Microbial physiology
Colonies, Turbid suspension, Biofilm
楊倍昌
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Where to find microbe? Everywhere!
Cell Phone Bacteria: http://www.youtube.com/watch?v=4lmwbBzClAc
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Learning Objectives
After reading this section, students will be able to...
• Explain how to make microbes accountable.
• Describe the pattern and requirement for bacterial
growth.
• Explain growth methods used to synchronize cells.
• Describe how microbes do catabolism to get
energy and metabolism to build structure.
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Microbial physiology
All before doing anything: If you do not separate
an individual from a mix population, you can not
really known who is it, not to say how it works.
By making photomicrographs, I can reveal the bacteria true to
nature and free of subjective misinterpretation ---- Robert Koch
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Counting the viable cells:
Dilution (兩種物理特性)
科學的第一步在於數字化: 定量
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Finding a solid medium
What can be a replacement?
Agar's first use discovered by Frau Franny Eilshemius (a physician's wife)
whose husband told Robert Koch, who is credited with the discovery!
Dr. Walther and Fanny Hess
Agar is derived from Gracilaria (Gelidium species) a bright
red sea vegetable with the botanical name of Gleidium
purpurascens. Agar due to its high gelling properties is
considered the queen of gelling agents.
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Growth measurement

Cell count: microscopic
observation; flow
cytometer (direct)

Colony formation:
Measure the living cell
(direct)

Biomass determination:
dry weight; essential cell
component (indirect)

Turbidity (indirect)
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1, 2, 4, 8, 16, 32, 64, 128, 256, ….
Doubling time
 Escherichia coli:12.5 min
 Vibrio cholerae : 13 min (can kill a man within 12 h)
 Mycobacterium tuberculosis : 24 h (develop
symptom after months)
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Growth and survival

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Lag phase: adaptation to the environment
Exponential logarithmic growth: machine in full rum
Stationary phase: nutrition exhausted, toxin increased
Decline: cell die (steady biomass) or lysis (decrease
biomass)
Dormant as spore, non-viable state
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In bioreactors
in 37oC, pH 5.1 ; in 45oC, pH 6.2
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Factors affecting Growth
• The orderly increase in the sum of all the
components of an organism
Affected by:

Nutrients

pH: neutrophils, acidophils, alkalophis

Temperature: psychrophils; mesophils; themophils

Aeration

Pressure

Ionic strength and osmotic pressure: halophils,
osmophils
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The biochemical reactions in living cells which allow
them to assimilate food to provide energy for their
growth and reproduction, are termed metabolism.
The ideal temperature range for
maximum reproduction rate of
most bacteria is between 80° F
(27oC) and 105°F (40.5oC).
The ideal pH range for
maximum bacterial growth
of most strains is 6.5 to 8.5.
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Thermophiles
70o-110o
Mesophiles
10o-50o
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Thermophils

visible in hot springs when singlecelled photosynthetic bacteria
(cyanobacteria) form dense layers of
biomass called bacterial mats.
 contained "environmentally friendly"
enzymes that were stable under high
temperatures and could be used in
place of more dangerous chemicals
that have been concocted by modern
industry.

