26 J. A. Bentley 



1. Oscillatoria spp. (Cyanophyceae) from Dr. A. J. Brook, Fresh- 

 water Fisheries Laboratory, Pitlochry. A sample of approximately 150 

 g. fresh weight was collected from a small Scottish lake, where it 

 occurs as a persistent bloom and as practically a unialgal culture. 



2. Chlorella pyrenoidosa (Chlorophyceae). 118 g. fresh material 

 was received frozen from Dr. G. E. Fogg, University College, London. 

 This was an 11-day-old culture and was extracted immediately. 



3. Ochromonas malhamensis (Chrysophyceae). Two samples, con- 

 sisting of 3.3 and 2.1 g., respectively, of freeze-dried cells, were re- 

 ceived from Dr. J. E. Ford, National Institute for Research in Dairy- 

 ing, Reading. 



EXPERIMENTAL TECHNIQUES 



Extraction 



1. Oscillatoria spp. The inaterial was frozen ( — 30° C), then 

 thawed, acidified to pH 5.0, and extracted with ether. The dried 

 ether extract was partitioned between 95 per cent methanol and pe- 

 troleum ether (40° to 60° C), and the petrol fraction rejected. The 

 methanol fraction was separated into acidic and neutral components. 



2. Chlorella pyrenoidosa and Ochromonas malhamensis. The ma- 

 terial was extracted at pH 4.0 by stirring with 70 per cent aqueous 

 ethanol. After neutralization, the alcohol was distilled in vacuo and 

 the re-acidified residue extracted with petroleum ether (40° to 60° C.) 

 and then ether. The ether extract was separated into acid and neutral 

 fractions. The aqueous residues were saponified with A'' NaOH (15 

 min. at 15 lb. pressure), extracted with ether and the ether extract 

 separated into acid and neutral fractions. 



Chromatography 



Extracts were usually purified by preliminary paper chromatogra- 

 phy using water as eluant; the pigments remained on the starting 

 line, which was rejected. The remainder of the paper was flushed 

 with ethanol and the recovered material chromatographed usually in 

 isopropanol-ammonia-water (10:1:1) or water. Chromatograms were 

 examined under filtered ultraviolet light (2537A) and used either for 

 color tests [usually Ehrlich, Salkowski and nitrous-nitric acid reagents, 

 using the techniques of Jepson (6)] or for bioassay. Chromatograms 

 to be bioassayed were cut into ten equal portions and the portions 

 eluted with water for testing. 



Assay Technique 



The Avena straight-growth method was used (2), with some modi- 

 fications which give greater sensitivity. It is possible to use 10 sec- 



