Soil 6 - Methods of studying Flashcards
What does the scientific method help us achieve, ideally?
predictive power!
What are the 5 steps of the scientific method?
- observe
- hypothesize
- predict
- experiment
- repeat
Why is it hard to do science with microbial communities
- extremely high diversity
- culturing = inherently isolating (specific conditions)
What are the 2 methods of plate culturing?
- Spread plate - spread diluted bacterial suspension on plate, get colonies on surface
- Pour plate method - mix bacterial suspension with agar, pour into plate (colonies on surface and in agar)
How dWhat is the Great Plate Count Anomaly?
- plate cultures are NOT representative of communities
- from DNA and microscopy, found that the number of organisms appearing in plates is MUCH lower than cell numbers (lots of undescribed life in soil that can’t be cultured)
- lots of microbes = viable but not culturable, and ENRICHMENT BIAS is common in plates
What are the benefits of using microscopy?
- easy/cheap (only need a light microscope)
- can determine the form of soil particles
What are the disadvantages of using microscopy?
- non-quantitative
- difficult to distinguish microbes from soil
- thin sections - hard to truly observe soiol organisms
What are the advantages of epifluorescence?
- relatively easy
- no need to culture organisms (no media bias)
- all specimens can be counted
- separate soil from cells
What are the disadvantages of epifluorescence?
- physiological groupings are unknown
- lots of background fluorescence (stuff other than cells gets stained)
- slime layers make it hard to separate cells from soil
- expensive!
What are the 2 types of electron microscopy?
- transmission electron microscopy
- scanning electron microscopy
How does transfer electron microscopy work?
- e- beam passes through a thin sample
- e- pass through sample onto a transfer surface, creating an image of the opaque/clear areas of a cell
How does scanning electron microscopy work?
- samples mounted on a stub, coated
- electrons scan over sample, display image on a screen
What is the eye of the needle through which all organic matter that enters the soil must pass?
microbial biomass!
What are the 2 types of methods for counting microbe biomass?
- physiological methods (CFI, GSR)
- Chemical methods (CFE, PLFA)
What is the CFI method of measuring microbial biomass?
- Chloroform Fumigation Incubation
- kill most of the microbes in the soil
- after CHCl3 is removed, remaining microbes metabolize the other microbe remains
- flush in CO2 and NH4 (respiration) can be measured
What are the advantages of the CFI method of measuring microbial biomass?
- can recover surviving microbes for analysis
- isotopic compositions of CO2 and NH4 can be used for tracer studies
- inexpensive
- can be used to estimate microbial N
What are the disadvantages of method of the CFI measuring microbial biomass?
- no proper control
- can’t be used in acid, calcareous, or fresh soils
- only measuring CO2 flux, doesn’t necessarily mean microbial mass
What is the GSR method of measuring microbial biomass?
Glucose-stimulated respiration
- when glucose is added, microbse create a flush of CO2 proportional to community size
What are the advantages of the GSR method of measuring microbial biomass?
- quick and simple
- can recover living microbes (for analysis)
- can be modified to measure relative contribution of fungi/bacteria
What are 4 disadvantages of the GSR method of measuring microbial biomass?
- CO2 release is temperature-sensitive
- optimal glucose amount depends on soil
- timing of maximum respiration rate varies
- can’t be used to estimate microbial N
What is the CFE method of measuring microbial biomass?
Chloroform Fumigation-Extraction
- fumigation kills most of the microbes in the soil
- cell contents can be recovered as SOC (soil organic carbon) and SON
What are 4 advantages of the CFE method of measuring microbial biomass?
- rapid
- can be used in many soil types
- can estimate microbial N
- can trace isotopic composition
What are 3 disadvantages of the CFE method of measuring microbial biomass?
- CFE is calibrated against CFI
- Kce and Kne not defined?
- need special equipment to determine SOC (soil organic carbon) and SON
What is the PLFA method of measuring microbial biomass?
specific Phospholipid Fatty Acids
- PLFAs are characteristic of different BActeria classes
- some = common to all microorganisms, bacteria, or fungi
- some = indicative of specific groups (ex gram-negative bacteria)
What are 3 advantages of the PLFA method of measuring microbial biomass?
- can be used to characterize microbial structural biodiversity
- correlates with other methods of biomass determination
- can be used to estimate ratios of different microbe groups
What are 3 disadvantages of the PLFA method of measuring microbial biomass?
- time-consuming and expensive
- many hazardous chemicals required
- different equations for forest floor/mineral soils
What is the difference between ribose and deoxyribose (in the DNA/RNA sugar backbone?)
- ribose has a OH group on carbon 2, where deoxyribose has a H
What are the groups of nitrogenous bases in genetics?
- pyrimidines: Cytosine, Thymine, Uracil (RNA)
- Purines: Adenine, Guanine
What is Chargaff’s rule?
In nucleotide pairing:
A=T
C=G
due to complementary H bonds between pairs
What is the central dogma of molecular biology?
- DNA to RNA = transcription
- RNA to proteins = translation
- DNA to DNA = replication
What is metagenomics?
the genetic material of a community
What does a UV spectrophotometer do in DNA quantification?
indicate purity of sample
What do DNA vs RNA tell us about a soil sample?
DNA: who is there?
RNA: who is potentially active? (denatures quickly)
How does gel electrophoresis work?
- negatively charged DNA moves through a matrix toward a positive electrode
- separates according to size
How does Sanger Sequencing work? (original sequencing technology!)
1 amplification
2. primer extension
3. separation
what is new in ‘next gen’ DNA sequencing?
more automated
