium and equipment- proper handling

1

2

34

Confluent mixtureIsolated colony

Bacterial growth: basic concepts

Anabolism = biosynthesis

Catabolism = reactions to recover energy (often ATP)

Precursors

Bacterial growth: basic concepts

Chemolithotroph = inorganic compounds as energy source

Chemoorganotroph = organic compounds as energy source

troph = „to feed“ (where does energy come from?)

inconsistent nomenclature! Autotrophy = CO2 can be sole C-source

Microbial nutritionNutrients = chemical „tools“ a cell needs to grow/replicateMacronutrients = chemicals needed in large amountsMicronutrients = chemicals needed in small/trace amounts

50%

12%

% of dry weight

(sometimes non-essential)(sometimes non-essential)

Microbial nutrition: Micronutrients-Trace amounts required by all organisms- only considered if media are made from highly purified substances

Microbial nutrition: Growth factors

- organic compounds requiredby some bacteria

- vitamins, amino acids, purines, pyrimidines

- Streptoccus, Lactobacillus, Leuconostoc (lactic acid bacterium): complex vitamin requirements

Microbial growth media- chemically defined: highly purified inorganic and organic compounds in dest. H2O

- complex (undefined): digests of casein, beef, soybeans, yeast, ...

Microbial growth media

Media PurposeComplex Grow most heterotrophic organismsDefined Grow specific heterotrophs and are often mandatory for

chemoautotrophs, photoautotrophs and for microbiological assays

Selective Suppress unwanted microbes, or encourage desired microbesDifferential Distinguish colonies of specific microbes from othersEnrichment Similar to selective media but designed to increase the numbers of

desired microorganisms to a detectable level without stimulating the rest of the bacterial population

Reducing Growth of obligate anaerobes

MacConkey Agar:

Regeneration of NAD+

Fermentation RespirationNo added terminal e--acceptor Oxidant = terminal e--acceptor

ATP: substrate level phosphorylation ATP: (e--transport) oxidative phosphoryl.

Glucose

2 Glyceraldehyde-3-P 2 ATP2 NADH

2 Pyruvate

2 Lactate+ 2 H+

Acetaldehyde+2 CO2

2 Ethanol

Acetate+ Formate

H2 + CO2

Glucose2 ATP2 NADH

2 Pyruvate

2 Acetyl-CoACO2

Citric acidcycle

CO2

GTPNADH, FADH

Cytoplasmic membrane

out

inATP

H+H+H+H+H+H+

O2H2O

1 Glucose 2 ATP 1 Glucose 38 ATPSlow growth/low biomass yield Fast growth/high biomass yield

Alternate modes of energy generation

(H2S, H2, NH3)(in autotrophs)

Bacterial growth

Growth = increase in # of cells (by binary fission) generation time: 10 min - days

Growth rate = cell number/time or cell mass/time 1 generation

Bacterial growth: exponential growth

Generation time = 30 min

Cell volume = 5 m3

.... 5 ml total cell volume

80 h 7 x 1036 m3 (> earth)

Bacterial growth: exponential growth

Semilogarythmic plot

Straight line indicates logarithmic growth

Bacterial growth: calculate the generation time

g =tn

t = time of exponential growth (in min, h)g = generation time (in min, h)n = number of generations


Nt = N0 x 2nNt = number of cells at a certain time pointN0 = initial number of cellsn = number of generations

g =tn



Nt = N0 x 2nNt = number of cells at a certain time pointN0 = initial number of cellsn = number of generations

g =tn


logNt = logN0 + n x log2

logNt - logN0= n x log2

n =logNt - logN0

log2

n = 3.3 x (logNt - logN0)


Im Erlenmeyerkolben wurde eine E. coli Kultur angesetzt. Die Kultur befindet sich in der exponentiellen

Wachstumsphase. Die Geschwindigkeit des bakteriellen Wachstums wurde gemessen:

9.00 Uhr 10.00 Uhr104 Bakterien/ml 8 x 104 Bakterien/ml

Generationszeit = ?


