Maintenance of Trichoplax adherens culture

There are two components to maintaining a Trichoplax colony. First, establishing the algae foodstock, and second, setting up and feeding the Trichoplax dishes. Algae must be prepared 7-10 days before it is needed in order to grow a properly dense culture.

1.    Algae

Stock culture is a Cryptomonas sp. (stock number LB 2423) that is bought from the University of Texas:

UTEX

The Culture Collection of Algae

The University of Texas at Austin

1 University Station A6700

Austin, TX 78712-0183

USA

512-471-4109  (phone)

512-471-0354  (fax)

Algae need to be cultured at room temperature with a light/dark cycle (12:12). If faster growth is required they can be kept under permanent light and supplemented with CO₂.  Growth medium (F/2) contains 150 ul of component A and B in 500 ml of 0.02um filtered, autoclaved seawater. Use different pipette tips for each solution so that they don’t get cross contaminated.

In parallel to the working stock described above, small, sterile quantities of algae stock solution are maintained in cloning tubes that can hold approximately 20 ml of solution. These need to be inoculated with sterile algae solution and lightly capped so that air exchange can take place. These are the clean stocks used to prepare the feeding culture. A new culture is started weekly (or when needed) by adding, under sterile conditions,  1 ml of living cells from the old algae culture to the new tube of growth media. It is generally good practice to make two new cultures at a time in case one crashes. These cultures are used to prepare the Trichoplax feeding solution.

The algae culture for feeding is maintained in a sterile 1 liter bottle. Prepared growth medium (500 ml) and the contents of one of the small cloning tubes are combined in this bottle. A 2ml transfer pipet is connected to an air hose and used to bubble air through the algae culture. The mouth of the 1 liter bottle is covered with parafilm. This culture is prepared a week before it is needed in order to grow algae to the proper density. Once algae reach the right density, remove pipette and cap with orange bottle cap. Place the bottle into the fridge for 1-2 weeks.

2.    Trichoplax

A.  Water.

The Trichoplax colony is maintained in artificial seawater using a commercial Red Sea salt mix or instant ocean.  The target salt concentration is 35 ppt, but a range of 30-35 ppt should be ok. It is important that soap or bleach never touches the glassware used for culturing. Cleaning involves washing with tap water and rinsing 2 times with deionized water. A special brush is kept in the wetlab to use on glassware; it is not used with bleach or soap!

B.  New Trichoplax cultures

The target is to make about 2 new cultures per month. If the animals are growing well more cultures can be setup. Colonies are maintained in clean, autoclaved Pyrex 90 mm x 50 mm evaporating dishes (P/N 3140). Dedicated Eppendorf pipetters (200 ul and 1000 ul) with filtered tips are used to measure and transfer small solutions.

Fill a clean, autoclaved Pyrex dish 80% with filtered seawater and 2 ml of algae feeding solution.  Then add 10-15 Trichoplax. Note that the algae and Trichoplax are both added the same day. A plastic Petri dish is used to cover the Pyrex dish and the date of inoculation and each water change written on it. Water for the colony should be changed about every two weeks. Do no seal the dishes!