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(Source: W. Driever and C. Boggs from Zebrafish Book 5th Edition)

Culture of Paramecia

1.   Grow paramecia cultures under "cell culture" conditions. Make passages in a sterile tissue culture hood. 
2.   Use 500 ml or 1000 ml media bottles (e.g., with orange caps) and culture at 28°C in the dark, for example in a cabinet in your fish room. 
3.   Dilute the starter culture (a clear suspension of paramecia) 1:1 with fresh (milky) medium and incubate with the cap loosened one half turn to provide air.  After 2‑3 days, the culture will clear and you should be able to observe clouds of paramecia.
4.   Expand the culture by diluting it 1:1 with fresh medium.  If you add twice the volume of fresh medium, it takes about 3 days to clear.  Use clear cultures for harvesting paramecia or store for 3 to 5 days at about 20°C.

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Use Dryls solution (1 volume clear culture to 5 volumes Dryls) to prepare a backup stock in case the original culture becomes contaminated.  Paramecia live about 10 days in this solution in the dark at 18‑20°C.  Prepare the solution from autoclaved components and add the CaCl2 last or it will precipitate.

Long-term Paramecium Storage Medium Storage Medium

Component

Stock

Amount (ml)

NaH2PO4

0.01M 

10

NaH2PO4

0.01M 

10

Sodium citrate

0.1M

20

CaCl2

0.1M

15

dH20

 

945



Harvesting paramecia from clear cultures

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1.   Choose bottles of cleared parame­cia cultures for harvest.  Approximate­ly 40‑50 ml of culture will feed up to 100 larval fish per day. 
2.   Place the funnel in a flask and filter the paramecia stock through the folded cotton handkerchief to remove precipi­tates of the Schönefeld's medi­um.
3.   Filter the liquid through fluted cir­cles until the flow decreases to a slow drip.
4.   Rinse the paramecia off the filter paper into a flask using embryo medi­um. Repeat until all the stock is filtered.
5.   Refilter the paramecia through fluted circles rinsing with embryo medium until most of the yellow color has disap­peared.
6.   Collect the paramecia in a clean bottle making sure that the yellow color is gone.  Rinse again if neces­sary.  Some paramecia pass through the fluted fil­ters.  If they are needed for feeding, they can be collected by filtering through the What­man No. 1 circular filter.  However, filtering through a Whatman 1 is very slow. 
7.   Adjust the volume with embryo medium to equal ½ of the original culture volume.  Store in darkness at 20°C until needed for feeding.