Im Erlenmeyerkolben wurde eine E. coli Kultur angesetzt. Die Kultur befindet sich in der exponentiellen

Wachstumsphase. Die Geschwindigkeit des bakteriellen Wachstums wurde gemessen:

9.00 Uhr 10.00 Uhr104 Bakterien/ml 8 x 104 Bakterien/ml

Generationszeit = ?

g =tn

n = 3.3 x (logNt - logN0) = 3.3 x (log8 x 104 – log104)

= 3.3 x (4.9 – 4) = 3

=1 h3 = 20 min

Bacterial growth: batch culture

Batch culture: Lag phase

no Lag phase:Inoculum from exponential phase grown in the same media

Lag phase:

Inoculum from stationary culture (depletion of essential constituents)After transfer into poorer culture media (enzymes for biosynthesis)Cells of inoculum damaged (time for repair)

Batch culture: exponential phase

Exponential phase = log-phase

„midexponential“: bacteria often used for functional studies

Maximum growth rates

Batch culture: stationary phase

Bacterial growth is limited:

- essential nutrient used up- build up of toxic metabolic products in media

Stationary phase:

- no net increase in cell number- „cryptic growth“- energy metabolism, some biosynthesis continues- specific expression of „survival“ genes

Batch culture: death phase

Bacterial cell death:

- sometimes associated with cell lysis- 2 Theories:

- „programmed“: induction of viable but non-culturable- gradual deterioration:

- oxidative stress: oxidation of essential molecules- accumulation of damage- finaly less cells viable

Measurement of microbial growth

A. Weight of cell massB. number of cells:

- Total cell count- Viable count- Dilutions- turbidimetric

total cell count

A. Sample dried on slideB. Counting chamber:

Limitations:- dead/live not distinguished- small cells difficult to see- precision low- phase contrast microscope- not useful for < 106/ml

viable cell countsynonymous: plate count, colony count1 viable cell 1 colonycfu = colony forming unitAdvantage: high sensitivity; selective mediaOptimal: 30 – 300 colonies per plate ( plate appropriate dilutions)

spread plate method:

pour plate method:Bacteria must withstand 45°C briefly

dilutionsExample:3 h culture of E. coli in L-brothHow do I determine the actual number?

Turbidimetric measurements

Relationship between OD and cfu/ml must be established experimentallyExponential culture of E. coli in L-broth: 1 OD = ca. 2-3 x 109 cfu/ml

Turbidimetric measurements

Limits of sensitivity at high bacterial density„rescattering“ more light reaches detector

Continuous culture: the chemostat

steady state = cell number, nutrient status remain constant

Control:1. Concentration of a limiting nutrient2. Dilution rate3. Temperature Independent control of:

- Cell density- Growth rate


1. Concentration of a limiting nutrient

Results from a batch culture


2. Dilution rate

Factors affecting microbial growth

• Nutrients• Temperature• pH• Oxygen• Water availability

Factors affecting microbial growth: Temperature

3 cardinal temperatures:

Usually ca. 30°C

Maximum temperature

- Covalent/ionic interactions weaker at high temperatures.- Thermal denaturation: covalent or non-covalent

reversible/ irreversible- heat-induced covalent mod.: deamidation of Gln and Asn

Thermal protein inactivation:

- Missense mutations: reduced thermal stability (Temp.-sens. mutants)- Heat shock response: proteases, chaperonins (i.e. DnaK ~ Hsp70)

Genetics:

Factors affecting microbial growth: TemperatureMinimal temperature:

Proteins:- Greater -helix content- more polar amino acids- less hydrophobic amino acids

Membranes: - temperature dependent phase transition

Thermotropic Gel: Hexagonal arranged

- homoviscous adaptation

„Fluid mosaic“

Membrane proteinsinactive (mobility/insertion)

Protein function normal

Tm

Growth at low Temperatures: „Homoviscous adaptation“

Homoviscous adaptation = adjustment of membrane fluidity

- lowered Tm

- More cis-double bonds- Reduced hydrophobic interactions

- high Tm

- Few cis double bonds- optimal hydrophobic interactions

Fatty acid composition of plasma membrane as % total fatty acidsE. coli grown at: 10°C 43°CC16 saturated (palmitic) 18 % 48 %C16 cis-9-unsat. (palmitoleic) 26 % 10 %C18 cis-11-unsat. (cis-vaccinic) 38 % 12 %

- thermophiles- mesophiles

„Temperature classes“ of organisms

Psychrophilic vs. Psychrotolerant

Psychrophiles

Maximum: <20°COptimum: <15°CMinimum: <0°CHabitats:

- deep sea- glaciers

Psychrotolerant

Maximum: >20°COptimum: 20-40°CMinimum: <0-4°CHabitats: much more abundant than psychrophiles

- soil in temperate climate- foods- grow slowly even in fridge!

