We keep the algae in two different kinds of cultures: agar tubes, in which the algae grows relatively slow and for a long time; and glass petri dishes, which are used to feed the worms.
Please download and read the general MacLab guidelines thoroughly and make sure that they are followed when attempting to culture Macrostomum and Nitzschia.
| culture / process |
method | used for |
| agar tubes (stock cultures) |
For 20 tubes: Prepare 20 test glass tubes (we use 21 ml, with nickel-steel caps) in an autoclavable rack. Prepare 200 ml of agar (some will be lost). Pour 8-10 ml of agar in each tube, while still warm. Cover the tubes with the metal caps, and autoclave for 20 minutes at 120 degrees Celcius. IMPORTANT: Let autoclave pressure decrease by itself, but remove tubes from the autoclave while still warm (the agar must still be liquid), place the rack with the tubes on a stable surface so that the tubes are at an angle of about 12 degrees (the agar will harden leaving a longer surface for the algae to grow). Don't move until cool. Choose two algae-stock tubes with nicely grown algae (at least three-weeks old). Work under sterile conditions, either in a sterile chamber, or using a burner. With a blunt glass pipette that has been sterilised, inoculate the fresh tubes with the stock algae as follows: First, move the blunt pipette gently through the stock algae, taking care of not breaking the agar. Now spread the algae onto the new agar, taking care of not breaking the agar. Cover the tube immeadiately with the cap, label, and store at 20° C under good light conditions. We use Osram LUMILUX BIOLUX T8 light bulbs, and change them once per year. Allow to grow about two-three weeks before use. Check regularly and discard immediately if any contamination is observed. Usually in good condition until about 12 weeks |
petri-dishes stock cultures |
| petri dishes |
For two boxes (12 100mm glass dishes each): Optional: Dry-sterilize the glass petri-dishes (closed!) for at least 30 minutes at 160°C. Clean the plastic box (we use a polyestiren transparent box, 31x22x5 cm) with alcohol. Filter about 600 ml of f/2. (20 ml per dish plus loss) Chose two algae-stock tubes with nicely grown algae (between three and nine weeks old). Work under sterile conditions, either in a sterile chamber, or using a burner. Prepare the stock solution: add 12.1 mL of f/2 into a small beaker. Pipette three ml of this f/2 into the algae-stock tube, and gently detach the algae from the agar using a blunt glass pipette. Resuspend (e.g. with the help of a pipette) and pour into the solution. Repeat with next tube. Add 16-20 mL of f/2 to each petri dish. Add 0.5 ml of the stock solution to each dish, cover, and carefully place in an A4 box. Label and store at 20° C under good light conditions. We use Osram LUMILUX BIOLUX T8 light bulbs, and change them once per year. Allow to grow two-three weeks before use. Check regularly and discard immediately if any contamination is observed. Best used within 3 to 5 weeks If you use small glass petri dishes (18 dishes per box), the procedure is the same, the quantities vary slightly. |
worm cultures algae solutions |
| algae solutions |
In order to feed in a standardized fashion, we prepare an algae solution and distribute it among the worms. The amount of algae petri dishes to use depends on the amount of worms to feed, and the density of the algae in the dishes. 1 large dish for 100 worms should suffice for a week. Always check how much algae your cultures yield! Example with 6 dishes: Choose 6 algae-petri-dishes with nicely grown algae. Remove the lid, gently shake the medium and discard (if most algae are not attached, see procedure below!). Pour some freshly filtered f/2 and gently detach the algae with the algae scraper. Discard the medium from the next dish (again shaking it gently) and pour the f/2 with algae from the first dish into the second. Scrape the alge and go onto the next dish. Every time you will get a much denser solution, which you can dilute to the desired concentration. Sometimes the algae is not adhered to the petri-dishes. In this case, scrape the algae directly and go on as described above without discarding the medium. After collecting the algae from all the dishes, pour into a small erlenmeyer and allow the algae to sink (2-3 hours). Decant as much of the medium as possible (a pipette helps) and refill with fresh f/2. To control the amount of cells in the solution, count the cells in a counting chamber. A good protocol is described here. |
iso-female lines experiments feeding |
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last uptaded: 29.02.2008