Biomass Measurement I. Particle and Volumetric Concentration Date Prepared: 2/8/1995

Transcription

1 PARTNERSHIP FOR THE DELAWARE ESTUARY Science Group Biomass Measurement I. Particle and Volumetric Concentration Date Prepared: 2/8/1995 Prepared By: Danielle Kreeger Suggested Citation: Kreeger, D Biomass Measurement I. Particle and Volumetric Concentration. Partnership for the Delaware Estuary. PDE Method No pp. PDE-Method (02/1995) Page 1

2 Biomass Measurement I. Particle and Volumetric Concentration Partnership for the Delaware Estuary (PDE) Method Danielle Kreeger Description Procedures are described for counting particle concentrations using either a hemacytometer or Coulter Counter. Hemacytometers are relatively simple to use, do not need calibration and will need to be used when a Coulter Counter is unavailable (e.g. at Lacawac) or when interspecific counts are needed for mixed assemblages of particles. Coulter Counters are more rapid, accurate and precise than hemacytometers (if calibrated and used properly), and when used with a Channelyzer or Multi-sizer, they can provide much more information (e.g. volumetric concentrations, size-specific concentrations). One purpose of counting particle concentrations will be to calculate how much algae to deliver to zooplankton in feeding experiments. Since the size of algal cells can vary dramatically under different culture conditions, it is important to calculate algal rations on the basis of their volumetric concentration and not cellular concentration - cell volume has a closer relationship with cell ash-free dry weight (AFDW). We will establish volume:afdw regression equations for each algal species and culture condition. For characterizing the particle size distribution of natural seston with the Coulter Counter, 50 ml of water should be sampled and analyzed as soon as possible. If the interval between collection and analysis is more than 4 hours, samples should be kept in the dark (wrapped with foil if needed) and in a cooler with ice or ice packs. Samples should never be frozen. If the interval between collection and analysis will be more than 24 hours, samples can be fixed with Lugol s solution (request methods). Note that fixatives can alter the cell size and may even cause some cells to burst and so some fresh samples should always be analyzed and compared to splits that are fixed to account for such losses. The precision of this technique will depend on the carefulness of weighings on an analytical balance. Since only 1 mg or less of sample might need to be used, the balance should be set at 5 places (± 10 μg) and calibrated before and during (after every 50 samples) use. Weights should be measured only on desiccated samples, and it is important to strictly adhere to the drying and ashing times. Procedures 6.1 Hemacytometer Procedure Wash the hemacytometer and cover slip with soapy water (e.g. 7X), rinse three times with tap water, three times with Milli-Q water and dry with a Kimwipe; place cover slide on hemacytometer. PDE-Method (02/1995) Page 2

3 6.1.2 Mix the algal or lake sample by shaking. Use Pasteur pipet to transfer a drop of the mixed suspension to the edge of the cover slide of the hemacytometer, and capillary action will pull the drop into the counting chamber. Repeat for the other side of the hemacytometer, but take care not to overfill. Count the number of cells on 8 squares of each side (try always to count the same 8 squares). The number of cells in 16 squares x 104 equals the cell concentration per ml Repeat particle count in step three times. 6.2 Coulter Counter Procedure The Coulter Counter contains a vacuum pump attached to a narrow glass tube with a mercury manometer. When a sample is placed under the aperture nozzle, and a stopcock is opened on the instrument panel, the vacuum pump pulls the mercury out of its equilibrium position and resets the LED particle counter. After the valve is closed, the mercury gradually recedes to its equilibrium position, and while doing so, it gradually pulls approximately 750 μl of sample through a small opening on the aperture nozzle. Electronic sensors attached to the mercury manometer start and stop the counting of particles so that exactly 500 μl of sample is counted. As the water sample is drawn through the small aperture, particles disrupt the electronic field around the aperture and these disruptions are translated into counts on the LED display. Since the Coulter Counter operates on a semi-continuous flow basis, it is important to check the inflow and outflow reservoirs before and after using the instrument. The inflow and outflow reservoir should be kept between 1/4 and ¾ full Calibrate the instrument for particle size at least monthly. This is easily done with a suspension of uniform, pre-defined size particles (e.g. monolatex beads). If possible, use a particle size that is similar to the size particle being routinely counted. On the setup screen of the Channelyzer, enter the diameter of this particle under the calibration sub-menu. Run the sample and view the particle size distribution on the Channelyzer graph. Position the left and right cursor to surround the peak of the bead distribution, and press the "calibrate" button on the front of the Channelyzer. The particle diameter (x-axis) corresponding with the peak abundance (y-axis) should now equal the nominal bead size supplied by the manufacturer Dilute each sample of algae by adding 1 ml algae to 15 ml Isotone and mix by shaking. Dilute each sample of natural seston by adding 10 ml of sample to 10 ml Isotone (at least 50% electrolyte is required for freshwater samples but saline samples can be analyzed without dilution) and mix by light shaking. Repeat twice to give 3 samples. Zoom in on the appropriate Channelyzer window. If the Multi-sizer is being used, prepare the software for data capture. Press the reset button on the front of the Channelyzer. Shake each sample to mix just prior to counting. Put PDE-Method (02/1995) Page 3

