L13: Culture Based Methods Flashcards
List 6 Examples of Samples to Study
Plant Roots
Plant Leafs
Animal Feces
Sediments
Wastewater
What is the importance of sampling methods in environmental bio
- as soon as it is retrieved biotic and abiotic parameters change => sampling must minimise change by replicates, in situ + chemical anaylsis
What are the 4 objectives of Sampling
- How many microbes/ biomass
- Who + What
- What are they doing, functionality
- What are the ecological interactions
What are the types of analysis
Culture-based & culture independent (both broad)
What is culture-based analysis + pro/cons
- anything that requires growing, in vitro
PRO: best way to understand physiology + genetics
CONS: only ~1%
What is culture independant + pro/cons
- anything that does not require growing: nucleic acids, microscopy, in vivo
PRO: samples entire community
CON: information is correlative with methodological bias
What are the steps in classical culture based analysis
- minimise transportation + storage
- store at in situ temp or freeze but thawing could be lethal (-80/20)
- disturbances in sample (like soil) could have downsteam effects
- use aseptic technique + equipment
What is the goal of Molecular Biology Analyses
- maintain TOTAL composition to provide a snap shot by
- minimise transport + storage
- store -80/20 in sutu
- perform nucleic acid extraction in a site of freeze with liquid N, dry ice or ethanol baths
- use DNA preservation reagent
How do you conduct an enumeration of microorganisms
A. direct microscope counts: flu micro, nucleic acid /protein/viability strains
B. Culture-dependant: heterotrophic plate counts
How does electron microscopy counts work
estimates length and width
How do nucleic acid stains work
AODC orange: binds to NA, and differenetiates between live and dead, lot of background
DAPI: binds to dsDNA, precisse and good for sediment and seawater
Hoechst: binds to AT rich regions
When can SYBR green dye be used
- microscopy
- gel electrophoresis
- flow cytometry
How do protein stains works
FITC: labels biomolecules like immunoglobin, nucleic acids, nucelotides
DTAF: proteins stain but gives live and dead, so use for low background
How do viability stains work
- dead vs alive, use Sytox green (intact + damaged membrane && propdium (damaged membrane)
Explore more staining techniques
Naladixic Acid: direct viability count
* counts growing cells, inhibits gram neg dicision, makes long dormat/dead cells, automatic
AODC + int
* very fast , total number + cells respiring
* INT becomes INTO formazon by respiring bacteria
PRO: quick, fast good estimate
CONS: subjective, expensive
How does Fluorescence microscopy Work
- specimen absorbs light of defined wavelegnths ==> emits the light of lower energy/long wavelength ==> shiny
Hw does optical system + fluorecne overlap
- optical systems for fluo uses colour filters to help limit light incident of exiction + emision
What is a micropy way to do direct counts
PHC for bacteria
Hemocytometer for euks
What are culture dependant tehcniques
- viable counts: aerobic heterophic plate counts
- viable is usually <1-10% of direct count
- depdnant on medium + incubation conditions
- usually low nutrient concentration, slow growing and adapted to those conditions ==> usually general purpose media is too rich and will kill or inhibit soil microorganisms
What are two methods of viable count + PRO/CONS
- spread plate: sample on agar, spread (incubate) count
- pour plate: into the sterile plate, add medium (solidification + incubation) count, no mix
pro: allows for subsequent isolation of culture bac+ detection/percentage of interesting genotypes by colony hybridization
con: only ~1% microorg can be cultured + only detect fast-growing isolates which dominate easily
What is another method of viable counts
What is the second viable count method
- most probable number techqniues
- avoiding agar which could contaminate
- successive dilution to point of extinction
- stat anlysis MPN gives extimate of number of viable micronial organsims in sample
- good for anerobic
What is seletive vs different media
- enchance growth of one group while inhibting growth of other- Antibiotuc
differential media: add reagant that allow for visual differentation - EMB
