Transcript Generation time

General Microbiology Laboratory
Bacterial Generation Time
Microbial Growth
 Binary fission
 Generation time
 Growth curve
 Enumeration of bacteria
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Reproduction and cell division
HOW DO BACTERIA REPRODUCE?
• Binary fission
• Budding
• Spore formation
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Microbial Growth
Growth= an increase in the number of cells, not an
increase in size
Bacterial species only maintained if population
continues to grow
Generation=growth by binary fission
Growth rate = cell number/time
or cell mass/time
Generation time= time it takes for a cell to divide
and the population to double; most are 1-3 hours.
Generation times vary markedly with the species of
microorganism and environmental conditions; they
can range from 10 minutes for a few bacteria to
several days with some eucaryotic microorganisms.
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Generation Times
Generation Time
Bacterium
Medium
(minutes)
Escherichia coli
Glucose-salts
17
Bacillus megaterium Sucrose-salts
25
Streptococcus lactis Milk
26
Streptococcus lactis Lactose broth
48
Staphylococcus
Heart infusion broth 27-30
aureus
Lactobacillus
Milk
66-87
acidophilus
Rhizobium
Mannitol-salts-yeast
344-461
japonicum
extract
Mycobacterium
Synthetic
792-932
tuberculosis
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Treponema pallidum Rabbit
testes
1980
What occurs during binary fission?
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The Growth Cycle
 The population growth is studied by analyzing the
growth curve of a microbial culture.
 The standard bacterial growth curve describes
various stages of growth a pure culture of bacteria
will go through, beginning with the addition of cells
to sterile media and ending with the death of all of the
cells present.
 Usually analyzed in a closed system called a batch
culture; plotted as the logarithm of cell number
versus the incubation time.
 Bacteria added to fresh media typically go through
four more-or-less distinct phases of growth: lag,
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exponential, stationary,
and
death.
Typical growth curve for a bacterial population
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Phases of Growth
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Lag phase
• The period of apparent inactivity in which the cells are
adapting to a new environment and preparing for reproductive
growth.
• Cells are usually synthesizing new components.
• In practice, bacteria from one medium to another, where there
are chemical differences between the two media, typically
results in a lag in cell division.
• This lag in division is associated with a physiological
adaptation to the new environment.
• Cells may increase in size during this time, but simply do not
divide (by binary fission).
• Lag phase varies considerably in length depending upon the
condition of the microorganisms and the nature of the
medium.
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Log (exponential) phase
• The period in which the organisms are growing at the
maximal rate possible given their genetic potential,
the nature of the medium, and the conditions under
which they are growing.
• Generation time can be easily obtained from the
exponential phase of a growth curve
• The population is most uniform in terms of chemical
and physical properties during this period.
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Stationary phase
• Eventually population growth decrease, and
the growth curve becomes horizontal.
• steady-state equilibrium
• rate of cell growth = rate of cell death
• Cell death may result from Nutrient
limitation & Toxic waste accumulation (e.g.
acid buildup from fermentation); as well as O2
depletion, critical population level reached.
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Death phase
• Stationary phase, in a standard bacterial
growth curve, is followed by a die-off of cells,
called Death phase.
• It is the period in which the cells are dying at
an exponential rate.
• Some of the reasons are: continued
accumulation of wastes, loss of cell's ability to
detoxify toxins, etc.
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Balanced and Unbalanced Growth
• Balanced (exponential) growth occurs when
all cellular components are synthesized at
constant rates relative to one another.
• Unbalanced growth occurs when the rates of
synthesis of some components change relative
to the rates of synthesis of other components.
This usually occurs when the environmental
conditions or nutrient levels change.
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Measurement of Bacterial Growth
Cell numbers
Cell mass
Metabolic rate
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Cell numbers
1- Direct microscopic count
• accomplished by direct microscopic
observation on specially etched slides (such
as Petroff-Hausser chambers or
hemocytometers) or by using electronic
counters.
2- Viable count (colony forming units)
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Cell mass
Dry weight versus wet weight
Volume of cells after centrifugation
Measurement of total N or protein
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Cell mass / numbers
 Turbidity or optical density
• it is one of the optical methods for counting cells; can estimate
cell numbers accurately by measuring visible turbidity.
• Light scattered is proportional to number of cells.
• Use a spectrophotometer to accurately measure absorbance,
usually at wavelengths around 400-600 nm.
• Accurate measure of cells when concentration not too high.
Easy and quick to measure (can measure a sample in less than
a minute).
• This technique measures the total mass of organisms
and does not distinguish between dead and viable
organisms.
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Metabolic rate
02 uptake, C02 and ATP production
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Turbidity measurements of microbial growth
Optical Density (Counting by
Spectrophotometer)
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Materials
TSB of Nonpathogenic Escherichia coli
Inoculating loop
Spectrophotometer
Black markers
Test tube rack
2 tubes of TSB
gloves
lab coat or apron
37C Incubator
Bunson burner
Surface disinfectant
Paper towels
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Procedure
1.
Wash hands and put on gloves.
2.
Assemble equipment and materials and prepare work area.
3.
Set the spectrophotometer at 450nm and let it warm up 30 minutes
before performing the readings. The meter on the spectrophotometer
should read “0% Transmittance”.
4.
Obtain 2 tubes of TSB. Label one C (uninoculated) for control and
one S (inoculated) for sample.
5.
Place tube C into the sample holder of the spectrophotometer. Turn
the light control knob until the meter needle is on “100%
Transmittance”. Remove the control tube. The spectrophotometer is
now standardized.
6.
Flame and cool the loop.
7.
Mix the culture of E. coli and inoculate tube S with 5 loopfuls of the
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broth.
8.
Flame and cool the loop.
9.
Start the turbidity readings by placing the S tube into the sample well
and read the OD (absorbance). Record the results in the data section.
10.
Take OD reading over the time period specified in the data section and
return the TSB tube to the 37 C incubator between each reading.
11.
Turn off the spectrophotometer upon completion of the readings.
12.
Plot the readings on the graph paper as absorbance vs. time.
13.
Clean work area with disinfectant.
14.
Remove gloves and wash hands with disinfectant.
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Data
Record the absorbance value for each sample
reading starting at 0 time.
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 Plot the absorbance values vs. time on graph
paper. Label the x and y axis. Each of the
readings will represent the growth of the E.
coli in the culture.
 Label each part of the growth curve: Lag
phase, Exponential growth phase, and the
Stationery phase.
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End of lecture
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