Advanced Microbiology Flashcards
What are the levels of organisation?
From smallest to largest
Individuals Populations Guilds Community Ecosystem
What are the 4 processes that cause change in a microbial community?
Diversification = Random, generation of genetic variation
Dispersal = Random, organisms movements in space
Drift = Random, changes in relative abundance through time
Selection = Deterministic, change caused by fitness differences
Explain “the great plate count anomaly”
CFU counts are far lower than we would expect from the environment, this is partially because of viable but non-culturable bacteria
Explain the “Enrichment” culture method.
Growth conditions that are selectively favourable for desired organisms.
Explain the “Dilution to extinction” isolation method.
Repeatedly dilute your enrichment sample to isolate the more abundant microbes.
This favours microbes that grow the fastest.
Explain “Single cell isolation”
Single cell isolation methods are ways to isolate single cells.
Laser tweezers use high infared laser beams to grab single cells.
Explain “Flow cytometry”
Flow cytometry selects for cells based on fluorescence or charge. This involves flowing the cells past a detector which then filters them based on fluorescence or charge.
Explain how “I-chips” are used
Isolation-chips are chips with microscopic holes designed to isolate single cells in each hole. Incubation occurs in the environment and isolates become more “domesticated” with each cycle.
Non-specific fluorescent markers tend to target what macromolecule?
DNA
Specific markers target the other macromolecules.
What is FISH and what does it stand for?
Fluorescent In-Situ Hybridisation
Fluorescently labeled oligonucleotide probes targeted to species specific genes.
What is CARD-FISH?
Similar to FISH but the oligonucleotides are tagged with a readout enzyme such as horse radish peroxidase which amplifies the readout signal.
What is DGGE?
Denaturing Gradient Gel Electrophoresis
This method separates DNA by the melting points of it’s fragments which is determined by the fragments G-C contents.
Describe Second generation sequencing
Second generation sequencing is amplification based and gives many many short reads for high coverage of the DNA.
Describe Third generation sequencing
Third generation sequencing does a few very long read of the DNA and gives high quality alignments and assemblies.
Describe a Next Generation Sequencing (NGS) workflow.
Start with sample DNA extraction.
Amplification using 16S primers and multiplexing (Barcode) then run the PCR.
Clone library prep which involves attaching sequence adapters + pooling the libraries and sequencing the clones.
Data analysis which involves preprocessing which is the removal of primers, demultiplexing and filtering for quality clones.
