Transcript Continuous and Batch Culture

Continuous and Batch Culture
Culture media: general
• For general use in isolating and growing
micro-organisms, fairly crude media may
be used
• These may be based on meat extract,
blood, milk, yeast extract, or other
nutrient-rich sources, and usually contain
a wide range of nutrients such as sugars,
amino acids and vitamins.
• Nutrient broth and nutrient agar are
good examples
Culture media: synthetic
• For more detailed study of microbial
growth and metabolism, microbiologists
usually use synthetic media of precisely
known composition
• Because the contents of synthetic culture
media are known and controlled, valid
comparisons can be made between the
growth of different species, or of the
same species under different conditions
Culture media: minimal
• Biochemists studying metabolic pathways in microorganisms can use minimal media to supply or
withhold specific nutrients and observe the effects
• Minimal media provide the bare minimum of
nutrients required by the species under
investigation: for many heterotrophs, this may
consist of the necessary mineral ions, with perhaps a
single sugar as the only carbon and energy source
• Use of minimal media can quickly establish which
sugars a particular organism can and cannot
metabolise. Other types may not grow in minimal
medium unless specific amino acids or growth
factors are added, thus giving important information
about their synthetic capabilities.
Culture media: selective
• The nutrient content of the medium, and its
pH, can be adjusted to favour the growth of
the particular organism being studied and
exclude others. Media like this are called
selective or narrow-spectrum media
• For example to isolate nitrogen fixing bacteria
from soil you would inoculate a small sample
of soil into a synthetic medium containing
sugars but no nitrogen compounds; nitrogen
fixers such as Azotobacter will grow, but
bacteria unable to fix nitrogen will not
Culture media: selective and indicator
• MacConkey agar is commonly used to differentiate between various gramnegative rod-shaped organisms. MacConkey agar is both a selective and an
indicator medium, in that
 it contains bile salts and favours the growth of the gram-negative rods
which inhabit the gut
 it can differentiate between lactose fermenters and non-fermenters.
Many facultative anaerobes in the intestine are lactose fermenters (e.g.,
Escherichia coli), while several well-known pathogens are unable to
ferment lactose (e.g., Shigella, Salmonella).
Culture media: solid or liquid
• Solid media usually based on agar, a seaweed extract
containing polysaccharides
• Nutrients are added to molten agar, and the mixture sets into
a jelly on which micro-organisms can grow
• Because most micro-organisms are unable to digest agar, it
remains solid even when they are growing on the nutrients it
contains
• Liquid media include various broths or synthetic media
• Microbiologists may choose to use solid media or liquid media
depending on the purpose of a particular investigation
• In the table that follows, state whether you would choose a
solid or a liquid medium in each case, and give your reasons.
Culture media: solid or liquid
Investigation
Solid or liquid
Reasons
Isolation and identification of bacterial species
in a soil sample
Solid
Separate colonies allow
isolation
Total cell count only possible
in liquid
Measuring the growth of a yeast population
over a period of time by periodic total cell
counts
Liquid
Measuring the growth of a bacterial
population over a period of time by periodic
viable counts
Liquid for
culture, solid for
counting
Viable cell count by counting
colonies
Measuring the growth of a mould by
measuring colony diameter over a period of
time
Solid
Separate colonies allow
measurement
Measuring the growth of a yeast population by
sampling and measuring dry mass
Liquid
Producing a large mass of micro-organisms or
their products for chemical analysis
Liquid
Periodic sampling and
separation from medium
Bulk growth in liquid (in 3
dimensions)
Sending a sample of a newly isolated bacterial
species to a colleague in Sydney
Solid
Safer / more stable
Distinguishing between species on the basis of
gas production
Liquid
Formation of bubbles
Pouring plates
• Solid media are usually used in Petri dishes; these may be glass
(sterilised by autoclaving), or the disposable plastic variety supplied
in packs which are sterile until opened.
• Medium is made up in liquid form, and may be dispensed into small
screw-top bottles before being autoclaved.
• After autoclaving, the medium can be kept liquid until required by
placing the bottle in a water bath at 60oC.
• Before pouring the plate, the agar should be cooled to 50 - 55oC.
• To avoid contamination, the plate should be poured quickly with
the lid of the Petri dish held ajar.
• While the agar in the dish is still hot, the lid may safely be left ajar
as the outflow of steam will prevent airborne organisms from
entering: this helps to reduce problems of condensation on the
Petri dish lid.
• After cooling, the closed dish is kept upside down. If condensation
is still a problem, the dish can be kept overnight in a warm
incubator to remove it.
Culture vessels for liquid media
Any piece of glassware that can be
sterilised and plugged can be used for
growing micro-organisms, but for
serious study a fermenter or
bioreactor of some kind is commonly
used:
Air in
Air out
Air filter
Air filter
Syringe for adding
buffer, nutrients
etc.
Syringe for
withdrawing
samples
Sparger to increase
efficiency of aeration
Culture
medium
Culture vessels for liquid media
A fermenter of this kind has a number of advantages over
a plain culture tube or bottle:
•air can be passed in and out through lines fitted with
filters: this provides oxygen for aerobes, and also stirs the
culture, improving contact between micro-organisms and
nutrients. A sparger on the air inlet produces a large
number of very tiny bubbles, increasing the total surface
area of air in contact with water and thus the rate of entry
Air out
of oxygen into solution.
•a magnetic stirrer can be used as well, further improving Air filter
mixing of the culture.
•syringes can be fitted for inoculation of the medium with
micro-organisms, for the addition of materials during the
growth of the culture, or for the taking of samples for
analysis or population estimation.
Air in
Air filter
Syringe for adding
buffer, nutrients
etc.
Syringe for
withdrawing
samples
In addition, the culture vessel is usually maintained at
constant temperature, either in an incubator or in a
thermostatically controlled water bath.
Sparger to increase
efficiency of aeration
Culture
medium
Continuous and batch culture
•The fermenter shown here is set up for a batch
culture: that is, it is ready to be inoculated with a
culture of micro-organisms, which will multiply,
changing the conditions in the medium by using up
the nutrients and producing their own waste
products. Eventually conditions in the culture will
become too unfavourable for the organisms to
survive, and the population will die out.
Air out
•Batch culture is suitable for most school-based
Air filter
experiments on microbial growth, and is used
industrially in many processes that harvest the
secondary metabolites of micro-organisms (such
as antibiotics).
•Secondary metabolites are produced by metabolic
processes which are not essential to the organism's
short-term survival, and are often not produced in
large amounts when the organism is growing at its
fastest; industrial production is therefore most
efficient if the organisms are allowed to reach
maximum population size and stop growing before
Sparger to increase
the product is harvested
efficiency of aeration
Air in
Air filter
Syringe for adding
buffer, nutrients
etc.
Syringe for
withdrawing
samples
Culture
medium
Continuous and batch culture
Continuous culture, by contrast, aims to
keep a culture growing indefinitely. This can
be done if:
•fresh nutrients are continually supplied
•accumulated cells and waste products are
removed at the same rate
•conditions such as temperature and pH are
kept at their optimum values.
A culture vessel designed for continuous
culture is called a chemostat:
Continuous culture is important in
industrial processes that harvest
the primary metabolites of microorganisms as their products.
(Primary metabolites are produced
in greatest quantities when the
organisms are growing at their
fastest rate).
In addition to the features
shown, the culture vessel
would probably be fitted with
temperature and pH probes
for monitoring growth
conditions.