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major components of microbial ecology
biodiversity & microbial activity
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Population
bacteria that derived from a single cell
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Guild
Populations that are metabolic related
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Microbial community
set of guilds
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Ecosystem
Communities of organisms and their natural environment
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Biogeochemical Cycle
Biological and chemically mediated chemical transformation of elements
"cycling of elements'
typically by oxidation-reduction rxn
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Niche
growth and function at optimal levels, habitat,
Physical habitat -determines the niche
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Mutualism
Commensalism
Ammensalism
Parasitism
Symbiosis
- Mutualism: + / +
- Commensalism: + / 0
- Ammensalism: 0 / -
- Parasitism: + / -
- Symbiosis: -share the same ecosystem
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Polpulation of microbes vs individual microbes
- Ubiquity - Biodiversity
- Abundance
- Metabolic Power - microbial activity
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3 ways to isolate to obtain a pure culture
- 1. streak - drag to a single colony
- 2. agar shake - molton agar, double layer- colonies embedded in agar.
- 3. Liquid Dilution - serially dilute until no growth. also called MPN
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MPN
- Most Probable Number analysis
- a repeat dilution series
- used for estimating the number of microorganisms in foods, waster, and other samples
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What is the criteria for Purity
- 1. microscopy - gram stain
- 2. observation of colony characteristics on plate or shake tubes
- 3. growth in media in which the culture grows poorly but contaminates grow well.
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What are the 2 drawbacks in studying pure cultures?
- 1. microorganisms will change characteristics to benefit them in a lab setting
- ex - pathogens lose virilence - may have different antigens
- 2. microbial communities are too small (3mm)
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Winogradsky Column
- algae & cyanobacteria
- purple non sulfur bacteria
- sulfur chemolithotrophs
- purple & green sulfur bacteria
- Anoxic decomposition & sulfate reduction
gradient: O 2 to H 2S & light gradient
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Growth Rates of microorganisms in the lab
- 1. faster growht than natural habitat because of feeding all the good stuff (food scarce in nature)
- 2. slower growth, microorganism may need another factor that is unknown.
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How do we quantify microbes in a microbial habitat ?
- 1. PCR and DGGE
- 2. Metagenomics
- 3. Staining methods - fluorescence
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1. Viability Staining
- use 2 dyes:
- 1. green fluorescening dye - penetrates all cells
- 2. red dye (Propidium iodide) - penetrates cells that dont have cell membranes (aka dead cells)
commercial name: BacLight
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2. Fluorescent staining using DAPI or Acridine Orange
- -stain binds to DNA and strongly fluorescent when exposed to UV lights.
- -problem: stains both live and dead cells
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3. Fluorescent Antibody Probes
- -good tracking a specific organisms within a habitat
- -specific antibody attaches to specific site
- -good for diagnosing infectious diseases and other lab work :-)
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4. GFP as a cell Tag
- -insert GFP into the geome and add to the natural population.
- -tags green (but not always green)
- -reporter gene - replace gene with GFP
- -problem: need oxygen
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5. FISH - Fluorescence in situ hybridization
- -nucleic acid probe
- -obligonucleotide complementary to a sequence in a targe gene or RNA -hybridize)
- -hybridized to cells in a natural environment
- -doesnt kill microbe
- -can measure gene expression
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PCR
DGGE
PCR - polymerase chain rxn: artifical amplification of a DNA sequence by repeated cycles of strand separation & replication
DGGE - denaturing gradient gel eletrophoresis: separating nucelic acid fragments of the same size that differe in base sequence
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phylogenetic staining
ex - red fluorescent dye tag bacillus, green dye tag staph, etc.
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Metagenomics
- the total genetic complement of all cells present in a particular environment
- -detect as many as genes as possible that encode recognizable proteins & determine the phylogenetic groups to which it belongs to
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Measuring microbial activities in nature
(4 ways)
- 1. Chemical Assays
- 2. Radioisotopes
- 3. Microelectrodes
- 4. Stable Isotopes
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Chemical Assay of microbial activities in nature
- direct chemical measurements of microbial rxn
- ex- fate of lactate oxidation by sulfate-reducing bacteria H2S
- - not good for highly sensitivity
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Radioisotopes in microbial activites in nature
- - good for:
- 1. when high sensitivity is required
- 2. turnover rates need to be determined
- 3. fate of portions of a molecules needs to be followed.
- problem: some transofrmations might be due to abiotic prcesses
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killed cell control
- -account the abiotic process in radioisotopes
- -yields proper control in experiment
- -ex formalin
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Microelectrodes in measuring microbial activities in nature
- -small glass electrodes
- -measures: pH, Oxygen, N2O, CO2, H2, or H2S
- ex- shine the light, add electrodes = measures the amount of oxygen being generated
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microautoradiography
- -MAR
- -method that cells are exposed to a radioisotope and slided - photographic emulsion. darkness-decay =silver grains in the emulsuion
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Stable Isotope probing
a method for characterizing an organism that incorporates a particular substrate by feeding the subsrate in 13C form and then isolating 13C-enriched DNA and analyzing the gene
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Isotopic fractionation
- -one of two stable isotpe methods
- -discrimination by enzymes against the heavier isotope of various isotpes of C or S, leading to enrichment of the ligher isotopes.
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