The Zebrafish Science Monitor Vol 3(5)
CULTURING Tetrahymena AS AN ALTERNATIVE BABY FOOD TO PARAMECIABy M. Gerson and D. Stainier Department of Biochemistry and Biophysics, UCSF School of Medicine, San Francisco, CA 94143-0554, USA
Encouraged by the early report of Speksnijder and Bijmolt, we took advantage of local expertise (i.e. Liz Blackburn's lab) to optimize Tetrahymena culture conditions. Tetrahymena are pear-shaped, ciliate protozoa, about 30-100 micrometers in size. Although they are not parasites, Tetrahymena can be associated with sick or weakened fish, found on skin lesions where they feed upon bacteria and fungi.
As a baby food, Tetrahymena offer the advantage of being easier to culture and maintain than paramecia since the media is much less complicated to prepare, and cultures rarely crash. Tetrahymena can be used as an exclusive food source for babies until they start eating brine, or can be used in conjunction with paramecia or rotifers. The protocol we use is adapted from one given to us by Cathy Strahl (Blackburn lab) and has provided us with a reliable supply of Tetrahymena for the past 6 months.
Media Stock Solution (1 liter of 10X PPYS)
Component Amount Source proteose peptone 200.0 g Fisher #DF0120-17-6 yeast extract 20.0 g Fisher #DF0127-17-9 sequestrene (NaFe form) 0.3 g Ciba-Geigy1. Combine proteose peptone, yeast extract, and sequestrene in a large flask. Adjust to 1000 ml with warm ddH20 and stir until dissolved.
2. Spin at 7K gsA rotor for 20 min.
3. Pour off supernatant into 200 ml aliquots. (Note: a small amount of solid material in the media will not affect the culture.)
4. Store at -20C.
Before use:
1. Thaw one aliquot of media and adjust to 1000 ml with ddH20. Aliquot into smaller volumes if desired. Autoclave 20 min.
2. Add P/S/F (10 ml for a 1000 ml culture; P/S/F not required for small cultures). P/S/F = penicillin/streptomycin/fungizone (Gibco/BRL #15240-013, 100 ml bottles). This is 100x for use in PPYS. Can be used at 5x to cure a mold infection in Tetrahymena cultures.
Note: medium: 2X for growth +P/S/F 1X for stock tube (strain storage) (no P/S/F)
Tetrahymena Culture
Stock tubes
Tetrahymena can be obtained through Ward's Biology and Lab Supplies, Connecticut Valley Biological, and other biological supply companies. The strain we use is Tetrahymena Thermophila.
1. Maintain Tetrahymena in 10 ml stock tubes of 1X PPYS (no P/S/F).
2. About 2x monthly take 0.2-0.5 ml from top of stock tube (healthiest cells) and transfer to ~5 ml 1X PPYS. Store at room temperature loosely capped for aeration.
Growth for Harvest
1. Inoculate 0.5-1 ml stock into 50 ml.
2. Grow at 30C O/N shaking at about 100 rpm (if no shaker is available, aerate/bubble at room temperature) for about 24 h.
3. To inoculate a 1 liter culture, prepare a 50 ml culture ahead of time (about 15-25 ml is enough to inoculate a 1 liter culture). Incubate as for 50 ml culture.
Harvest
1. Prepare egg H2O (0.3 g Instant Ocean or similar salts/ 1000 ml ddH2O)
2. For small cultures, spin cells in 50 ml conical tubes, 5-10 minutes, 20C at 3000 rpm clinical centrifuge. For large cultures, spin cells in 500 ml tubes, 10 minutes, 20C at 5000 rpm gsA rotor.
3. Resuspend pellet in egg H2O and spin again
4. Resuspend pellet in original volume in egg H2O. Note that cells swim after spinning, so supernatant must be poured off immediately.
Storage and Use
We dilute 1 liter of Tetrahymena into 2 liters of egg H2O since they seem to keep better when less concentrated. Storage at room temperature also prolongs their life. (Live Tetrahymena swim in the water column, while the dead ones fall to the bottom). We generally add 10 ml of Tetrahymena per "100 ml" of babies.
Zebrafish Science Monitor Vol 3(5)
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