MATERIALS AND METHODS 



The observations reported here come from natural phytoplankton assem- 

 blages collected and fixed under field conditions, natural assemblages 

 brought into the laboratory and subjected to experimental nutrient and heavy 

 metal additions, and populations isolated from the lakes and maintained in 

 the laboratory. 



Culture Conditions 



Natural assemblages used for experiments were returned to the laboratory 

 within 5 hours of collection in 20-£ prerinsed plastic containers. Contain- 

 ers were placed in an insulated, light-tight box for transport to avoid 

 temperature and light shock. In the laboratory experimental material was 

 maintained in a culture chamber at the temperature of collection (+ 1.0°C), 

 and 200 y Ein m"2 sec-^ of illumination on an alternating 16-hr day, 8-hr 

 night cycle. 



Cultured material was grown in FM medium (Lin and Schelske 1978) at 15°C 

 at the same illumination and daylength conditions used for natural assem- 

 blages. 



Light Microscopy 



All observations reported were made with a Leitz Ortholux microscope 

 with irmiersion objectives furnishing numerical aperature of at least 1.30. 

 Cells were stained for polyphosphates by the method of Ebel et al^. (1958) 

 and were observed and photographed either in temporary aqueous mounts or in 

 permanent mounts embedded in Epon prepared by the same method used for elec- 

 tron microscopy. Photographs were taken with a Leitz Orthomat photo appara- 

 tus. 



Electron Microscopy 



Material was fixed with 3% (vol. /vol.) biological grade glutaraldehyde 

 in 0.05 M cacodylate buffer (pH 7.2) for one hour at 4°C and post-fixed in 

 1% OSO4 for 1 hour. Cells were dehydrated in a graded ethanol -propylene 

 oxide series and embedded in Epon (Luft 1961). 



Thin sections were cut with a diamond knife, collected on 300 mesh grids 

 and stained with uranyl acetate (Stempak and Ward 1964). Sections were exa- 

 mined on a Zeiss EM 9S-2 electron microscope. Microscope magnification 

 calibrations were made by use of a grating replica. 



X-Ray Analysis 



Sections for X-ray analysis approximately 60 nm thick were cut with a 

 diamond knife and collected on 75X300 mesh titanium grids. Sections were 

 examined at 100 KV in STEM mode in a JEM lOOC electron microscope equipped 

 with a KEVEX series 7000 energy dispersive X-ray analysis system. The 

 specimen was tilted 30° toward the detector. Specimen to detector distance 

 was 18 mm. Spot analysis of inclusions was made with a spot size of 50 A. 



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