Microbial Ecology/Communities 1 Flashcards
(23 cards)
What is DAPI
A general fluorescent stain
What does DAPI bind strongly to & thus what does it stain
Binds strongly to adenine - thymine (AT) rich regions in DNA & thus stains DNA
What is DAPI used for
Used for identifying microorganisms in natural samples
What does the dye in the live bacterial gram stain kit bind to
It binds specifically to N-acetylglucosamine in the peptidoglycan layer of gram pos bact
What will gram pos bact stained using this live bacterial gram stain kit show as
Fluorescent blue interior & fluorescent red cell surface
What will gram neg bact stained using this live bacterial gram stain kit show as
Gram neg bact will be stained with blue fluorescence only
Viability PCR can be used to distinguish between what kinds of cells
Alive and dead cells
Steps in viability PCR
- The dye PMAxx is dead cell specific
- Once inside the dead cell, they bind to DNA
- Intense light exposure causes the dye to become reactive and covalently bind DNA
- DNA modification prevents amplification in subsequent PCR rxns
What is PMAxx highly useful in detecting
Selective detection of live bacteria by PCR. (Dead cells are not amplified in the PCR rxn, only live cells)
Steps of variability PCR (vPCR) worflow (5)
- Addition of dye
- Incubation in the dark
- Exposure to intense light
- DNA extraction/purification
- Performance of qPCR (quantitative PCR)
Bioluminescent meaning
Glows in the dark
Fluorescent bioluminescent meaning
Glows in response to UV light
What is GFP
Naturally occuring 27kDa protein derived from jellyfish that fluoresces green when exxposed to blue light
What can GFP (green fluorescent protein) be used for
GFP makes cells autofluorescent & is a means of tracking cells introduced into the environment
Main principals of these culture independent techniques (3)
- Forgoes need for culture of microbes
- No isolations of microbes
- No microscopic identification or quantification
How does PCR based phylogenetic analysis work
- Phylogenetically distinct microbes are present in a population
- This allows phylotypes be described
Pros of culture independent analyses of microbial communities
- Forgoes need for culture of microbes
- No isolations of microbes
- No microscopic identification or quantification
Purpose of DNA polymerase in PCR reactions
DNA polymerase adds nucleotides in the same order as those of the gene of interest to the primers
3 steps of PCR rxn
- Denaturation - rxn heated to 94-98 C for 20-30 secs to break hydrogen bonds between strands
- Annealing - rxn temp lowered to 50-65 C for 20-40 secs to allow primers anneal to template strands
- Elongation - temp increased for addition of dNTPs
What is phylogenetics
The analysis of molecular sequencing data to study evolutionary relationships among groups of organisms
What ribosomal subunit is suitable for study in prokaryotes
The 16s ribosomal RNA
What ribosomal subunit is suitable for study in eukaryotes
The 18s rRNA
The 16s or 18s rRNA gene sequencing process
- Isolate DNA from organism
- Heat to separate strands
- Add primers
- Primer extension w DNA polymerase
- Repeat many PCR cycles to yield multiple copies of 16s ribosomal RNA gene
- Run agarose gel & check for correct size PCR product
- Purify PCR product
- Sequence
- Compare sequence to database
- Identify bacterial species
