General Growth and Maintenance of Streptomyces sp.
General growth and maintenance of Streptomyces species
Streptomyces are multi-nuclear filamentous organisms that undergo several different stages of growth. This section will detail some of the practices that will help maintain healthy stocks of Streptomyces strains in the lab.
Streaking for single colonies
It may seem like a simple thing, but it is important when acquiring a strain for the first time to streak for single colonies before preparing your own spore stocks. The reason for this is that Streptomyces can be quite heterogeneous. Some of the colonies on a streakout plate may display abnormal morphology, such as developmental defects or pigment overproduction. It is important to pick colonies which are consistent with the population majority. This is especially important following any mutagenesis protocol, even though it may be tempting to assume that it is a mutant because it displays a phenotype.
##Pictures of Streptomyces plate with aberrant colonies will be included shortly##
Streptomyces life cycle
The time taken to complete a full life cycle varies between Streptomyces strains but is generally between 3 and 10 days. Following inoculation of agar media with Streptomyces spores, small shiny colonies will appear. This is vegetative mycelia which grows into the media following germ tube formation after spore germination. The next stage of development sees aerial hyphae emerge. For most Streptomyces strains, this phase is characterised by a white fluffy appearance. The final stage of development sees a transition from aerial hyphae to highly hydrophobic spore chains, often characterised by the production of a spore pigment.
##Pictures of Streptomyces development will be included shortly##
Growth of Streptomyces in liquid culture
Most Streptomyces grow at 30oC; on solid and in liquid culture. Cultivating liquid cultures of Streptomyces with consistency can be problemlatic however due to the tendancy of many strains to form clumps of mycelium which rapidly fall out of suspension, making it difficult to quantify. There are exceptions to this, Streptomyces venezuelae is able to grow more consistently in liquid culture than most. Even with Streptomyces venezuelae, it is recommended to grow Streptomyces in liquid culture with sterile springs or glass beads, in order to break up the mycelium. Liquid cultures should always be grown in a shaking incubator, usually between 200-250 RPM.
##Pictures of Streptomyces cultures in spring flasks will be included shortly##
Preparing Spore Stocks
Long term storage of filamentous Actinobacteria at -80oC requires the aseptic harvest of spores from a confluent lawn of the bacteria on an agar plate. For this you will need:
- Sterile cotton buds
- Autoclaved syringes with a wad of cotton wool at the end
- A confluent lawn of your strain to store
- Sterile 40% glycerol
- Sterile distilled water
- Add 5 mL dH2O over your lawn
- Displace the spores using a cotton bud, suspending them in the water
- Transfer the spore suspension into the cotton stuffed syringe and use this to filter the suspension into a universal tube. This clears hyphal fragments displaced by the cotton buds
- Pellet the spores by centrifuging at 4000g for 10 minutes and aspirate as much water as possible from the pellet
- Resuspend the spores in 1-2 mL 40% glycerol and store at -80o or -20oC