Bacteria and Archaea live in hot
springs heated by geothermal reactions
deep in the Earth. Some springs reach
temperatures of 80 C (177F).
Taq polymerase
Distinct membrane
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There is no O2 in the earth in ancient
times. What is the advantage of growth
in O2-containing environment?
Aerobic respiration produce free radicals.
To detoxify oxygen, you need:
Catalase: H2O2 --- H20 and 02
Superoxide dismutase (SOD): oxygen radical --- H20 and O2
Growth pattern
1.
2.
3.
4.
5.
Obligate aerobe
Obligate anaerobe
Microaerophile
Aerotolerant anaerobe
Facultative anaerobe/aerobe
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Air requirement:O2
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Aerobe: A microorganism whose growth requires the
presence of air or free oxygen
Anaerobe: A microorganism that grows only or best in the
absence of free oxygen. Organisms utilize bound oxygen
Microaerophile: A microorganism that grows best in the
presence of low concentrations of oxygen
Facultative anaerobe/aerobe: A microbe that adjusts its
metabolism to depending on the oxygen concentration in
which it is growing
Aerotolerant anaerobe: an organism that always grows in
an anaerobic mode -- it ignors the presence of oxygen.
Capneic microbe: An organism that requires 3 to 10% CO2
for growth
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Create an environment to
grow bacteria
Capneic microbe: An
organism that requires 3% to
10% CO2 for growth
Obligate anaerobes. These will
not grow in presence of O2 Some
find oxygen very toxic, even at
short exposures. Example:
Bacteroides spp.
Q: How they grow in nature?
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Growth/Culture condition
Plating technique of Robert Koch for
single colony isolation
Microbiol. Mol. Biol. Rev. 64:847-867 (2000)
The majority of microbes persist
attached to surfaces within a
structured biofilm ecosystem
and not as free floating
organisms.
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Differences in culture:
when cells grow as biofilm
1. On metabolism
resistance
2. Drug
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Two types of biofilms
Environmental
Disease-associated
 Symbioses
 Dental plaque
 Termite, ruminant
 Endocarditis
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digestion
Sewage treatment
bioreactors
Water pipes
Dental units
Contact lens cases
 Cystic Fibrosis
 Otitis media
 Urinary catheter
 Implants
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Technique: continuous cultivation
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Synchronization
What for?
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Starvation
Temperature shock
Refresh from stationary phase
Filter binding/release
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Viable But Non-culturable
(VBNC)
Bacteria
Bacteria in the VBNC state fail to grow on the routine bacteriological
media on which they would normally grow and develop into colonies, but are
alive and capable of renewed metabolic activity (Oliver, 2000b).
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http://nihroadmap.nih.gov/hmp/
This initiative will begin with the sequencing of up to 600
genomes from both cultured and uncultured bacteria, plus several
non-bacterial microbes. Combined with existing and other
currently planned efforts, the total reference collection should
reach 1000 genomes.
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Run a store for life:
6 major tasks
Please refer to biochemistry course
1.
Nutrition uptake
2.
Metabolism: synthesis and catabolism
3.
Energy generation
4.
Discard garbage/toxin
5.
Reproduction
6.
Adaptation
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Metabolism: key of physiology
a very short summary
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Bacteria feed by absorption through their cell
membranes
In the first stage, they secrete enzymes (extra-cellular)
which break down the large particulates and solids.
In the second stage, the reduced particles are absorbed
through the membrane where cellular enzymes break
down the extra cellular enzymes.
This process will produce CO2 and H2O and seed
bacteria since cell division occurs when sufficient food
is processed.
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Energy and building block generation
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Glycolysis
 Two molecules of ATP are use to
phosphorylate glucose and start
glycolysis.
 The phosphorylated molecule is then
broken down in a series of reactions
into two three carbon molecules (lysis).
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Pentose phosphate shunt
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When reducing power are needed
Sensitive to the level of NADP+
Did not generate ATP
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Metabolism & Bacterial Identification
Some microbes can metabolize certain molecules while others can’t.
When molecules are metabolized, specific waste products are created
such as acids. Those waste products can be labeled by color.
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http://www.genome.jp/kegg/pathway.html
這個網頁中有很完善的整理資料 (KEGG PATHWAY Database)
Current knowledge on molecular interaction networks,
including metabolic pathways, regulatory pathways,
and molecular complexes
• 你如果要把所有的生理代謝路
徑背起來, 保證你一整年都還背
不完全。
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Life is a kind of chemical reaction
Swedish chemist Arrhenius discovered how temperature affects the rate of
chemical reaction and it can apply to cell growth
Arrhenius plot of growth rate of E. coli.
Individual data points are marked with
corresponding degrees Celsius. (Herenden et al 1979)
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Key messages
•
Cell counting is not always as obvious. There are 4 surrogate
ways: Cell counting (direct), Colony formation (direct) , Biomass
determination (indirect), Turbidity (indirect)
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Environmental factors affecting growth include: Nutrients, pH,
Temperature, Aeration, Pressure, Ionic strength and osmotic
pressure.
•
Cell growth can be divided into 4 phases: Lag phase (adaptation
to the environment), Exponential logarithmic growth (machine in
full run), Stationary phase (nutrition exhausted, toxin increased),
Decline (cell or lysis)
•
Microbes are useful tools in research because of their rapid life
cycle, their simple growth requirements, and their small size. Due
to this simplicity, microbes have been essential in understanding
core questions in biology.
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