Sierra Nevada

Chlamydomonas nivalisThe snow algaered spores

Limit: Freezing- Inhibits bacterial growth- freezing: often liquid pockets- many bacteria survive- cryoprotectants (DMSO, glycerol)

Growth at high temperatures

<65°C

Thermophilic:optimum > 45°CSoil in sun often 50°CFermentation: 60-65°C

Hyperthermophilic:optimum > 80°COnly in few areas:Hot springs: 100°CSteam vents 150-500°CDeep sea hydrothermal vents

Growth at high temperaturesMolecular adaptations in thermophilic bacteria

- Protein sequence very similar to mesophils- 1/few aa substitutions sufficient- more salt bridges- densely packed hydrophobic cores

Proteins

- more saturated fatty acids- hyperthermophilic Archaea: C40 lipid monolayer

lipids

- sometimes GC-rich- potassium cyclic 2,3-diphosphoglycerate: K+ protects from depurination- reverse DNA gyrase (increases Tm by „overwinding“)- archaeal histones (increase Tm)

DNA

Bacterial growth: pH

(extremes: pH 4.6- 9.4)

Most natural habitats

Growth at low pH

Fungi: - often more acid tolerantthan bacteria (opt. pH5)

Obligate acidophilic bacteria:Thiobacillus ferrooxidans

Obligate acidophilic Archaea:SulfolobusThermoplasma

Most critical: cytoplasmic membraneDissolves at more neutral pH

Bacterial growth: high pH- Few alkaliphiles (pH10-11)- Bacteria: Bacillus spp.- Archaea- often also halophilic- Sometimes: H+ gradient replaced by Na+ gradient (motility, energy)- industrial applications (especially „exoenzymes“):

-Proteases/lipases for detergents (Bacillus licheniformis)-pH optima of these enzymes: 9-10

Bacterial growth: Osmosis

Water acitvity Osmotic pressure

aw =ppo

aw: rel. Water activityp: vapor pressure of a solutionp0: vapor pressure of water

p = n x R x T

V

p: osmotic pressuren: number of dissolved particlesR: universal gas constantT: temperatureV: volume of the solution

low awhigh aw high plow p

Semipermeable membrane

Bacterial growth: Osmosis

Soil: water activity = 0.9 – 1.0In general: bacteria normally have higher osmotic pressure than environment

= „positive water balance“

Osmophiles: - grow in presence of high sugar concentrationXerophiles - grow in „dehydrated“ environments

Bacterial growth: HalophilesHalophiles: - requirement for Na+

- grow optimally in media with low water activity- Mild: 1-6 % NaCl- Moderate: 6-15 % NaCl- extreme: 15 – 30% NaCl

most other organismswould be dehydrated

Bacterial growth at low aw: compatible solutes

Strategy: increase internal solute concentration

a. Pump inorganic ionsb. Synthesize organic solutes

Solute must be „compatible“ with cellular processes

Bacterial growth: OxygenO2 as electron sink for catabolism toxicity of Oxygen species

Aerobes: growth at 21% oxygenMicroaerophiles: growth at low oxygen concentrationFacultative aerobes: can grow in presence and absence of oxygenAnaerobes: lack respiratory systemAerotolerant anaerobesObligate anaerobes: cannot tolerate oxygen (lack of detoxification)

Bacterial growth: toxic forms of Oxygen

triplet oxygen: ground statesinglet oxygen: reactive

inactivated by carotenoids produced by light, biochemically

Bacterial growth: Oxygen detoxificationCatalase assay

Bacterial growth: Anaerobes

Methods to exclude/reduce oxygen:

- Closed vessels- reducing agents (i.e. thioglycolate broth)- anaerobic jar (H2-generation + Pd catalyst)- glove box (oxygen free gas) a

ir air air air air

obl

. ae

robe

obl

. an

aer

ob

e

fac.

ae

robe

mic

roa

ero

phile

aer

oto

lera

nta

nae

robe

O2

bacterial growth

Documents