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Didier Stainier Didier_Stainier.BIOCHEM at QUICKMAIL.UCSF.EDU
Mon Jul 10 11:32:54 EST 1995

Culturing Tetrahymena as an Alternative Baby Food to Paramecia
Marina Gerson and Didier Stainier
Department of Biochemistry and Biophysics, UCSF School of Medicine, San
Francisco, CA 94143-0554

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
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)	 0.30 g		Ciba-Geigy

1. 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
	material in the media will not affect the culture.)
4. Store at -20#161#C.

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,
	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)
	transfer to ~5 ml 1X PPYS.  Store at room temperature loosely capped	for

Growth for Harvest
1. Inoculate 0.5-1 ml stock into 50 ml.
2. Grow at 30#161#C 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.

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,
20#161#C at 
	3000 rpm clinical centrifuge
      - For large cultures, spin cells in 500 ml tubes, 10 minutes, 20#161#C
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

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.

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