Growth and Cultivation of microorganisms

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Growth and Cultivation of micro-organisms

• by
• E. Börje Lindström

This learning object has been funded by the
European Commissions FP6 BioMinE project
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Growth
Definition

• Growth implies that all building blocks of the
  cell increases with the following consequences

Growth Normally   Increase in cell
number     Multi cellular
Uunicellular organisms
organisms     increases size
increase number of organism
of organisms
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Mathematical representation

• The bacteria divide binary usually perpendicular
  to the length axis and thereby two new cells are
  produced

• For a unicellular bacterium the cell number
  increase exponentially with base 2 as seen in the
  table below

Cell no.
Exponential expression

• n no. of doublings

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21

• time for each doubling g (min, hr)

22
23
Etc.
2n

• The following mathematical expression is then
  obtained

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Math. cont.
Nt N0 x 2n (1)
Where

• Nt the cell number at time t
• N0 the cell number at t 0 and
• n represents the number of doublings
  (generations)

• if g time for a generation and
• t total time, then

Nt N0 x 2n N0 x 2t/g (2)

• set m 1/g

• where m is the specific growth rate constant
• inserted in (2) gives

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Math. cont.
Nt N0 x 2tm (3)

• take the logarithm of equation (3), which gives

log Nt log N0 t x m x log 2 (4)

• in a semi-logarithmic graph this is a strait line

log Nt
slope m x log 2
t
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Cultivation
Cultivation is normally performed batch-wise or
continuously.

• Batch cultivation

- the growth medium and the bacteria (inoculum)
are added to the growth vessel once at the start
of the experiment!
- any growth vessel can be used shake flasks,
stirred tank reactors etc.
- batch-wise cultivation is chosen to rapidly
obtain growth data

• During batch cultivation of a bacterial culture
  you can have
• four (4) growth phases as shown below

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Growth curve during batch cultivation
Stationary phase
Death phase
Lag-phase
Log-phase
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Properties of the growth phases

• Lag-phase

- the cell devision is delayed due to how the
inoculum has been treated
- the previous medium
- the temperature, etc.

• Log-phase

- exponential growth
- as fast as the soluble nutrients permit
- the doubling time, g, can be determined here
- in bioleaching there is often no exponential
phase due to that the energy source is a particle
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Properties, cont.

• Stationary phase

- growth is stopped due to changes in the medium
- an essential nutrient has ceased
- pH-changes due to end products
- dissolved oxygen for aerobic organisms

• Death phase

- an exponential curve

• due to some toxic substance excreted from the
  bacteria

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Quantitative methods for measuring bacterial
growth

• The growth of the bacterial population can be
  followed either by the changes in number of
  cells or weight of cell mass.

• In the following table a comparison of a few
  methods are found.

Sensitiveness (cells/ml)
Method
Note
Parameter
Gravimeter
Direct method
108
Cell mass (dry weight/ml)
Turbid meter (O.D.)
107
Indirect method
(depending on the compound)
Indirect method
Chemical analysis
Microscopy
Direct method
Cell number, total
106
 Cell number, viable
1-10
Indirect method
Viable count (V.C.)
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Comments to the quantitative methods

• Some of the methods are noted as direct or
  indirect. The direct methods show the cell mass
  or cell number directly in the sample. In the
  indirect methods you need a standard curve
  comparing a direct and an indirect method.

• If e.g. during growth the same sample is
  measured by the direct method gravimeter and the
  indirect method turbid meter and those values are
  plotted in a diagram you will have a standard
  curve for use in later experiments.

Cell mass (dry weight/ml)
Turbid meter, (O.D.)
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Comments, cont.

• The method gravimeter uses ordinary balances
  after removal of the water content of the sample.
  Given a sample size of one ml and assuming that
  an average dry bacterium is weighing 10-12 g and
  that a ordinary balance can detect 10-4 g this
  means that you must have gt108 bacteria per ml in
  the sample to be able to use weighing.

• The sensitivity given for any turbid meter
  e.g.ordinary spectrophotometers measuring optical
  density, is arbitrary.

• For microscopy the sensitivity value means that
  you have on average one cell in the smallest
  square on the special object glass used.

Depth
Grid in the bottom
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Comments, cont.
In viable count you usually pore out 0.1 ml of
the sample onto the surface of a nutrient agar
plate. If you get one colony after incubation
then you have had 10 bacteria per ml in the
sample.
0.1 ml
1 ml
Nutrient agar plate
10 bact/ml
1 bact/ml
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Balanced vs. Unbalanced growth

• The growth of a bacterial culture is related to
  the composition of the medium.

• In a minimal medium the growth is slower than in
  a complex medium.

• If all the essential nutrients are freely
  available the growth is balanced, which means
  that all the building bocks are synthezised with
  the same speed (see figure below).

• However, if the synthesis of one of the building
  blocks is stopped, the growth is terminated due
  to unbalanced growth, which often leads to death
  of the culture (see figure below).

Balanced
Unbalanced
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Enrichment and isolation
Enrichment

• When a special bacterial species is nutritional
  favoured during cultivation that species will be
  enriched in the culture.

• A small sample of this culture is then
  transferred to new fresh medium of the same type
  and the cultivtion is continued.
• This procedure is continued several times.

Isolation

• A small sample of the last enrichment culture is
  then spread on top of a agar plate with the same
  nutrient media as in the enrichment.

• Among those colonies appearing on the plate
  after proper incubation the wanted bacterial
  species will be. Testing these colonies will
  evetually result in the isolation.