Preparation and Isolation of Streptomyces venezuelae Spores from a Liquid Culture

Abstract

Source: Schlimpert, S., et al. Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device. J. Vis. Exp. (2016)

This video demonstrates the cultivation and isolation of spores from Streptomyces venezuelae. Spores, dormant cells that help bacteria survive stress, are produced during nutrient limitation and collected through centrifugation. The method ensures a pure, cold-stored spore suspension for downstream applications.

Protocol

1. Isolation of Fresh Streptomyces venezuelae Spores and Preparation of Spent Culture Medium

1. Inoculate 30 ml Maltose-Yeast Extract-Malt Extract (MYM) medium, supplemented with 60 µl R2 trace element solution (TE), with 10 µl spores of strain SV60 [attBϕBT1::pSS209 (PdivIVA-divIVA-mcherry PftsZ-ftsZ-ypet)]. For consistent growth and sporulation of the cells, use a baffled flask or a flask that contains a spring to allow for sufficient aeration.
2. Culture cells for 35-40 hr at 30 °C and 250 rpm.
3. Examine the cells by liquid-mounted phase-contrast microscopy. At this point, mycelial fragments and spores will be visible.
4. Centrifuge 1 ml of cells in a table-top centrifuge at 400 x g for 1 min to pellet the mycelium and larger cell fragments.
5. Transfer approximately 300 µl supernatant containing a suspension of spores to a new 1.5 ml tube and keep on ice. Keep the remaining culture medium
6. Dilute spores 1:20 in MYM-TE (final concentration: 0.5-5 x 10⁷ spores/ml) and keep on ice until needed.
   NOTE: Although the exact conditions that trigger sporulation in Streptomyces are not understood, using spent MYM-TE medium, including any unknown extracellular signals from a sporulating culture, stimulates sporulation.
7. Filter-sterilize 10 ml of the remaining culture medium to obtain spent MYM-TE that is free of spores and mycelial fragments. Use a sterile 0.22 µm syringe filter and transfer spent MYM-TE to a sterile 15 ml tube.
8. Keep the prepared spent MYM-TE for a few days at 4 °C if additional experiments using similar growth conditions are to be conducted.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Maltose-Yeast Exctract-Malt Extract (MYM)4 g Maltose4 g Yeast extract10 g Malt extractadd 1 L H2O using 50 % tap water and 50 % reverse osmosis water and supplement with 200 ml of R2 trace element solution per 100 ml after autoclaving			Sporulation medium used to culture Streptomyces venezuelae SV60
R2 Trace element solution (TE)8 mg ZnCl240 mg FeCl3-6H2O2 mg CuCl2-2H2O2 mg MnCl2-4H2O2 mg Na2B4O7-10H2O2 mg (NH4)6Mo7O24-4H2Oadd 200 ml H2OAutoclave and store at 4 °C			Add 0.002 volumes to MYM
PBS (phosphate buffered saline)	Sigma	P4417-100TAB	Used to refill inlet wells of unused lanes in B04A plates in order to prepare plate for short-term storage.
0.22 µm syringe filters	Satorius stedim	16532-K	Preparation of spent MYM-TE