For years 1992 -1994

2 ml of gluteraldehyde-preserved lake water was stained with 200 ul 100ug/ml
4'6 diamidino-2-phenylindole (DAPI), final concentration 10 ug/ml, in a 15 ml
Millipore filtration apparatus. After 5 minutes, the sample was vacuum filtered
through a 0.22 um pore size black Nuclepore 25mm filter. The filter was mounted
on a microscope slide using Cargille B immersion oil. Ten fields were counted
using a Zeiss epifluoresencce microscope.

Cell count= (x/field)(2.04 x 10^4 field/ml) = #/ml

For years 1995-2000

After transport to the laboratory at Woods Hole, MA, 2 ml of glutaraldehyde-preserved lake water from each depth was mixed with 2 ml 0.2 um filtered phosphate buffered saline to reduce humic interference and 0.1% Triton X-100 or Nonidet P-40 to improve cell permeability and stored overnight at 4*C. The sample was stained with 200 ul 100ug/ml 4'6 diamidino-2-phenylindole (DAPI), final concentration 10 ug/ml. After 5 minutes, the sample was vacuum filtered through a 0.22 um pore size black Nuclepore 25mm filter in a 15 ml Millipore filtration apparatus. The filter was mounted on a microscope slide using Cargille A immersion oil. Ten fields were counted using a Zeiss epifluoresencce microscope. Reference Citations: Hobbie, J.E., T.L. Corliss and B.J. Peterson, Seasonal patterns of bacterial abundance in anArctic lake. 1983. Arctic and Alpine Research 15:252-259.Porter, K.G. and Y.S.Feig The use of DAPI for identifying and counting aquatic microflora.1980. Limnology and Oceanography 25:943-948