Streptomyces:Protocols/Spore Prep: Difference between revisions
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Slightly thicker plates are needed when inoculating | Slightly thicker plates are needed when inoculating Streptomyces as they take longer to grow than other general laboratory bacteria, ~4-5 days. This is partially due to it having a more complex life cycle (spore → vegetative hyphae → aerial hyphae → spores) and its preferred incubation temperature is 28-30°C. If the stock is being made from a single colony, ensure it is well mixed and carefully streaked. Smoothly poured plates without bubbles and confluent growth will aid the harvesting process. | ||
Whilst harvesting, be careful not to dig into the agar. Use light pressure to begin, gradually increasing; removing the spores without breaking the surface. Agar pieces mean less spores will be collected and pipetting is difficult. When done properly the majority of the surface of the agar should be visible. To rub the spores from the edges of the plate, use a circular motion. | Whilst harvesting, be careful not to dig into the agar. Use light pressure to begin, gradually increasing; removing the spores without breaking the surface. Agar pieces mean less spores will be collected and pipetting is difficult. When done properly the majority of the surface of the agar should be visible. To rub the spores from the edges of the plate, use a circular motion. | ||
The cotton wool in the syringe helps to filter out any agar pieces and longer hyphal fragments. Filtered tips are needed to help prevent cross-contamination, as an aerosol of spores can enter the pipette chamber. | The cotton wool in the syringe helps to filter out any agar pieces and longer hyphal fragments. Filtered tips are needed to help prevent cross-contamination, as an aerosol of spores can enter the pipette chamber. | ||
Centrifuging at a lower- | Centrifuging at a lower-than-normal speed (~5000g) will keep the spores intact ready for further experimentation; it may also help to prevent hyphae from pelleting. | ||
Spore stocks are a precious material. Not only do they take time to prepare, but if the strain has been modified from the wild type a lot of effort has been put into achieving this. Spore stocks should be kept at least -20°C and when needed, thawed on ice; helping to prevent germination. Always use the stocks in a sterile environment, i.e. in a laminar flow hood or by using good technique, under a Bunsen flame. Decontamination of a spore stock is a laborious and unnecessary process. | Spore stocks are a precious material. Not only do they take time to prepare, but if the strain has been modified from the wild type a lot of effort has been put into achieving this. Spore stocks should be kept at least -20°C and when needed, thawed on ice; helping to prevent germination. Always use the stocks in a sterile environment, i.e. in a laminar flow hood or by using good technique, under a Bunsen flame. Decontamination of a spore stock is a laborious and unnecessary process. |
Latest revision as of 06:01, 14 August 2007
Protocols - Spore Prep
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Spore Prep - Inoculating & HarvestingDescription
Notes
Spore stocks are a precious material. Not only do they take time to prepare, but if the strain has been modified from the wild type a lot of effort has been put into achieving this. Spore stocks should be kept at least -20°C and when needed, thawed on ice; helping to prevent germination. Always use the stocks in a sterile environment, i.e. in a laminar flow hood or by using good technique, under a Bunsen flame. Decontamination of a spore stock is a laborious and unnecessary process.
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