Laboratory culture: pure culture

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Laboratory culture pure culture

• Contaminants other microorganisms present in
  the sample
• history of the pure culture
• Koch employed gelatin as solidifying agent
• Walter Hesse adopted agar
• Petri (1887) invented Petri-dish
• culture medium
• rich/selective
• growth inhibitors
• liquid/solid
• temperature
• Nutrients
• carbon, nitrogen, elements ...
• Aseptic technique
• sterilization of medium and equipment
• proper handling

Confluent mixture
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Isolated colony
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4
3
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Bacterial growth basic concepts
Precursors
Anabolism biosynthesis
Catabolism reactions to recover energy (often
ATP)
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Bacterial growth basic concepts
troph to feed (where does energy come from?)
Chemolithotroph inorganic compounds as energy
source
Chemoorganotroph organic compounds as energy
source
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Microbial nutrition
Nutrients chemical tools a cell needs to
grow/replicate Macronutrients chemicals needed
in large amounts Micronutrients chemicals
needed in small/trace amounts Autotrophy CO2
can be sole C-source
of dry weight
50
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(sometimes non-essential)
(sometimes non-essential)
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Microbial nutrition Growth factors

• organic compounds required
• by some bacteria

• vitamins, amino acids, purines, pyrimidines

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

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Microbial growth media

• chemically defined highly purified inorganic
  and organic compounds in dest. H2O

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

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Microbial growth media
Media Purpose Complex Grow most heterotrophic
organisms Defined Grow specific heterotrophs
and are often mandatory for chemoautotrophs,
photoautotrophs and for microbiological
assays Selective Suppress unwanted microbes,
or encourage desired microbes Differential Disti
nguish colonies of specific microbes from
others Enrichment Similar to selective media
but designed to increase the numbers of
desired microorganisms to a detectable level
without stimulating stimulating the
rest of the bacterial population Reducing Growth
of obligate anaerobes
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Bacterial growth
Growth increase in of cells (by binary
fission) generation time 10 min - days
1 generation
Growth rate Dcell number/time or Dcell
mass/time
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Bacterial growth exponential growth
Generation time 30 min
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Bacterial growth exponential growth
Semilogarythmic plot
Straight line indicates logarithmic growth
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Bacterial growth calculate the generation time
t
t time of exponential growth (in min, h) g
generation time (in min, h) n number of
generations
g
n
The generation time is the time needs the culture
population to double
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Bacterial growth calculate the generation time
t time of exponential growth (in min, h) g
generation time (in min, h) n number of
generations
t
g
n
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Bacterial growth calculate the generation time
t time of exponential growth (in min, h) g
generation time (in min, h) n number of
generations
t
g
n
Nt number of cells at a certain time point N0
initial number of cells n number of generations
Nt N0 x 2n
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Bacterial growth calculate the generation time
t time of exponential growth (in min, h) g
generation time (in min, h) n number of
generations
t
g
n
Nt number of cells at a certain time point N0
initial number of cells n number of generations
Nt N0 x 2n
logNt logN0 n x log2
logNt - logN0 n x log2
logNt - logN0
n
log2
n
3.3 x (logNt - logN0)
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Bacterial growth calculate the generation time
Im Erlenmeyerkolben wurde eine E. coli Kultur
angesetzt. Die Kultur befindet sich in der
exponentiellen Wachstumsphase. Die
Geschwindigkeit des bakteriellen Wachstums wurde
gemessen 12.00 Uhr 14.00 Uhr 103
Bakterien/ml 1.6 x 104 Bakterien/ml Generations
zeit ?
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Bacterial growth calculate the generation time
Im Erlenmeyerkolben wurde eine E. coli Kultur
angesetzt. Die Kultur befindet sich in der
exponentiellen Wachstumsphase. Die
Geschwindigkeit des bakteriellen Wachstums wurde
gemessen 12.00 Uhr 14.00 Uhr 103
Bakterien/ml 1.6 x 104 Bakterien/ml Generations
zeit ?
n
3.3 x (logNt - logN0)
3.3 x (log1.6 x 104 log103)
3.3 x (4.2 3) 4
2 h
t
0.5 h

g
4
n
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Bacterial growth batch culture
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Batch culture Lag phase
no Lag phase
Inocculum from exponential phase grown in the
same media
Lag phase
Inocculum from stationary culture (depletion of
essential constituents) After transfer into
poorer culture media (enzymes for
biosynthesis) Cells of inocculum damaged (time
for repair)
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Batch culture exponential phase
Exponential phase log-phase
Maximum growth rates
midexponential bacteria often used for
functional studies
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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

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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

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Measurement of microbial growth
A. Weight of cell mass B. number of cells
- Total cell count - Viable count - Dilutions -
turbidimetric
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total cell count
A. Sample dried on slide B. Counting chamber

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

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viable cell count
synonymous plate count, colony count 1 viable
cell ? 1 colony cfu colony forming
unit Advantage high sensitivity selective
media Optimal 30 300 colonies per plate (?
plate appropriate dilutions)
spread plate method
pour plate method Bacteria must withstand 45C
briefly
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dilutions
Example 3 h culture of E. coli in L-broth How do
I determine the actual number?
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Turbidimetric measurements
Relationship between OD and cfu/ml must be
established experimentally Exponential culture of
E. coli in L-broth 1 OD ca. 2-3 x 109 cfu/ml
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Turbidimetric measurements
Two typical growth curves in batch culture
Klett units
Limits of sensitivity at high bacterial
density rescattering? more light reaches
detector
Klett units
1 Klett unit OD/0.002
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Continuous culture the chemostat
steady state cell number, nutrient status
remain constant

