A method is described by which the contribution of Non-Living Particulate Organic Matter (NLPOM) to Total Organic Matter can be determined. The dye Trypan Blue stains only dead organic matter. The surface of the stained particles is used as an indirect measure of the NLPOMcarbon content. Applying this method, it is shown that the bivariate linear regression of POM on chlorophyll a may produce serious errors in the calculated NLPOM contribution when carbon which is neither triptonnor chlorophyll-related is present in appreciable quantities. INTRODUCTION easily obtained when enough samples containing the same type of tripton are available. In the euphotic zone, Non-Living Particulate Organic Matter (henceforth NLPOM) is usually determined by calculating the difference between TPOM MATERIAL AND METHODS (Total Particulate Organic Matter) and Living Particulate Organic Matter. The latter is calculated by multiIn our procedure we use the vital stain Trypan Blue. plying the ATP-content, chlorophyll a content, or cell This dye, which is widely used in animal cytology, has count by a conversion factor (Hagmeier 1961, Straththe interesting property of staining proteins in dead rnann 1967, Gassmann & Gillbricht 1982). This method cells or particles only. For our purpose we have modhas several disadvantages, all related to the determiified the original recipe published by Phillips (1973) as nation of the 'Living Carbon' (Banse 1977). The carbon follows. To obtain 100 m1 solution, 0.4 g Trypan Blue, to chlorophyll ratio is not a constant factor in living 0.81 g NaCl and 0.6 g KH,PO, are dissolved in distilled cells; neither is the cell volume to cell carbon ratio. water. After boiling and cooling, the pH is adjusted to There are individual (Laws & Wong 1978), species7.2 using NaOH. To stain the NLPOM (or rather its specific (Chan 1978), diurnal (Laws & Wong 1978), and proteins), 10 m1 of this solution is added to a sample of possibly geographical and seasonal variations. Since about 100 ml. After 5 min the mixture is filtered on a 47 the ATP-content of a cell is influenced by the phymm Whatman GF/C glassfiber filter which is aftersiological condition of the cell, the ATP to carbon ratio wards rinsed with filtered seawater. The filters may is variable as well (Mel'nikov 1976). All these probthen be preserved in a freezer in a Millipore Petrislide lems and drawbacks make the calculated Living Car(47 mm). bon, and therefore also the NLPOM data, rather impreFor determination of total NLPOM surface area, the cise. To compound the problem, the assumption that outline of each particle is drawn using a light microNLPOM equals TPOM minus chlorophyll-related C is scope with a camera lucida. Calculation of the surface not always justified. areas is performed using an Apple I1 graphics tablet, For this reason, we here propose a new method coupled to an Apple I1 microcomputer. The program involving the vital stain Trypan Blue. This dye makes which we used not only calculates the surface area of it possible to distinguish between living and dead the particles, but also the equivalent circular diameter particulate organic matter. Though the method does and, furthermore, produces histograms of surface and not by itself yield a quantitative measure of NLPOM in diameter distributions. terms of carbon, an empirical conversion factor is For determination of chlorophyll a content, replicate Q Inter-Research/Printed in F. R. Germany 108 Mar. Ecol. Prog Ser. 25: 107-109, 1985 samples were filtered on Whatman GF/C glassfiber filters. Chlorophyll was extracted with 90% acetone. After centrifugation, the extract was measured spectrophotometrically. Calculations were made using the Strickland & Parsons (1968) formula. Total Particulate Organic Carbon (TPOC) was measured with a Coulomat 701 Carbon Analyser. The total NLPOM surface area is used as an indirect measure of the NLPOM-carbon content. This measure, together with chlorophyll a and TPOC, may be fed into a multiple linear regression program with TPOC as the dependent variable. This produces a regression formula of the form: TPOC = constant + A (chlorophyll a) + B (NLPOM) (1) where B (NLPOId) = NLPOM carbon of interest. Using this formula, one also obtains an empirical estimate of the specific carbon content of the phytoplankton, viz. the A-factor. When there is no zooplankton present, the constant should theoretically equal zero. As will be seen in the results, however, this is not always true.