Transcript Continuous Culture

Chapter4
Microbial growth
Isolation of pure cultures
the growth curve
Measurement of microbial growth
The continuous culture of microorganisms
Isolation of pure cultures
• Pure culture: a population of cells arising from a single cell,
to characterize an individual species. They are required
for the careful study of an individual microbial species.
• Single colony
• How to get
1. Dilute-pour/spread plate technique:
dilutepour/spread isolated colonies
2. Streak plates: inoculating loop
Petri Disk Cultivation of a Pure Strain
The Streak Plate Technique:
to obtain a pure clone
Laboratory Culture of Microorganisms
1.5% Agar
Colony morphology and growth
individual species often form colonies of characteristic
size and appearance.
The growth of colonies on agar
• At the colony edge: cells grow at maximum
rates;
• In the center of colony: cells are lying; growth
is much slower
Cause: Oxygen, nutrients and toxic products
The colony center is much thicker than the edge.
Cell-cell communication and quorum sensing
Microbial growth
• Batch culture: When microorganisms are
grown in a closed system, population
growth remains exponential for only a few
generations and then enters a stationary
phase due to factors like nutrient limitation
and waste accumulation.
• Continuous Culture: If a population is
cultured in an open system with continual
nutrient addition and waste removal, the
exponential phase can be maintained for
long periods.
The growth curve
The logarithm of the number of viable cells
versus the incubation time.
The Growth Cycle of Populations
(a) Lag phase: cells begin to synthesize
inducible enzymes and use stored food
reserves.
(b) Logarithmic growth phase: the rate of
multiplication is constant.
(c) Stationary phase: death rate is equal to rate
of increase.
(d) Death phase: cells begin to die at a more
rapid rate than that of reproduction.
Lag phase
• no immediate increase in cell mumber. The
cell is synthesizing new compoments.
ATP, cofactor, ribosomes, enzymes.
• The lag phase varies in length with the
condition of the microorganisms and the
nature of the medium.
• How to shorten the lag phase?
Exponential phase/log phase
• Microorganisms are growing and
dividing at the maximal rate possible.
• The population is most uniform in
terms of chemical and physiological
properties.
• Such culture: biochemical and
physiological studies.
Stationary phase
• Balance between cell division and cell death.
Poplation growth ceases and the growth curve
becomes horizontal.
Bacteria: 109 cells per ml;
Protozoan and algae: 106 cells per ml.
• Reasons: 1. Nutrient limitation
2. The accumulation of toxic waste products
3. A critical population level is reached
Death phase
• The decline in the number of viable cells.
• Starvation can be a positive experience for bacteria.
1. decrease overall size: protoplast shrinkage;
nucleoid condensation.
2. Produce a variety of starvation proteins.
3. Increase peptidoglycan cross-linking and cell wall
strength.
4. The Dps protein protects DNA.
5. Chaperones prevent protein denaturation and
renature damaged proteins.
Cell life cycle in Eukaryotic cells
G1
Primary growth phase of the cell during which cell enlargement occurs, a
gap phase separating cell growth from replication of the genome
S
phase in which replication of the genome occurs
G2 Phase in which the cell prepares for separation of
the replicated genomes,
this phase includes synthesis of microtubules and condensation of DNA to
form coherent chromosomes, a gap phase separating chromosome replication
from miosis.
M
phase called miosis during which the microtubular apparatus is associated
and subsequently used to pull apart the sister chromosomes.
Eukaryotic cell:
G1
Prokaryotic cell:
G1
S
R
G2
D
M
generation time
The time required for a cell to divide (and its
population to double) is called the generation time.
Suppose that a bacterial population increases from103 cells
to 109 cells in 10 hours. Calculate the generation time.
Number of cells
Nt = No x 2n
G = t log2 / log Nt – log No
No = number of bacteria at beginning of time interval.
Nt = number of bacteria at end of any interval of time (t).
G = generation time
T = time , usually expressed in minutes
n = number of generation
Time
Generation time
• Example
100 bacteria present at time 0
If generation time is 2 hr
After 8 hr mass = 100 x 24
3 types of cell division
a. Binary Fission
b. Budding
c. Multiple Fission
Binary fision
Most bacterial cells reproduce asexually by binary fision, a
process in which a cell divides to produce two nearly equalsized progeny cells. Binary fision involves three processes:
Increase in cell size (cell elongation),
DNA replication
Cell division
Binary Fission (二分裂)
Attention: difference between cocci and bacilli in binary fission
Budding (芽殖)
Multiple Fission
Measurement of microbial growth
• Population mass
• Population number
No single technique is always best.
Measurement of cell numbers
• Total cell count (direct observation under
microscope)
• Viable count (plate count or colony count)
– Spread plate method
– Pour plate method
– The membrane filtration procedure
–The plates required to have between 30
and 300 colonies.
Direct counting
•The counting chamber
Disadvantages: 1. The microbial population
must be fairly large for accuracy because such
a small volume is sampled.
2. Difficult to distinguish between living and
dead cells
•Electronic counters: the Coulter Counter
for larger microorganisms such as protozoa,
algae, and nonfilamentous yeasts.
Viable count: Spread plate method
and Pour plate method
Problems:
1. Clumps of cells.
Three basic steps: colony forming units(CFU);
2. employed agar medium
• Dilution,
cannot support growth of all
• Plating
the viable microorganisms
• Incubation
present.
3. Hot agar:
spread plates>pour plates
Measuring viable bacteria
Colony forming units
colony
Viable Count: Dilution
Viable Count: Plating
Incubation (and counting)
Pour plate method
Spread plate method
The membrane filtration procedure
• Analyze aquatic samples
实验9：滤膜法测定水中大肠菌群数（设计性）
Measurement of cell mass
1. Dry weight: useful for measuring the
growth of fungi.
cells growing in liquid medium are collected
by centrifugation, washed, dried in an oven,
and weighed.
for bacteria, not very sensitive
2. Turbidity and microbial mass measurement
3. The amount of a substance
total protein or nitrogen
chlorophyII(algae)
Measuring bacterial mass (live + dead)
in liquid culture
Turbidity
(Cloudiness)
Measuring cell density:
Turbidity vs Cell density
The continuous culture of
microorganisms
• Continual provision of nutrients and
removal of wastes.
• A microbial population can be maintained
in the exponential growth phase and at a
constant biomass concentration for
extended periods in a continuous culture
system.
• The chemostat:
Chemostat used for continuous cultures,
rate of growth can be controlled either by
controlling the rate at which new medium
enters the growth chamber or by limiting a
required growth factor in the medium
• The turbidostat:
measures the absorbance or turbidity of the
culture in the growth vessel.
automatically regulated to maintain a
predetermined turbidity or cell density.
How to maintain Exponential Growth?
Questions
• How to obtain a pure culture? How to calculate
cell growth?
• How to describe the colony morphology?
• How cells divide themselves? How to do viable
count?
• What is the microbial cell growth cycle?
• How to measure the cell mass and number?
• What is continuous culture, chemostat,
turbidostat?
• How to maintain exponential growth?