• Control
• Concentration of a limiting nutrient
• Dilution rate
• Temperature
• Independent control of
• Cell density
• Growth rate

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Continuous culture the chemostat
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Continuous culture the chemostat
2. Dilution rate
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Factors affecting microbial growth

• Nutrients
• Temperature
• pH
• Oxygen
• Water availability

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Factors affecting microbial growth Temperature
3 cardinal temperatures
Usually ca. 30C
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Factors affecting microbial growth Temperature
Arrhenius equation
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Maximum temperature
Thermal protein inactivation
- Covalent/ionic interactions weaker at high
temperatures. - Thermal denaturation (covalent or
non-covalent) - heat-induced covalent mod.
deamidation of Gln and Asn - Thermal
denaturation reversible or irreversible.
Genetics
- Missense mutations reduced thermal stability
(Temp.-sens. mutants) - Heat shock response
proteases, chaperonins (i.e. DnaK Hsp70)
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Factors affecting microbial growth Temperature

• Minimal temperature
• Proteins
• Greater a-helix content
• more polar amino acids
• less hydrophobic amino acids

Membranes - temperature dependent phase
transition
Thermotropic Gel Hexagonal arranged
Fluid mosaic
Tm
??
Membrane proteins inactive (mobility/insertion)
Protein function normal
- homoviscous adaptation
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Growth at low Temperatures Homoviscous
adaptation
Homoviscous adaptation adjustment of membrane
fluidity
- high Tm - Few cis double bonds - optimal
hydrophobic interactions
- lowered Tm - More cis-double bonds - Reduced
hydrophobic interactions
- thermophiles
- mesophiles
Fatty acid composition of plasma membrane as
total fatty acids E. coli grown
at 10C 43C C16 saturated (palmitic) 18
48 C16 cis-9-unsat. (palmitoleic) 26 10
C18 cis-11-unsat. (cis-vaccinic) 38 12
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Temperature classes of organisms
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Psychrophilic vs. Psychrotolerant
Sierra Nevada
Psychrotolerant
Psychrophiles
Maximum gt20C Optimum 20-40C Minimum
lt0-4C Habitats much more abundant than
psychrophiles - soil in temperate climate -
foods - grow slowly even in fridge!
Maximum lt20C Optimum lt15C Minimum
lt0C Habitats - deep sea - glaciers

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

Chlamydomonas nivalis The snow algae
red spores
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Growth at high temperatures
Thermophilic optimum gt 45C Soil in sun often
50C Fermentation 60-65C
lt65C
Hyperthermophilic optimum gt 80C Only in few
areas Hot springs 100C Steam vents
150-500C Deep sea hydrothermal vents
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Growth at high temperatures
Molecular adaptations in thermophilic bacteria
Proteins

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

lipids

• more saturated fatty acids
• hyperthermophilic Archaea C40 lipid monolyer

DNA

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

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Bacterial growth pH
Most natural habitats
(extremes pH 4.6- 9.4)
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Growth at low pH Fungi - often more acid
tolerant than bacteria (opt. pH5) Obligate
acidophilic bacteria Thiobacillus
ferrooxidans Obligate acidophilic
Archaea Sulfolobus Thermoplasma Most
critical cytoplasmic membrane Dissolves at more
neutral pH
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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

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Buffers in bacterial culture media
pH range buffer system 1.1 - 3.5 glycine/HCl 2.2
- 4.0 KH-phtalate/HCl 3.6 - 5.6 Na-acetate/aceti
c acid 5.0 - 8.0 KH2PO4/Na2HPO4 5.0 -
6.6 Na-citrate/NaOH 7.2 - 9.0 TRIS/HCl 8.5 -
12.9 glycine/NaOH 9.2 - 10.7 Na2CO3/NaHCO3 10.9 -
12.0 Na2HPO4/NaOH
Sambrook et al., 1989, Molecular cloning, 2nd ed.
Küster Thiel, Rechentafeln für die chemische
Analytik, 1982, Walter de Gruyter
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Bacterial growth Osmosis
Water acitvity
Osmotic pressure
p po
n x R x T V
aw
p
p osmotic pressure n number of dissolved
particles R universal gas constant T
temperature V volume of the solution
aw rel. Water activity p vapor pressure of a
solution p0 vapor pressure of water
Semipermeable membrane
low aw
high aw
high p
low p
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Bacterial growth Osmosis
Soil water activity 0.9 1.0 In general
bacteria normally have higher osmotic pressure
than environment positive water balance
Osmophiles - grow in presence of high sugar
concentration Xerophiles - grow in dehydrated
environments
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Bacterial growth Halophiles
Halophiles - 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 organisms would be dehydrated
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Bacterial growth at low aw compatible solutes
Strategy increase internal solute concentration

• Pump inorganic ions
• Synthesize organic solutes

Solute must be compatible with cellular
processes
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Bacterial growth Oxygen
O2 as electron sink for catabolism ?? toxicity of
Oxygen species
Aerobes growth at 21 oxygen Microaerophiles
growth at low oxygen concentration Facultative
aerobes can grow in presence and absence of
oxygen Anaerobes lack respiratory
system Aerotolerant anaerobes Obligate anaerobes
cannot tolerate oxygen (lack of detoxification)
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Bacterial growth toxic forms of Oxygen
triplet oxygen ground state singlet oxygen
reactive inactivated by carotenoids
produced by light, biochemically
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Bacterial growth Oxygen detoxification
Catalase assay
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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)

air
air
air
air
air
aerotolerant anaerobe
obl. aerobe
fac. aerobe
obl. anaerobe
microaerophile