4 the sample under the aperture (be sure the foil sensor is immersed) and open the upper stop-cock one quarter turn. You should see the mercury column move and the LED counter reset to zero. Close stopcock, and the counter will automatically count the sample for seconds. Count each replicate sample twice and record the particle concentration within the windowed range of the Channelyzer. Repeat a count if the two values differ by >10 %. The count, which is the concentration of cells per 0.5 ml, should be between 500 and for best accuracy - if it is not, prepare new samples for counting using dilutions that are more appropriate and correct values to account for dilution factor. In addition to the algal samples, also count the particle concentration in the water used to suspend and dilute the algae as a blank. Subtract the particle concentration of the blank from the coincidence-corrected sample concentration, multiply by 2 to obtain the sample count per ml, and correct for the dilution (e.g. multiply by X times) Volumetric Counts Using Coulter Multi-sizer Although volumetric concentrations can be estimated using only the Channelyzer (tedious calculation of geometric mean volume for each of many channels), the Multisizer is much more accurate and fast. The software does nearly all of the work. Select the "edit listing" menu and check the boxes "differential" and "cumulative volume". Then select the function "listing" to view the cumulative volume data in column form, with each value corresponding to the total volume of particles with that particle size channel (256 channels measured per count). For seston samples, we might only want the volume of particles between 2 and 53 μm. For algal culture, we should pre-select a particle size window to quantify consistently so that we do not include any bacteria or other non-algal particulate material in our estimate of volume. Therefore, for each measurement, we will only want to record the volume of particulate material within a designated range rather than the whole 256- channel range recorded by the Multi-sizer. The cumulative volume associated with each size channel can be viewed from the listing" and recorded on a separate table (next page). The cumulative volume associated with the lower and upper limits of each sample's pre-selected range should be noted, and the difference between these values is equivalent to the total volume of particulate material within that size range. This particle volume should be divided by the volume of sample analyzed (i.e. each measurement counts 0.5 ml), and corrected for any dilution factor. The resulting "volumetric concentration" (VC) has the units volume per volume, usually μm3 per ml. The VC of a sample can then be divided by the corresponding concentration of ash-free dry tissue (units = mg per ml; from PDE- Method-07 Biomass Measurement II. Dry and Ash-Free Dry Weight) to calculate the relationship between sample volume and AFDW (units = μm3 per mg). We will establish a regression equation for this volume:afdw relationship for each type of algae. PDE-Method (02/1995) Page 4

5 Appendix Cell Number in AW Total Volume in AW (μm 3 ) Sample Initials Factor Analysis Window (AW) Replicate Raw (#/ 0.5 ml) Coincidence (#/ 0.5 ml) Blank & (#/ml) Raw (μm 3 /0.5 ml) Blank & "Vol. []" (μm 3 /ml) Mean Cell Volume (μm 3 ) Estimated Concen. of AFDW (mg ml -1 ) PDE-Method (02/1995) Page 5