PDF《algae》

10 t~g chlorophyll a l-t). The algal inoculum was then washed by adding gradually 30-100 ml of culture medium. Clamps C and D were closed when only a few ml remained in the syringe, and the vacuum pump and air supply were switched off. The contents of the syringe were transferred aseptically to a sterile test-tube. ISOLATION OF ALGAL CLONES Algal clones were isolated using the pipetting and washing techniques described by Droop (1969). Drops of filtered culture were placed on glass slides or plates containing agar culture medium with a micro-pipette. Single filaments or colonies of algae were then washed under a

171 172 S. I. HEANEY AND G. H. M. JAWORSKI [- I i D[I~ (/ FIG. 1. Filtration apparatus used to purify algal cultures. For explanation of letters see text. dissecting microscope through several drops of sterile liquid culture medium. The algal isolates were transferred finally to Erlenmyer flasks containing liquid culture medium or to clean agar plates and allowed to grow. TESTS FOR PURITY Algal cultures were examined for the presence of contaminating organisms before and after purification procedures and using old dying cultures. The cultures were examined by phase contrast and fluorescence microscopy. Samples of the cultures were treated with the fluoro- chromes acridine orange and euchrysine using the method of Jones (1974). Purity tests were also performed by inoculating the cultures into a variety of media including those contained in Fogg (1942), Collins &

Willoughby (1962) and MeCurdy &

Hodgson (1974). The cultures were incubated for at least

4 weeks at 8,

20 and 37°C. RESULTS PURIFICATION OF Aphanizomenon flos-aquae f. gracile (Lemm.) Elenk., and ASTERIONELLAFORMOSA Hass. This method has been used to isolate several pure clones of the blue-green alga Aphanizomenonflos-aquae from Lough Neagh, Ireland, and the diatom A techniquefor purifyingmicro-algae

173 Asterionella formosa from Rydal Water, a lake in the Windermere drainage basin. All the tests for purity failed to demonstrate the presence of other organisms, even after numerous subcultures, and the cultures were considered pure. On one occasion nine filaments of Aphanizomenon from a membrane filtered culture were isolated on to agar culture medium and

7 days later these had grown into visible colonies. Four of these colonies were without any apparent contamination and after transfer to liquid culture were found to be pure. Aphanizomenon was grown in ASM-1 medium (Gorham et al., 1964), modified by the addition of

200 mg 1-1 N-2-hydroxyethylpiperazine-N'

-ethanesulphonic acid (HEPES);

the pH was adjusted to 7.55, the pKa of this buffer, using 0.1 M sodium hydroxide. Agar medium was prepared using 1-5% m/V Oxoid bacterio- logical agar. The medium for growing Asterionellawas solution

10 of Chu (1942) modified as described in Lund et al. (1975). Cultures of both algae were grown at an irradiance between 1000-2000 lx [12.5-25/~E (400-700 nm) m-2 s-1] pro- vided by daylight fluorescent lamps. Aphanizomenon was grown at 20+ I°C and Asterionella at 16+ I°C. Both algae grew well in liquid culture media with maximum exponential growth rates similar to those obtained with cultures containing bacteria. The optimal doubling time of Asterionella in pure culture was just less than

12 h, the same as reported by Lund (1949) for cultures with bacteria. The optimal doubling time of Aphanizomenon, determined from optical density readings at

436 nm, was

21 h and was not significantly different from the value found by Smith &

Foy (1974) who used a non-axenic culture of the same algal isolate. Clonal cultures of these algae are maintained at the Ferry House Laboratory of the Freshwater Biological Association with the strain designations, Asterionella formosa Hass. L226A and B, and Aphanizomenonflos-aquae f. graeile (Lemm.) Elenk. L1, L2 and L3. A pure culture of one of the Aphanizomenon clones, strain designation L1401/1, is also kept by the Culture Centre of Algae and Protozoa,