Transcript Growth curve

Growth of bacteria in culture
Culture systems based on having:
1.
a pure culture
2.
suitable nutrient medium
3.
satisfactory growth conditions
4.
sterile technique to maintain pure culture
Different micro-organisms have different
nutrient requirements
•
Heterotroph- organism that uses
complex organic carbon compounds
as a source of carbon and energy
•
Photoautotroph- uses light as source
of energy and CO2 as carbon source
•
Chemoautotroph- uses oxidation of
simple inorganic compounds for
energy and fixes CO2 as a source of
carbon
Chemoautotrophic
bacteria appear purple,
Heterotrophic
E.coli
Phototrophic cyanobacteria
Bacteria can be grown on solid
media or liquid media:
•
agar in Petri plates
•
agar in glass bottles or deep
tubes (less dehydrating and
anaerobic conditions)
•
flat sided bottles allowing
growth as a monolayer
•
Liquid culture in a culture flask
Dynamics of growth usually studied in liquid culture
• Many cells can be grown as a homogeneous single celled
suspension e.g. many bacteria, yeast, some animal cells….
Need to be able to monitor the growth of organisms with time as
1. Want to make sure growth is occurring
2. Want to monitor for contamination
3. Want to follow stage of growth as some products produced
during growth, others at the end of growth
Batch culture
• Cells inoculated into a sterile vessel containing a fixed amount
of nutrients. Growth in the vessel normally follows the
predicable “growth curve”
• No nutrients are added or removed (a closed system)
• Cells grow by binary fission, as cell divides population
increases exponentially
A. Lag• No increase in cell number, cells
getting ready to grow and divide
• length related to how the cells used
as inoculum were previously
treated
B. Log/exponential• Cells growing at maximum rate for
these conditions.
• Growth is balanced i.e. nothing is
limiting
C. Stationary• Cell growth begins to be limited by
lack of particular nutrients or
accumulation of inhibitory
compounds
• No. Cells dividing are equal to cells
dying, a plateau is reached.
D. Death Phasecells dying faster than dividing
Measuring growth
1. Direct counts
e.g. using a haemocytometer
2. electronic
3. culture based
e.g. dilution and spread plating
4. using a spectrophotometer
Direct counts using a Hemocytometer
Direct counts using a
Hemocytometer
•Sample is viewed on special slide
with fixed volume
•Sample may be stained first to
increase visability or to discern
whether cells are viable
•A count of number of cells in a
primary square is made
•This is repeated for several other
squares
•An average cells per primary
square is calculated
•As volume counted is known cells
per ml can be calculated
Electronic counters
e.g. Coulter counter The device counts because whenever a particle goes
through the hole, the electronic system detects a sudden and momentary
increase in resistance (a partial interruption of current flow) and a green
vertical line appears on the screen, as shown at the top.
Issues with coulter counter
• Does not distinguish between live and
dead
• Clumping is a problem
• Interference is an issue
• expense
Other electronic devises include Flow
cytometers that incorporate fluorescence
and lasers to distinguish and count cells.
These are very expensive and require
dedicated technologists
Culture based methods
1. Pour plate- cells are mixed with molten
agar and poured into petri plate, allowed to
grow and colonies counted
2. Spread plate- cells are spread on the
surface of an agar plate allowed to grow
and colonies counted
Both methods assume one cell gives rise to
one colony
Colorimetric/spectrophotometric methods:
Turbidity is a measure of the # of microbes:
• measured by light scattering or absorbance in a
spec
• Cells must be single cells in suspension
• Clumping makes the relationship non linear
• Can make a standard curve for specific
organism of cell number versus absorbance so
can correlate absorbance with number of cells