Back to the basics: Streptomyces soil enrichments

Time flies when you are getting your PhD. Time also flies when you promise your brand new blog that you will brilliantly expound on lofty themes of evolution and ecology by diving into a massive body of literature (centuries worth?!?!?) in the coming weeks. Sigh. Rookie mistake. As per suggestion of my friend and colleague @chuck_pr (you should follow him and check out his blog), I’ve  instead decided to change focus and share some insider tips about my intimate encounters with Streptomyces over the last five years.

Let’s go back to the very beginning. So you want to isolate Streptomyces? You’re in luck because they are easy to come by and relatively easy to culture. For my research in the Buckley lab, we are interested in patterns of biogeography driven by dispersal limitations, so we aimed to minimize environmental variables by sampling predominately neutral-slightly acidic grassland soils. However, Streptomyces are ubiquitous in soil habitats (forests, beaches, etc.) and are also found in aquatic systems. While any enrichment culture-based strategy is unable to capture the true picture of standing diversity, we found our method recovers an appreciable amount of Streptomyces (known) taxonomic diversity. Here’s a rundown of our protocol:

  1. Air dry soil for 2-3 days. This will eliminate some non-spore forming microbes. Streptomyces spores are desiccant resistant but are not endospores, so don’t heat shock.
  2. Dilute a small amount of air dried soil with sterile PBS (e.g. 50 mg soil in 5 ml PBS) in a conical tube and shake it vigorously for 2-3 minutes to suspend the spores.
  3. Plate out 50-100 µl of the spore suspension. Depending on the sample you can expect anywhere from 10 to more than 100 Streptomyces colonies to form, so scale up accordingly.
  4. We use arginine-glycerol-salt (AGS) agar media (pH8.7). It’s a classic for selective isolation of aerobic Actinomycetes like Streptomyces (El-Nakeeb and Lechevalier, 1963). 
  5. Fungal contamination and overgrowth can be a problem. Anti-fungals cycloheximide (300 mg/L) and Rose Bengal (30 mg/L) are helpful. Rose Bengal makes your plates an awesome hot pink color.

    Streptomyces_soilplate
    Soil enrichment on AGS media with Rose Bengal, representative Streptomyces colonies circles in bright green.
  6. Incubate for 1-2 weeks at room temperature. I like to do this in a drawer or tupperware with some wet paper towels to keep things from drying out.
  7. Streptomyces are easy to spot for isolation. They are the sporulated colonies, generally small and circular and come in a range of colors from white to grey to yellow to green. They often produce pigments and can grow above the agar in various morphologies.
  8. Pick colonies of interest with a sterile toothpick and transfer onto new AGS plates (no need for anti-fungals at this point). I skip streaking for isolated colonies and instead do a “wedge” streak to make wheel plates (see photo).

    Wheel plate of Streptomyces isolates from soil.
    Wheel plate of Streptomyces isolates from soil.
  9. You may need to streak for purity a few times. It’s common for pure cultures to exhibit some heterogeneity in growth patterns, so don’t let this alarm you.
  10. Plates keep for months at 4˚C, but for longer storage you will want to make glycerol spore stocks for your -80˚C (stayed tuned for that post).

That’s all for now!

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