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
Confluent mixture
1
- growth inhibitors
Isolated colony
- liquid/solid
- temperature
-Nutrients:
- carbon, nitrogen, elements ...
-Aseptic technique:
- sterilization of medium and equipment
4
- proper handling
2
3
Bacterial growth: basic concepts
Precursors
Anabolism = biosynthesis
Catabolism = reactions
to recover energy (often
ATP)
Bacterial growth: basic concepts
troph = „to feed“ (where
does energy come from?)
Chemolithotroph =
inorganic compounds as
energy source
Chemoorganotroph =
organic compounds as
energy source
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%
12%
(sometimes non-essential)
(sometimes non-essential)
Microbial nutrition: Growth factors
- organic compounds required
by 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
*Complex
Purpose
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
Distinguish colonies of specific microbes from others
*Enrichment
stimulating
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
Bacterial growth
Growth rate = Dcell number/time
or Dcell mass/time
1 generation
Growth = increase in # of cells
(by binary fission)
generation time: 10 min - days
Bacterial growth: exponential growth
Generation time = 30 min
Bacterial growth: exponential growth
Semilogarythmic plot
Straight
line
indicates
logarithmic
growth
Bacterial growth: calculate the generation
time
t
g= n
The generation
time is the time
needs the culture
population to
double
t = time of exponential growth (in min, h)
g = generation time (in min, h)
n = number of generations
Bacterial growth: calculate the generation time
t
g= n
t = time of exponential growth (in min, h)
g = generation time (in min, h)
n = number of generations
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 = N0 x
2n
Nt = number of cells at a certain time point
N0 = initial number of cells
n = number of generations
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 = N0 x
Nt = number of cells at a certain time point
N0 = initial number of cells
n = number of generations
2n
logNt = logN0 + n x log2
logNt - logN0= n x log2
n=
logNt - logN0
log2
n = 3.3 x (logNt - logN0)
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
103 Bakterien/ml
14.00 Uhr
1.6 x 104 Bakterien/ml
Generationszeit = ?
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
103 Bakterien/ml
14.00 Uhr
1.6 x 104 Bakterien/ml
Generationszeit = ?
n = 3.3 x (logNt - logN0) = 3.3 x (log1.6 x 104 – log103)
= 3.3 x (4.2 – 3) = 4
2h
t
g = n = 4 = 0.5 h
Bacterial growth: batch culture
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)
Batch culture: exponential phase
Exponential phase = log-phase
Maximum growth rates
„midexponential“: bacteria often used for functional studies
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 mass
B. number of cells:
- Total cell count
- Viable count
- Dilutions
- turbidimetric
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 < 106/ml
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 45°C briefly
dilutions
Example:
3 h culture of E. coli in L-broth
How do I determine the actual number?
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
Turbidimetric measurements
Limits of sensitivity at high bacterial density
„rescattering“ more light reaches detector
Klett units
Klett units
Two typical growth curves in batch culture
1 Klett unit = OD/0.002
Continuous culture: the chemostat
steady state = cell number, nutrient status remain constant
Control:
1. Concentration of a limiting nutrient
2. Dilution rate
3. Temperature
 Independent control of:
- Cell density
- Growth rate
Continuous culture: the chemostat
1. Concentration of a limiting nutrient
Results from a batch culture
Continuous culture: the chemostat
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
Factors affecting microbial growth: Temperature
Arrhenius equation:
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)
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
Membrane proteins
inactive (mobility/insertion)
- homoviscous adaptation
Tm

„Fluid mosaic“
Protein function normal
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
- mesophiles
- thermophiles
Fatty acid composition of plasma membrane as % total fatty acids
E. coli grown at:
10°C
43°C
C16 saturated (palmitic)
18 %
48 %
C16 cis-9-unsat. (palmitoleic)
26 %
10 %
C18 cis-11-unsat. (cis-vaccinic)
38 %
12 %
„Temperature classes“ of organisms
Psychrophilic vs. Psychrotolerant
Sierra Nevada
Psychrophiles
Maximum: <20°C
Optimum: <15°C
Minimum: <0°C
Habitats:
- deep sea
- glaciers
red spores
Chlamydomonas nivalis
The snow algae
Psychrotolerant
Maximum: >20°C
Optimum: 20-40°C
Minimum: <0-4°C
Habitats: much more abundant than
psychrophiles
- soil in temperate climate
- foods
- grow slowly even in fridge!
Limit: Freezing
- Inhibits bacterial growth
- freezing: often liquid pockets
- many bacteria survive
- cryoprotectants (DMSO, glycerol)
Growth at high temperatures
Thermophilic:
optimum > 45°C
Soil in sun often 50°C
Fermentation: 60-65°C
<65°C
Hyperthermophilic:
optimum > 80°C
Only in few areas:
Hot springs: 100°C
Steam vents 150-500°C
Deep sea hydrothermal vents
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)
Bacterial growth: pH
(extremes: pH 4.6- 9.4)
Most
natural
habitats
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
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
Buffers in bacterial culture media
pH range
buffer system
1.1 - 3.5
2.2 - 4.0
3.6 - 5.6
5.0 - 8.0
5.0 - 6.6
7.2 - 9.0
8.5 - 12.9
9.2 - 10.7
10.9 - 12.0
glycine/HCl
KH-phtalate/HCl
Na-acetate/acetic acid
KH2PO4/Na2HPO4
Na-citrate/NaOH
TRIS/HCl
glycine/NaOH
Na2CO3/NaHCO3
Na2HPO4/NaOH
Küster Thiel, Rechentafeln für die chemische Analytik, 1982, Walter de Gruyter
Sambrook et al., 1989, Molecular cloning, 2nd ed.
Bacterial growth: Osmosis
Water acitvity
p
aw = p
o
aw: rel. Water activity
p: vapor pressure of a solution
p0: vapor pressure of water
Osmotic pressure
p=
nxRxT
V
p: osmotic pressure
n: number of dissolved particles
R: universal gas constant
T: temperature
V: volume of the solution
Semipermeable membrane
high aw
low aw
low p
high p
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
Bacterial growth: Halophiles
Halophiles:
- requirement for Na+
- grow optimally in media with low water activity
- Mild: 1-6 % NaCl
most other organisms
- Moderate: 6-15 % NaCl
would be dehydrated
- extreme: 15 – 30% NaCl
Bacterial growth at low aw: compatible solutes
Strategy: increase internal solute concentration
a. Pump inorganic ions
b. Synthesize organic solutes
Solute must be „compatible“
with cellular processes
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)
Bacterial growth: toxic forms of Oxygen
triplet oxygen: ground state
singlet oxygen: reactive
inactivated by carotenoids
produced by light, biochemically
Bacterial growth: Oxygen detoxification
Catalase assay
Bacterial growth: Anaerobes
air
air
air
air
air
obl. anaerobe
fac. aerobe
microaerophile
aerotolerant
anaerobe
- Closed vessels
- reducing agents (i.e. thioglycolate broth)
- anaerobic jar (H2-generation + Pd catalyst)
- glove box (oxygen free gas)
obl. aerobe
Methods to exclude/reduce oxygen: