Analysis for microbial growth
Dr. Shir-Ly Huang (黃雪莉)
Professor, Institute of Microbiology and Immunology
Professor, Institute of Food Safety and Health Risk Assessment
National Yang Ming Chiao Tung University
Taiwan
[Link]@[Link]
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Bacterial Growth
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Bacterial cell division: binary fission
Replication requires
1. extension of the cell wall
2. replication of the chromosome
3. septum formation.
Membrane attachnent of the DNA
pulls each daughter strand into a new
cell.
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Bacterial growth curves
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Generation time (g)
the time required for the cell population to double
N = N0 2 n
log N = log N0 + n log 2
n = 3.3 (log N – log N0)
g = t/n
t: growth time
n: number of generation
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Method of estimating the generation time (g)
Hour
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Generation times vary markedly with the species
of microorganism and environmental conditions
- they can range from minutes to days -
microbes species Generation time
E. coli 20 mins
Prokaryote Legionella pneumophila ~2 hrs
Mycobacterium tuberculosis days
Yeast
Eukaryote 1.25~2 hrs at 30°C
(Saccharomyces cerevisiae)
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Growth rate constant (k)
• The number of generations that occur per
unit time in an exponentially growing
culture (h-1)
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The bacterial growth curve
• Usually analyzed in a closed system called a
batch culture
• Usually plotted as the logarithm of cell number
versus the incubation time
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* Bacterial growth phases:
Lag Phase Log Phase Stationary Phase Death Phase
Intense activity Logarithmic, or Period of equilibrium; Population Is
preparing for exponential, microbial deaths balance decreasing at a
population growth, increase in production of new cells. logarithmic rate.
but no increase in population.
population.
The logarithmic growth in the
log phase is due to
reproduction by binary fission
(bacteria) or mitosis (yeast).
Staphylococcus spp.
(Bacteria are grown under controlled conditions
in pure culture)
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Bacterial populations follow a sequential series of
growth phases: the lag, log, stationary, and death phases
Knowledge of the bacterial growth curve is critical to understanding
population dynamics and population control in the course of infectious
diseases, in food reservation and spoilage, as well as in industrial
microbiology processes, such as ethanol production.
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Measure bacterial growth
[Link] count
[Link]
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Plate Count
▪ After incubation, count colonies on plates that have
25-250 colonies (colony forming units, CFUs)
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Estimating Bacterial Numbers
by Indirect Methods
▪ Turbidity by spectrophotometer
(分光光度計)
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Aseptic techniques (6 mins)
[Link]
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Bacterial culture & scale up
Plate
One single colony
5 mL broth in test tube
0.5~1 mL inoculum
50 mL broth in 250 mL flask
10 mL inoculum
500 mL broth in 2.8 L flask
100 mL inoculum
5 L fermentor 17
Bacterial Culture: Scale Up
colony on 5 ml in 50 ml in 500 ml in
agar plate 30-40 ml tube 250 ml flask 2800 ml flask
5 L fermenter
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Strategies to Increase dissolved oxygen
45°
Groove Baffle 19
Bioreactors
Stirred-tank Airlift Fix-bed stirred-tank
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Perfusion device coupled to a stirred tank bioreactor
• ATF alternating tangential flow
• The media and waste products are continuously exchanged and
product in harvested throughout the culture period
• A membrane device is used to retain the cells in the bioreactor, and
waste medium is removed from the bioreactor by the device
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Antibiotics produced as secondary metabolites
Primary metabolites are produced • Secondary metabolites are produced
during active cell growth, matches the near the onset of stationary phase e.g
growth in population of the organisms antibiotic chemicals.
Produced by all microorganisms
• Not produced by all microorganisms
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Aseptic technologies, scale up and growth phases
<Example> Preparation of competent cell for DNA transformation:
Competent cell:
A cell that is able to take up
external DNA and be transformed.
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Fresh competent E. coli prepared using calcium chloride
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Procedure
Scale up
Growth
curve
Aseptic
techniques
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