Cultures Flashcards
Why are bugs not isolated by enrichment techniques?
Laborious and expensive
Describe the process to getting a large scale culture
Isolate culture, test in a small scale, develop for large scale production
Describe the more focused approach of isolating cultures
Consider the product characteristics (what do I isolate and where do I look for it?)
Using ecophysiological methods of isolation and screening
Give two examples of ecophysiological methods of isolation and screening
Need an enzyme that works at moderately high pH and salinity and over a range of temps- look for microorganism in DESERT.
Need an enzyme to work at elevated temps- look in composts, hot springs and hydrothermal vents.
List ten general isolation guidelines
1) list the groups/types of microorganisms to be isolated
2) describe the ecosystem/habitat from which the samples are to be collected
3) group samples into types (eg plants and plant parts, soils, rocks etc)
4) list the environmental parameters to be considered (pH, salinity, temp, soil composition)
5) consider the constraints that large scale conditions can place on small scale conditions. Microbial numbers and species diversity are often a seasonal response to windows of opportunity that exist in nature.
6) list the available natural substrates in the ecosystem (lignin and cellulose in forest soils)
7) design the isolation techniques around the information collected in steps 1-6 (ie. diluents, substrates, natural extracts and incubation conditions)
8) evaluate the ecophysiological isolation methods by using standard methods as controls
9) modify known methods as determined by the ecological parameters of the material to be sampled.
10) use specific enrichment procedures for microbial groups that may be of interest to screen (eg. Actinomycetes, fungi)
Name the four processes involved in isolating a culture
Sampling
ecophysiological considerations and media
Isolation
Subculturing and purification
Describe some kinds of isolation media
The isolation media is based on the sample source and what needs to be isolated.
It can contain natural extracts (from plants, rocks and compost- added as growth factors). Anti fungal agents (cycloheximide and nystatin) can be incorporated for bacteria because they slow fungal growth without affecting bacterial growth.
For gram negative isolation, crystal violet or bile salts can be added.
For gram positive isolation, nalidixic acid can be added.
How does pretreatment of the maple help and give an example
It can help to increase the portion of particular groups of microorganisms.
Actinomycetes can be isolated by allowing the samples to air dry or 3-10 days (reduces the non-actinomycete population)
Describe 5 approaches to obtaining new microbial metabolites
Screening for the production of new metabolites with new isolated of microorganisms and/or new test methods
Chemical modification of known microbial molecules
Bio transformation (in which an enzyme/microorganism changes the structure of a molecule)
Protoplast fusions between species- allows for recombination of DNA from closely related strains (new/hybrid molecules can be generated).
Gene cloning- genes from a unrelated species may be introduced into a strain producing a known substance
Name 8 types of screening
Anti microbial screening (primary and secondary)
Enzyme-inhibitor screening for antibiotic discovery
Screening for anti fungal agents
Screening for pharmacologically-active agents
Antiviral screening
Screening for veterinary compounds
Screening for antitumour agents
Identification of active compounds arising from a screen
What is the process of primary screening
Take sample from the environment, obtain a pure culture, grow in different media for testing
Name two gram positive pathogenic bacteria
Staphylococcus aureus
Enterococcus faecalis
Name two gram negative bacteria for screening
Proteus vulgaris
Escherichia coli
Name an anaerobic gram negative pathogenic bacteria used for screening
Bacteroides fragilis
Which bacteria allows for elimination of known antibiotics during screening?
Bacteria resistant to different classes of antibiotics
Which bacteria allows for the identification of known antibiotic classes?
Bacteria hypersensitive to B-lactans, aminoglycosides and macrolides
Name three bugs sensitive to antitumour agents
Bacillus subtilis (rec-) Escherichia coli (rec-) Pencillium avellaneum
Why is Acholeplasma laidlawii used for screening?
Mycoplasma, sensitive to polyether antibiotics
What can be used to screen for anti fungal agents?
Chitin synthase inhibitors (chitin is unique to fungal walls)
Agents that affect the sterols in fungal membranes
Why is screening for antitumour agents hard?
The screen requires an in vivo model and human cells grow slower than bacterial cells.
Prescreens have been developed to give an indication of potential antitumour activity- microorganisms (penicillium avellaneum) or Acholeplasma ladilawii
What are the aims of strain development?
To increase the yield of product
Can eliminate undesirable products
Can adapt strains to cheaper/more desirable substrates
Can eliminate chemicaly closely-related compounds.
What does strain improvement involve?
Generation of genetic variation in the population (mutation)
Selection of individuals
Testing of individuals for desirable proerpties
Describe spontaneous and induced mutations
Spontaneous mutations are often point mutations in which a base in the DNA sequence has been changed or deleted. They happen less frequently than induced mutations.
The process of inducing mutations relies on the ability to induce DNA lesions and on the DNA repair system making errors
Describe two mutagenic agents
Radiation (UV, X-rays)
Chemical (nitrous acid, hydroxylamine, alkylating agents- most potent)
How has protoplast fusion been used in mutagenesis?
Used in actinomycetes and fungi to generate strain diversity.
Has been used to combine characteristics from divergent strains derived from the me parent strain by several rounds of mutation and selection.
Generates greater heterogeneity among the recombinants but one may have trouble successfully regenerating normal cells from the protoplasts.
Name two types of selection for mutants
Random screening- survivors are randomly selected and tested in small scale fermenters. The best 5-10 strains are then used a the parent strains for the next round of mutagenesis. Takes advantage of silent mutation which may show enhanced production at a later stage in the mutagenesis program.
Selective isolation- imposing conditions that promote the growth/early detection of mutants lea to isolation.
What are analogue resistant mutants?
Antimetabolite resistant mustants
What are auxotrophs?
These mutants lack the ability to produce an essential cellular molecule (a growth factor). Mutants with blocks in branching biosynthetic pathways can overproduce amino acids or nucleotides.
Treated cells are plated on complete media followed by a minimal medium which stops auxotroph growth because they cannot synthesise and essential growth factor
Name two reversion mutants
1) Reversion of non producing strain. By screening the non producing parent strains for activity, revertants regain the ability to produce the produce through a subsequent spontaneous mutation
2) Reversion of auxotrophs. Overproduction of a precursor model increases antibiotic production. This can be achieved by mutations that relieve feedback regulation in its biosynthetic pathway.
