Nov 13, 1991 - Chemistry, Memorial University ofNewfoundland, St. John's, Newfoundland AlB 3X7, Canada. Received ..... Philpott, R. (Anotec Separations).
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1992, P. 773-776
Vol. 58, No. 2
0099-2240/92/020773-04$02.00/0 Copyright © 1992, American Society for Microbiology
Use of Inorganic Membrane Filters (Anopore) for Epifluorescence and Scanning Electron Microscopy of Nanoplankton and Picoplanktont CYNTHIA H.
McKENZIE,9*
ROBERT HELLEUR,2
AND
DON DEIBEL'
Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland AlC 557,1 and Department of Chemistry, Memorial University of Newfoundland, St. John's, Newfoundland AlB 3X7, Canada Received 12 August 1991/Accepted 13 November 1991
Inorganic membrane filters (Anopore) were examined qualitatively by epifluorescence and scanning electron microscopy to determine their suitability for the study of nanoplankton and picoplankton. Compared with filters currently used, the Anopore filters allowed for increased resolution of the specimen with epifluorescence microscopy because of filter flatness and increased illumination caused by the large number of pores cm-2. The inorganic filters had a lower filtration rate than polycarbonate filters. For scanning electron microscopy, the metal oxide (Anopore) filters were efficient support for the plankton, with little charging of cells or background.
Clarity of image and high resolution are required to and identify the nanoplanktonic (2- to 20-,um) and picoplanktonic (0.2- to 2.0-,um) elements of the plankton. The ability to clearly define the dimensions of these organisms will become increasingly important as more attention is paid to critical biomass estimates. Clarity will be particularly important as image analysis systems become more automated. Track-etched polycarbonate filters (Nuclepore) have become the standard for epifluorescence microscopy (6). To enhance the visibility of autotrophic and heterotrophic flagellates, cyanobacteria, and bacteria, various stains are used. Acridine orange and 4',6-diamidino-2-phenylindole (DAPI) are used for bacterial counts and for heterotrophic and autotrophic ratios (3, 9). Filters are also stained with Irgalan Black to enhance the contrast of the stained bacteria against the background (6). Proflavine is used to increase visibility and identifiability, since it stains the whole cell and flagella (4). Primulin has been compared with these stains and was found to enhance visibility while not masking chlorophyll autofluorescence (2). Hewes and Holm-Hansen (5) suggest a filter-transfer-freeze method for transferring cells from filter to slide, which can then be viewed both with epifluorescence and transmitted light for species identification and accurate measurement. Inorganic membrane filters (Anopore) were significantly more effective than track-etched polycarbonate membrane filters (Nuclepore) for direct counts of bacteria stained with acridine orange because of the increased visibility of the bacteria (7). There is no published information on filtration rate or the quality of resolution by using inorganic membrane filters with epifluorescence and transmitted light for the study of nanoplankton and picoplankton. Nanoplankton samples prepared for the scanning electron microscope have been routinely filtered onto Nuclepore filters and critical-point dried (1). However, there are inherent problems with charging in the sample as well as the background, no matter how well grounded the filter or how much silver paint is applied. This problem is not only frustrating but also expensive in terms of time and man-
when attempting to find suitable specimens for photomicrographs. The objective of this study is to determine qualitatively the suitability of inorganic membrane filters (Anopore) for use on both epifluorescence and scanning electron microscopes for the study of nanoplankton and picoplankton. Anopore filters (Anodisc 25, 0.2-,um pore size) were purchased from Scientific Products and Equipment, Concord, Ontario, Canada, at a price of $46.00 Canadian for 50 filters. The price has since increased to $61.00 for 50 filters. These filters were used with natural phytoplankton samples from Logy Bay, Newfoundland, Canada, for both epifluorescence and scanning electron microscopy. For each sample, 25 ml of seawater was filtered by using a vacuum of
