Flashcards in Semester 1 Deck (58):
When bacteria has colonised the whole plate.
Isolate colonies derived from single cells in a mixed culture
Streaked onto surface to produce well-separated colonies
Measure viable cell count
Inoculum is added to the melted, cooled agar mixed, is gently mixed and then poured into the plates.
This is useful for the isolation of bacterial cultures from a mixed inoculum or when heaved even growth (lawn) is needed.
As in pour plates but a thin layer is poured on top of already solid medium.
Grow a large number of cell lines in a limited space
Surface are of the medium is increased
Grow large amounts of bacteria, but not discrete colonies, e.g. for DNA isolation.
A straight needle in plunged vertically deep into the medium to anaerobically culture microorganism.
Large yellow/gold colonies
Clusters of cocci
Large white colonies with diffuse edges.
Rods, short chains
Small, white-grey colonies
Cocci, short chians
Preparation and fixing of films
Degrease the microscopic slide by passing through a bunsen flame
Prepare the films by using a sterile wire to transfer microbial culture
Heat fixation by passing through a gently burning bunsen.
General staining with methylene blue
To examine the general form of the individual cells and their arrangement
It is a positive stain: the organism and other material fixed on the slide is coloured.
A negative stain colours the area outside the organisms and other material fixed on the slide.
Stain with methyl violet
Rinse with iodine
Wash with alcohol
Add counter-stain (carbol fuchsin)
Gram positive retain methyl violet in their thick peptidoglycan cell walls, they appear purple-blue
Gram negative have a thin peptidoglycan layer so alcohol removes the violet stain, they appear pink-red.
Escherichia, Pseudomonas, Neisseria.
Chromosomes treated with trypsin (digests protein)
Giemsa stain (stains transcriptionally silent regions of DNA containing AT-rich sequences, creating dark bands)
Colchicine is added to growing cells stopping microtubules from polymerising, preventing spindle formation.
Cells are harvested
Buffer with low salt concentration is added to lyse the cells
Lysed cells are fixed to slides.
Submetacentric: between middle and end
Acrocentric: near one end
Telocentric: at the end.
Levelling off of graphs
Less substrate available so decreases
Product could act as a competitive inhibitor
Product could affect pH and as it builds up could prevent enzyme action occurring at a higher rate.
Release from living cells
Released from disintegrating cells
Hydrolyse sulphate esters with an aromatic radical
Measuring the growth of microrooganisms
-Pros: very quick and easy; cons: not accurate above about 0.6 and no information on cell viability
Counting cell number
-Pros: accurate determination of biomass; cons: time consuming and no information on cell viability
Serial Dilution and Plating
-Pros: most accurate and determines number of viable cells only; cons: time consuming must wait at least overnight before getting results.
Measurement of cell constituent e.g. protein or chlorophyll
-Pros: very quick and easy; cons: need to assume that the proportion of the cell made up by the cell constituent does not change, no information on cell viability
Anaerobic test tube conditions
Leaving a tube unshaken provides anaerobic conditions as all the oxygen will be used early in the incubation.
Dunaliella: 10 x 5 µm *largest
B. megaterium: 8 x 2 µm
S. cerevisae: 10-20µm in diameter
Prophase of meiosis I
Condensation of chromosomes
Homologous chromosomes associated in bivalents by the process of synapsis
The bivalents shorten and thicken, crossing over occurs but it is not visibly detectable.
The two homologous chromosomes within each bivalent separate over most of their length including the centromeres, but are held together by chiasmata.
-Sister chromatids remain attached at their centromeres.
Chromosomes undergo further shortening and thickening.
The parents of the affected individuals are themselves unaffected.
At least one individual would have an affected parent.
Each affected female would have an affected father
-They would need an affected father and an unaffected mother.
Affected males are much more common than affected females
-Each affected male receives the disease allele on the X-chromosome he receives from his heterozygous mother.
-Because males only possess one X-chromosomes, while females have two.
Migration of macromolecules through a polymer matrix (agarose or polyacrylamide) in response to an applied electric field.
DNA fragments separate in an agarose gel according to their size.
-The smaller pieces travel the fastest and the furthest through the gel.
-The larger pieces have more difficulty moving through the gel matrix and thus move more slowly through the gel.
The movement of DNA fragments in the gel is logarithmic, they migrate at a rate inversely proportional to he logarithm of the molecular weight.
