Link to this post | posted 03 Jul, 2017 15:53

joycestamm

Apologies for the newbie question. I'm hoping to try to use Streptomyces as a new host this fall, and would like to do a run-through with my summer research students. I've read through the updated versions of the instructor guide and the Streptomyces specific phage discovery guide, but haven't found the answer - how do I make a spore suspension? Also, I read the previous thread on this forum with interest. Do you have an opinion as to whether I should use the spread technique or top agar? The phage discovery guide says top agar, because a germination spore preparation is not practical for large scale use, but have you found that to be true? Thanks in advance for your help.

Link to this post | posted 03 Jul, 2017 21:50

lhughes

Hi Joyce: At UNT we currently prepare spore suspensions by the following procedure: Spore Harvest Note: Prior to use, all solutions and apparati should be sterilized by autoclaving. 1. Pipet 6-8ml dH2O to a well-sporulating plate (2-3ml for slant cultures). 2. Scrape of growth with a sterile loop to release spores. 3. Collect solution off the top of the plate using a pipet and transfer all liquid into a sterile 15 conical tube. 4. Mix vigorously on a vortex mixer for 1-2 minutes. 5. Filter by vacuum through fiberglass wool to remove mycelial fragments. 6. Centrifuge at 1800 x g for 10 minutes in a Sorvall H1000B rotor (3000 RPM using the Sorvall centrifuge in the HHMI lab) 7. Carefully pour off the supernatant (leave about .5ml or last few drops in) 8. Add 1ml dH2O and mix well. 9. Transfer the mixture to a screw-top vial containing 0.5ml of 80% (w/v) glycerol to achieve a final concentration of 20% glycerol. 10. Store in the freezer at -20 degree celsius. Steve Caruso at UMBC has a slightly different protocol (maybe he can post his as well). On your second question, we use only spread plates for our lawns (the Streptomyces Phage Discovery Guide shouldn't say top agar anywhere - unless I missed an edit somewhere) with good success. We also work from liquid cultures for the spread plates (there are other protocols out there that use spores and top agar, but most require a spore germination step that takes a lot more time). I am assuming that you are planning to use S. griseus (each species has their own quirks). Hope this helps, Lee

Link to this post | posted 05 Jul, 2017 21:47

scaruso

Joyce, I use a slightly different protocol that I found online and liked, but it's only different in the details, the idea is the same. I would use whichever works best, or easiest, for you. For mine, you will want some cotton/glass/fiberglass wool, as above, which you can put in a funnel and cover in foil for autoclaving. It makes a nice long term dispenser that you can re-cover with the foil. 1 - Spread 100 uL of a growing culture on two plates and incubate ~4-5 days at 30ºC. 2 - Assemble the filter - place a sterile 10 mL syringe in a sterile 50 mL conical tube and remove plunger, use sterile forceps to push wool into bottom of syringe. 3 - Add ~5 mL sterile ddH2O to the each confluent plate. 4 - Using sterile cotton applicators (or spreaders as Lee uses), gently rub spores off surface of plates 5 - Transfer the liquid using a filtered pipette tip to the 10 mL syringe, and push through the wool using the plunger 6 - Remove the syringe, replace cap, and spin the conical tube 5' @ ~5kg 7 - Carefully remove supernatant, then resuspend the pellet in the minimum volume of sterile 20% glycerol (~500 uL) 8 - Transfer to a 2 mL screw cap tube and store at -20ºC. I'm not sure what lab the original came from, but a longer version of the protocol can be found here: Protocols - Spore Prep Edited 05 Jul, 2017 21:49

Link to this post | posted 08 Jul, 2017 15:12
scaruso	Also, check out: Shepherd MD, Kharel MK, Bosserman MA, Rohr J. 2010. Laboratory maintenance of Streptomyces species. Curr Protoc Microbiol Chapter 10:Unit 10E.1. PMID: 20812215 PMCID: PMC2950629 doi: 10.1002/9780471729259.mc10e01s18 It also talks about regular frozen stocks of the mycelia, which work fine. But make several, as they don't freeze/thaw well it seems. Steve Edited 08 Jul, 2017 15:14

Link to this post | posted 08 Jul, 2017 20:08
lhughes	Thanks for the link, Steve.

Link to this post | posted 08 Jul, 2017 20:14
joycestamm	Thanks, Steve and Lee for your help! To check if I'm understanding you correctly - I would make the spore suspensions in lieu of 'regular' freezer stocks, correct? Edited 08 Jul, 2017 20:15

Link to this post | posted 11 Jul, 2017 17:08
scaruso	They are supposed to be more stable, though I haven't tested it myself.