Laboratoryculture:pureculture

- 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
Confluent mixture
- growth inhibitors
Isolated colony
- liquid/solid
- temperature
- minimum: - source of energy
- sources of carbon, nitrogen, ...
-Aseptic technique:
- sterilization of medium and equipment
4
- proper handling

Bacterialgrowth:basicconcepts

Precursors

Anabolism = biosynthesis
Catabolism = reactions
to recover energy (often
ATP)

Bacterialgrowth:basicconcepts

troph = to feed (where

does energy come from?)

Chemolithotroph =
inorganic compounds as
energy source

Chemoorganotroph =
organic compounds as
energy source

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

Microbialnutrition
Nutrients = chemical tools a cell needs to grow/replicate
Macronutrients = chemicals needed in large amounts
Micronutrients = chemicals needed in small/trace amounts

% of dry
weight
50%

12%

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

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

Microbialnutrition:Growthfactors
- organic compounds required
by some bacteria
- vitamins, amino acids, purines,
pyrimidines
- Streptoccus, Lactobacillus,
Leuconostoc (lactic acid bacterium):
complex vitamin requirements

Microbialgrowthmedia
- chemically defined: highly purified inorganic and organic compounds in dest. H2O
- complex (undefined): digests of casein, beef, soybeans, yeast, ...

Microbialgrowthmedia
Media
Complex
Defined
Selective
Differential
Enrichment
Reducing

Purpose
Grow most heterotrophic organisms
Grow specific heterotrophs and are often mandatory for
chemoautotrophs, photoautotrophs and for microbiological
assays
Suppress unwanted microbes, or encourage desired microbes
Distinguish colonies of specific microbes from others
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
Growth of obligate anaerobes

MacConkeyAgar:

Fermentation

Respiration

No added terminal e--acceptor

Oxidant = terminal e--acceptor

ATP: substrate level phosphorylation

ATP: (e--transport) oxidative phosphoryl.

Glucose

Glucose
2 Glyceraldehyde-3-P

2 ATP
2 NADH

2 Pyruvate
Regeneration of NAD

2 Lactate
+ 2 H+

Acetaldehyde
+2 CO2

2 Pyruvate
CO2
2 Acetyl-CoA
Citric acid
cycle

Acetate
+ Formate

H2O

in

2 Ethanol

H2 + CO2

CO2
GTP
NADH, FADH
O2

ATP

Cytoplasmic membrane
out

1 Glucose 2 ATP
Slow growth/low biomass yield

2 ATP
2 NADH

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

1 Glucose 38 ATP
Fast growth/high biomass yield

Alternatemodesofenergygeneration

(H2S, H2, NH3)

(in autotrophs)

Bacterialgrowth

1 generation

Growth = increase in # of cells

(by binary fission)
generation time: 10 min - days
Growth rate = cell number/time
or cell mass/time

Bacterialgrowth:exponentialgrowth
Generation time = 30 min

Cell volume = 5 m3
.... 5 ml total cell volume

80 h

7 x 1036 m3 (> earth)

Bacterialgrowth:exponentialgrowth
Semilogarythmic plot

Straight line
indicates
logarithmic
growth

Bacterialgrowth:calculatethegenerationtime
t
g= n

t = time of exponential growth (in min, h)

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

Bacterialgrowth:calculatethegenerationtime
t
g= n

t = time of exponential growth (in min, h)

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

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

t
g= n
Nt = N0 x 2

Nt = number of cells at a certain time point

N0 = initial number of cells
n = number of generations

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

t
g= n
Nt = N0 x 2

Nt = number of cells at a certain time point

N0 = initial number of cells
n = number of generations

logNt = logN0 + n x log2

logNt - logN0= n x log2
n=

logNt - logN0
log2

n = 3.3 x (logNt - logN0)

Bacterialgrowth:calculatethegenerationtime
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
104 Bakterien/ml

10.00 Uhr
8 x 104 Bakterien/ml
Generationszeit = ?

Bacterialgrowth:calculatethegenerationtime
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
104 Bakterien/ml

10.00 Uhr
8 x 104 Bakterien/ml
Generationszeit = ?

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

= 3.3 x (4.9 4) = 3
1h
t
g = n = 3 = 20 min

Bacterialgrowth:batchculture

Batchculture:Lagphase
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)

Batchculture:exponentialphase

Exponential phase = log-phase

Maximum growth rates
midexponential: bacteria often used for functional studies

Batchculture:stationaryphase
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

Batchculture:deathphase
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

Measurementofmicrobialgrowth
A. Weight of cell mass
B. number of cells:
- Total cell count
- Viable count
- Dilutions
- turbidimetric

totalcellcount
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

viablecellcount
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

dilutions
Example:
3 h culture of E. coli in L-broth
How do I determine the actual number?

Turbidimetricmeasurements

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

Turbidimetricmeasurements

Limits of sensitivity at high bacterial density

rescattering more light reaches detector

Continuousculture:thechemostat
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

Continuousculture:thechemostat
1. Concentration of a limiting nutrient

Results from a batch culture

Continuousculture:thechemostat
2. Dilution rate

Factorsaffectingmicrobialgrowth

Nutrients
Temperature
pH
Oxygen
Water availability

Factorsaffectingmicrobialgrowth:Temperature
3 cardinal temperatures:

Usually ca. 30C

Maximumtemperature
Thermal protein inactivation:
- Covalent/ionic interactions weaker at high temperatures.
- Thermal denaturation: covalent or non-covalent
reversible/ irreversible
- heat-induced covalent mod.: deamidation of Gln and Asn

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

Factorsaffectingmicrobialgrowth:Temperature
Minimal temperature:
Proteins:
- Greater -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

GrowthatlowTemperatures: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:
10C
43C
C16 saturated (palmitic)
18 %
48 %
C16 cis-9-unsat. (palmitoleic)
26 %
10 %
C18 cis-11-unsat. (cis-vaccinic)
38 %
12 %

Temperatureclassesoforganisms

Psychrophilicvs.Psychrotolerant
Sierra Nevada
Psychrophiles
Maximum: <20C
Optimum: <15C
Minimum: <0C
Habitats:
- deep sea
- glaciers

red spores

Chlamydomonas nivalis
The snow algae

Psychrotolerant
Maximum: >20C
Optimum: 20-40C
Minimum: <0-4C
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)

Growthathightemperatures
Thermophilic:
optimum > 45C
Soil in sun often 50C
Fermentation: 60-65C

<65C

Hyperthermophilic:
optimum > 80C
Only in few areas:
Hot springs: 100C
Steam vents 150-500C
Deep sea hydrothermal vents

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

Bacterialgrowth:pH

(extremes: pH 4.6- 9.4)

Most
natural
habitats

GrowthatlowpH
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

Bacterialgrowth:Osmosis
Water acitvity
p
aw = p
o
aw: rel. Water activity
p: vapor pressure of a solution
p0: vapor pressure of water

Osmotic pressure
nxRxT
p=
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

Bacterialgrowth: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

Bacterialgrowth: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

Bacterialgrowthatlowaw:compatiblesolutes
Strategy: increase internal solute concentration
a. Pump inorganic ions
b. Synthesize organic solutes

Solute must be compatible

with cellular processes

Bacterialgrowth: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)

Bacterialgrowth:toxicformsofOxygen
triplet oxygen: ground state
singlet oxygen: reactive
inactivated by carotenoids
produced by light, biochemically

Bacterialgrowth:Oxygendetoxification
Catalase assay

Bacterialgrowth: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:

O2