doi: 10.1101/pdb.prot5148Cold Spring Harb Protoc;  Nicole King, Susan L. Young, Monika Abedin, Martin Carr and Barry S.C. Leadbeater 

CulturesMonosiga brevicollisStarting and Maintaining

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Starting and Maintaining Monosiga brevicollis Cultures

Nicole King,1,2,3 Susan L. Young,1 Monika Abedin,1 Martin Carr,4 andBarry S.C. Leadbeater5,6

1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94704, USA2Department of Integrative Biology, University of California, Berkeley, Berkeley, CA 94704, USA3Canadian Institute for Advanced Research, Toronto, Ontario M5G 1Z8, Canada4Department of Biology, University of York, York YO10 5YW, United Kingdom5School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

INTRODUCTION

Choanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present inmarine and freshwater environments as well as in hydrated soils. Because they are the closest livingrelatives of the metazoa, the study of their cell biology and genomes promises to provide new insightsinto metazoan ancestry and origins. Most, if not all, choanoflagellates are heterotrophs that prey onbacteria. Thus, all choanoflagellate media provide nutrition for bacteria, which are in turn consumedby the choanoflagellates. If a culture is axenic or has low bacterial content, it can be supplementedwith a specific bacterial strain that has been grown separately. On the other hand, if a culture alreadycontains a well-established flora of bacteria, it can be cocultured with the choanoflagellate isolate. Ineither case, it is common to enrich the medium with an organic extract, such as liver extract, cerealgrass infusion, or a mixture of proteose peptone and yeast extract. This protocol describes how to startcultures of the marine choanoflagellate species,Monosiga brevicollis, from frozen stocks. These culturescan then be maintained and expanded in preparation for DNA or RNA isolation or cell biologicalassays.

RELATED INFORMATION

For a more extensive discussion on the background, husbandry, and potential uses of this species, seeThe Choanoflagellates: Heterotrophic Nanoflagellates and Sister Group of the Metazoa (Kinget al. 2009a). For alternative media (e.g., for freshwater choanoflagellate species), see Isolation ofSingle Choanoflagellate Cells from Field Samples and Establishment of Clonal Cultures (Kinget al. 2009b). Preservation of stocks is described in Long-Term Frozen Storage of ChoanoflagellateCultures (King et al. 2009c). Additional information on culturing choanoflagellates can be foundthrough the American Type Culture Collection (http://www.atcc.org/) and Culture Collection of Algaeand Protozoa (http://www.ccap.ac.uk/) websites.

© 2009 Cold Spring Harbor Laboratory Press 1 Vol. 4, Issue 2, February 2009

6Corresponding author (b.s.c.leadbeater@bham.ac.uk)This article is also available in Emerging Model Organisms: A LaboratoryManual, Vol. 1. CSHL Press, Cold Spring Harbor, NY, USA, 2009.Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5148 www.cshprotocols.org

Protocol

MATERIALS

CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with <!>, andrecipes for reagents marked with <R>.

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METHOD

To avoid contamination, perform all steps in a sterile tissue culture hood if possible.

Starting Cultures from Frozen Stock

1. Place one grain of sterile rice into a standard tissue culture dish. Using a sterile 15-mL pipette, add14 mL of choanoflagellate growth medium.

2. Thaw a vial of frozen M. brevicollis cells at room temperature or by warming in the hands.

3. When thawed, immediately pipette 1 mL of cells into the dish.

4. Incubate the culture in a 25°C incubator or at room temperature.Initially, the culture might look overgrown with bacteria, but within 2-3 d, the choanoflagellates should out-compete their bacterial food.

Subculturing and Maintaining Cultures

5. Grow the culture of M. brevicollis to maximum density (i.e., 106-107 cells/mL).

6. Using a cell lifter, scrape the bottom of the dish to resuspend any adherent cells.

7. Using a 1-mL pipette with a filtered tip, gently pipette the medium up and down to disrupt clumpsof cells.

8. Place one grain of sterile rice into a standard tissue culture dish. Add 14 mL of choanoflagellategrowth medium.

9. Add 1 mL of the cells (from Step 7) to the dish.

10. Incubate the culture in a 25°C incubator or at room temperature.

11. Monitor the growth and viability of cells on an inverted microscope at 200X-400X.

12. Every 2-3 d, split the culture (as in Steps 7-9) to fresh choanoflagellate growth medium.See Troubleshooting.

Reagents

<R>Choanoflagellate growth mediumM. brevicollis stock, frozen

Stocks are available from the American Type Culture Collection (ATCC 50154; http://www.atcc.org/).Alternatively, stocks preserved as described in Long-Term Frozen Storage of Choanoflagellate Cultures (Kinget al. 2009c) can be used.

Rice grains, sterileSterilize rice by autoclaving in a capped test tube.

Equipment

Cell lifterDish, tissue culture, polystyrene, 100- × 20-mmHood, tissue culture, sterileIncubator preset to 25°C (optional; see Steps 4 and 10)Microscope, inverted, equipped with 200X-400X magnificationPipette, glass or disposable, sterile, 15-mLPipette tips, 1-mL, equipped with filtersPipettor, 1-mL

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TROUBLESHOOTING

Problem: The culture contains an overgrowth of co-cultured bacteria.[Step 12]Solution: Bacterial growth is a fact of life in choanoflagellate cultures. If bacteria begin to dominate theculture and interfere with choanoflagellate proliferation, start over with a freshly thawed stock ofcells. M. brevicollis has an ~6-h doubling time during log phase and should reach a maximaldensity of 106-107 cells/mL.

REFERENCES

King, N., Young, S.L., Abedin, M., Carr, M., and Leadbeater, B.S.C.2009a. The choanoflagellates: Heterotrophic nanoflagellates andsister group of the metazoa. Cold Spring Harb. Protoc. (this issue).doi: 10.1101/pdb.emo116.

King, N., Young, S.L., Abedin, M., Carr, M., and Leadbeater, B.S.C.2009b. Isolation of single choanoflagellate cells from field sam-

ples and establishment of clonal cultures. Cold Spring Harb. Protoc.(this issue). doi: 10.1101/pdb.prot5147.

King, N., Young, S.L., Abedin, M., Carr, M., and Leadbeater, B.S.C.2009c. Long-term frozen storage of choanoflagellate cultures.Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5149.

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