were leached with either: (1) 0.5 N acetic acid/sodium acetate 

 ( pH 5) buffer, (2) 0.2 M oxalic acid/ammonium oxalate ( pH 3) 

 buffer, or (3) hot 8 M HC1 (Froelich, Bender, and Heath 1977). 

 The three classes of P released by these procedures are 

 operationally defined as CaC03-associated plus loosely bound P 

 (acetate extract), hydrogenous metal-associated P (oxalate 

 extract) and total P (HC1 extract). The latter treatment would 

 also include mineral-P. After a reaction period of 4 hours, the 

 tubes were centrifuged (1500 x g), diluted 1000-fold in 

 distilled water and measured using the SRP procedures given 

 previously. Only a small residue (1-5% of the original material) 

 was observed in the HC1 extracted sample. This colorless fine- 

 grained particulate matter is believed to be siliceous material. 



The remaining two preparations contained approximately 

 5-10 percent and 90-100 percent of the original mass of the 

 pulverized material for the oxalate and acetate preparations, 

 respectively. Samples of the dried, pulverized bacterial mat 

 were also prepared for analysis by electron microprobe and for 

 x-ray diffraction. 



Microbiological Measurements and Metabolic Rate Determinations 



Bright-field, epif luorescence and electron microscopy 



A subsample ( 100 ml ) of each vent water and bacterial mat 

 sample collected at Pele's Vent was immediately preserved in 

 electron microscopy grade glutaraldehyde ( final concentration, 

 2%) and stored at 4°C for subsequent laboratory processing. For 

 bright-field viewing, wet mounts of the bacterial mats were 

 prepared and examined at 100, 400, and lOOOx magnification with 

 a Zeiss Standard 18 compound microscope equipped with planapo 

 objectives. Photographs were taken with a 35 mm Olympus camera 

 and Kodak high speed ektachrome film. For epif luorescence 

 microscopy, a 5 ml portion of each preserved sample was stained 

 with acridine orange (0.05% final concentration), filtered onto 

 an irgalan-black stained 0.2 pm Nuclepore membrane filter and 

 viewed at lOOOx magnification with a Zeiss Standard 18 

 epif luorescence microscope equipped with a 100 W mercury lamp, 

 neofluar objectives and an A-0 filter set (Hobbie, Daley, and 

 Jasper 1977). Quantitative cell counts were determined for 

 selected Pele's Vent water samples. 



For transmission electron microscopy (TEM) of the bacterial 

 mat samples, a portion of the glutaraldehyde-f ixed particulate 

 material was first collected by high speed centrifugation 

 (10,000 x g), embedded in noble agar (2% wt/vol ) , washed in a 

 cacodylate buffered filtered seawater solution (0.1 M, pH 7.7), 

 post-fixed in an OSO4 (1%) cacodylate-seawater solution, 

 desalted in graded seawater/distilled water solutions (to 0% 

 seawater), dehydrated in graded distilled water/ethanol 

 solutions (to 100% ethanol ) , embedded in an epoxy resin and 



195 



