Transcript Growth and Cultivation of micro-organisms

Growth and Cultivation of microorganisms
by
E. Börje Lindström
This learning object has been funded by the European Commissions FP6 BioMinE project
Growth
Definition:
• Growth implies that all building blocks of the cell
increases with the following consequences:
Growth
Normally
Increase in cell number
Multi cellular
organisms
increases size
of organism
Uunicellular
organisms
increase number
of organisms
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
20
• n = no. of doublings
21
22
23
Etc.
2n
• The following mathematical expression is then obtained:
• time for each doubling
= g (min, hr)
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
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)
log Nt
slope = m x log 2
t
• in a semi-logarithmic graph this is a
strait line
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:
Growth curve during batch
cultivation
log Nt
Stationary phase
Death phase
Lag-phase
Log-phase
t
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
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
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.
Parameter
Cell mass
(dry
weight/ml)
Cell number, total
Cell number,
viable
Method
Sensitiveness
Note
(cells/ml)
Gravimeter
108
Direct method
Turbid meter
(O.D.)
107
Indirect method
Chemical analysis (depending on the
compound)
Indirect method
Microscopy
106
Direct method
Viable count
(V.C.)
1-10
Indirect method
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.
(dry weight/ml)
Cell mass
• 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.
Turbid meter, (O.D.)
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 >108 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
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
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).
Protein
Protein
RNA
RNA
DNA
DNA
Time
Balanced
Time
Unbalanced
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.