Electrophoresis loading buffer
DNA is added to a loading buffer which contains glycerol, and a dye.
The heavy glycerol facilitates loading of the sample by allowing the sample to sink to the bottom of the well, whereas the dye helps visualise the migration of the sample during electrophoresis.
Properties of DNA allowing electrophoresis
Negatively charged phosphate backbone.
Ethidium bromide/midori green intercalate between stacked bases of DNA.
A plasmid is defined as an independently replicating, extra-chromosomal element of DNA.
They often confer upon their host the capacity for neutralising toxic antibiotics or degrading exotic metabolites.
-Thus, there is generally a selective advantage to be gained by the host bacterium in harbouring such plasmids.
They are relatively small and present in a high copy number per cell so that they can be purified in large amounts.
A typical cloning vector includes:
Multiple cloning sites, which can be used to insert foreign DNA
Antibiotic resistance site, as a means of selection.
Origin of DNA replication.
Differences between plasmid and chromosomal DNA
Plasmids carry no vital genes necessary for the cell
Plasmids are always double stranded
Plasmids, thousands of bps, chromosomal DNA, millions of base pairs.
-Fold difference: 1000 fold
Topology of DNA molecules
Linear, dsDNA molecules migrate during electrophoresis at a rate inversely proportional to the log of their molecular weight
Small plasmids are predominantly isolated as the monomeric supercoiled form, whereas large plasmids are often extensively nicked and are rarely isolated in the supercoiled form.
It is generally true that the more compact molecules will migrate faster through the agarose gel.
-A supercoiled plasmid migrates faster through the gel than nicked DNA, which migrates faster than linear DNA.
-Sizing of the plasmid can therefore only be performed after it has been converted into a linear molecule, done by digestion with a restriction endonuclease with a single recognition site within the plasmid sequence.
Lyse cells with lysozyme to digest peptidoglycan in the cell wall. NaOH is used to denature DNA into single strands.
Centrifuge to remove cell debris
Neutralise the lysate. Potassium acetate neutralises the solution, allowing DNA strands to renature, precipitating the SDS from the solution.
Wash the DNA pellet with ethanol and then resuspend the plasmid DNA.
Use of EDTA (in DNA extraction)
EDTA binds divalent cations in the lipid bilayer, weakening the cell envelope. After cell lysis, EDTA also limits DNA degradation by binding Mg2+, a cofactor for bacterial nucleases.
Use of lysozyme (in DNA extraction)
To digest the peptidoglycan in the cell wall
Use of NaOH(in DNA extraction)
The NaOH denatures both the chromosomal and plasmid DNA into single strands, the two stands of intact plasmid DNA remain intertwined.
Use of SDS (in DNA extraction)
SDS is a detergent that dissolves the lipid contents of the cell membrane and cellular proteins.
SDS = sodium dodecyl sulphate
Dissolves lipids and proteins
Use of acetate (in DNA extraction)
Potassium acetate neutralises the solution so the DNA can renature. The long strands cannot rehybridise perfectly (forming a partially hybridised tangle). It precipitates the SDS (with its lipids and proteins) from the solution.
The SDS/lipid/protein precipitate traps the tangled chromosomal DNA forming a pellet after centrifugation. Shorter plasmid DNA strands are able to rehybridise correctly, remaining in solution.
Use of ethanol (in DNA extraction)
Ethanol precipitates nucleic acids and a wash helps remove salts and any remaining SDS which can interfere with a restriction digest.
Use of agarose (in electrophoresis)
Provides a matrix for DNA to travel through.
Use of midori green (in electrophoresis)
Midori green replaces ethidium bromide. It intercalates into DNA and is visible under UV light.
Lactose and β-galactosidase
Converted to allolactose, which binds to lac repressor.
Is also broken down to glucose and galactose by β-galactosidase.
Lactose shouldn't be as effective, as it is degraded in the cell whereas IPTG can be reused.
IPTG and β-galactosidase
Binds to the lac repressor and is not broken down by β-galactosidase.
ONPG and β-galactosidase
ONPG does not bind to lac repressor but is degraded by β-galactosidase, to form ONP and galactose. A colour change occurs from colourless to bright yellow.
Blocking protein synthesis in the lac operon
The induction is dependent on protein synthesis. If we block protein synthesis by adding chloramphenicol, there is no more induction.