L13: Culture Based Methods

Question 1

List 6 Examples of Samples to Study

Answer 1

Plant Roots
Plant Leafs
Animal Feces
Sediments
Wastewater

Question 2

What is the importance of sampling methods in environmental bio

Answer 2

• as soon as it is retrieved biotic and abiotic parameters change => sampling must minimise change by replicates, in situ + chemical anaylsis

Question 3

What are the 4 objectives of Sampling

Answer 3

1. How many microbes/ biomass
2. Who + What
3. What are they doing, functionality
4. What are the ecological interactions

Question 4

What are the types of analysis

Answer 4

Culture-based & culture independent (both broad)

Question 5

What is culture-based analysis + pro/cons

Answer 5

• anything that requires growing, in vitro
  PRO: best way to understand physiology + genetics
  CONS: only ~1%

Question 6

What is culture independant + pro/cons

Answer 6

• anything that does not require growing: nucleic acids, microscopy, in vivo
  PRO: samples entire community
  CON: information is correlative with methodological bias

Question 7

What are the steps in classical culture based analysis

Answer 7

1. minimise transportation + storage
2. store at in situ temp or freeze but thawing could be lethal (-80/20)
3. disturbances in sample (like soil) could have downsteam effects
4. use aseptic technique + equipment

Question 8

What is the goal of Molecular Biology Analyses

Answer 8

1. 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

Question 9

How do you conduct an enumeration of microorganisms

Answer 9

A. direct microscope counts: flu micro, nucleic acid /protein/viability strains

B. Culture-dependant: heterotrophic plate counts

Question 10

How does electron microscopy counts work

Answer 10

estimates length and width

Question 11

How do nucleic acid stains work

Answer 11

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

Question 12

When can SYBR green dye be used

Answer 12

1. microscopy
2. gel electrophoresis
3. flow cytometry

Question 13

How do protein stains works

Answer 13

FITC: labels biomolecules like immunoglobin, nucleic acids, nucelotides

DTAF: proteins stain but gives live and dead, so use for low background

Question 14

How do viability stains work

Answer 14

• dead vs alive, use Sytox green (intact + damaged membrane && propdium (damaged membrane)

Question 15

Explore more staining techniques

Answer 15

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

Question 16

How does Fluorescence microscopy Work

Answer 16

• specimen absorbs light of defined wavelegnths ==> emits the light of lower energy/long wavelength ==> shiny

Question 17

Hw does optical system + fluorecne overlap

Answer 17

• optical systems for fluo uses colour filters to help limit light incident of exiction + emision

Question 18

What is a micropy way to do direct counts

Answer 18

PHC for bacteria
Hemocytometer for euks

Question 19

What are culture dependant tehcniques

Answer 19

1. 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

Question 20

What are two methods of viable count + PRO/CONS

Answer 20

1. spread plate: sample on agar, spread (incubate) count
2. 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

Question 21

What is another method of viable counts

Answer 21

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

Question 22

What is seletive vs different media

Answer 22

• enchance growth of one group while inhibting growth of other- Antibiotuc

differential media: add reagant that allow for visual differentation - EMB