microbial physiology

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Microbial physiology 楊楊楊 Colonies, Turbid suspension, Biofilm 1

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Microbial physiology. 楊倍昌. Colonies, Turbid suspension, Biofilm. 略. Where to find microbe? Everywhere!. Cell Phone Bacteria: http://www.youtube.com/watch?v=4lmwbBzClAc. Learning Objectives. After reading this section, students will be able to... Explain how to make microbes accountable . - PowerPoint PPT Presentation

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Page 1: Microbial physiology

Microbial physiology

楊倍昌

Colonies, Turbid suspension, Biofilm

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Page 2: Microbial physiology

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Where to find microbe? Everywhere!

Cell Phone Bacteria: http://www.youtube.com/watch?v=4lmwbBzClAc

Page 3: Microbial physiology

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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Page 4: Microbial physiology

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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Page 5: Microbial physiology

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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Page 6: Microbial physiology

Finding a solid medium

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.

Dr. Walther and Fanny Hess

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!

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Page 7: Microbial physiology

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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Page 8: Microbial physiology

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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1, 2, 4, 8, 16, 32, 64, 128, 256, ….

Page 9: Microbial physiology

Growth and survival

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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Page 10: Microbial physiology

in 37oC, pH 5.1 ; in 45oC, pH 6.2In bioreactors

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Page 11: Microbial physiology

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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Page 12: Microbial physiology

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 pH range for maximum bacterial growth of most strains is 6.5 to 8.5.

The ideal temperature range for maximum reproduction rate of most bacteria is between 80° F (27oC) and 105°F (40.5oC).

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Page 13: Microbial physiology

Mesophiles10o-50o

Thermophiles70o-110o

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Page 14: Microbial physiology

Thermophils visible in hot springs when single-

celled 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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Page 15: Microbial physiology

Aerobic respiration produce free radicals. To detoxify oxygen, you need:

Catalase: H2O2 --- H20 and 02

Superoxide dismutase (SOD): oxygen radical --- H20 and O2

There is no O2 in the earth in ancient times. What is the advantage of growth in O2-containing environment?

Page 16: Microbial physiology

1. Obligate aerobe2. Obligate anaerobe3. Microaerophile4. Aerotolerant anaerobe5. Facultative anaerobe/aerobe

Growth pattern

Page 17: Microbial physiology

Air requirement:O2

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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Page 18: Microbial physiology

Create an environment to grow bacteria

Obligate anaerobes. These will not grow in presence of O2 Some find oxygen very toxic, even at short exposures. Example: Bacteroides spp.

Capneic microbe: An organism that requires 3% to 10% CO2 for growth

Q: How they grow in nature?18

Page 19: Microbial physiology

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Growth/Culture conditionPlating technique of Robert Koch for

single colony isolation

The majority of microbes persist attached to surfaces within a structured biofilm ecosystem and not as free floating organisms.

Microbiol. Mol. Biol. Rev. 64:847-867 (2000)

Page 20: Microbial physiology

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Differences in culture: when cells grow as biofilm

1. On metabolism 2. Drug resistance

Page 21: Microbial physiology

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Two types of biofilms

Symbioses Termite, ruminant

digestion Sewage treatment

bioreactors Water pipes Dental units Contact lens cases

Dental plaque Endocarditis Cystic Fibrosis Otitis media Urinary catheter Implants

Disease-associated Environmental

Page 22: Microbial physiology

Technique: continuous cultivation

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Page 23: Microbial physiology

Synchronization

Starvation Temperature shock Refresh from stationary phase Filter binding/release

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What for?

Page 24: Microbial physiology

Viable But Non-culturable (VBNC) BacteriaBacteria in the VBNC state fail to grow on the routine bacteriologicalmedia 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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Page 25: Microbial physiology

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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.

Page 26: Microbial physiology

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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Page 27: Microbial physiology

Metabolism: key of physiology a very short summary

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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Page 28: Microbial physiology

Energy and building block generation

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Page 29: Microbial physiology

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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Page 30: Microbial physiology

Pentose phosphate shunt

When reducing power are needed Sensitive to the level of NADP+ Did not generate ATP

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Metabolism & Bacterial Identification

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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

• 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。

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

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Page 33: Microbial physiology

Life is a kind of chemical reactionSwedish 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) 33

Page 34: Microbial physiology

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)

• 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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