CHAPTER 3 - EMBRYONIC AND LARVAL CULTURE
(Source: W. Driever and C. Boggs)
Preparation of paramecia culture medium:
The classical hay infusions are often unreliable (and tend to stink!) and some people have mixed results with mung beans or wheat. The following is a modification of an axenic method for growing paramecia (Schönefeld et al. J. Protozool. 33:222).
Schönefeld's Medium for growing paramecia:
Component; Concentration; Source
Powdered Skim Milk; 8.5 g/l; Any grocery store
Ribonucleic Acid; 1.0 g/l; Sigma R-6625
MgSO4; 0.5 g/l; Sigma M-2643
Phosphatidylcholine; 250 mg/l; Sigma P-5638
Stock Solution I; 5 ml/l
Stock Solution II; 2.5 ml/l
1. Prepare the solution of primary components. Make sure the dry ingredients are well mixed before adding them with stirring to the water or the ribonucleic acid and the phosphatidylcholine will lump together and not dissolve. This solution can be heated slightly to help dissolve small lumps. Then add Stock Solutions I and II.
Paramecia Medium Stock Solution I:
Component; g/l in dH2O; Source
Calcium pantothenate; 1; Sigma P-2250
Nicotinamide; 1; Sigma N-3376
Riboflavin; 1; Sigma R-9881
Pyridoxamine HCL; 1.16; Sigma P-9158
Folic Acid; 0.5; Sigma F-7876
Thiamine HCL; 3; Sigma T-4625
Biotin; 0.00125; Sigma B-4639
(make 100x stock) Store at -20°C; the riboflavin is dispersed through-out the solution and will settle to the bottom. Shake well before using
Paramecia Medium Stock Solution II
Component; g/l in ETOH; Source
Stigmasterol; 2; Sigma S-6126
Lipoic Acid (Dl-6,8-thioctic acid); 0.02; Sigma T-1395
Palmitic Acid; 3; Sigma P-5917
Stearic Acid; 2; Sigma S-4751
Oleic Acid; 0.4; Sigma 0-4379
Linoleic Acid; 0.2; Sigma L-1376
Linolenic Acid; 0.06; Sigma L-2376
(Note: Blow nitrogen gas over the solution and store at -20°C)
2. Adjust the pH of the medium to 7.2 at room temperature.
3. Autoclave for 15 minutes in 500 ml aliquots (Some coagulation may occur, which is ok, but if too much of the milk precipitates, the paramecia will not grow).
4. Test whether the medium is sterile and store in a refrigerator for up to several months.
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