2.0 Definition
Microzooplankton are defined sensu lato, following Dussart (1963), as phagotrophic organisms that are <200 mm in length. For the sake of operational convenience, the microzooplankton include the pico- and nanozooplankton (0.2-2 and 2-20 mm respectively) of Sieburth et al., (1978) although the latter are treated separately in section 7.

Microzooplankton biomass is defined as the quantity of microzooplankton organic carbon per unit volume of sea-water. The units of this are mgC liter ^-1 .

3.0 Principle
Microzooplankton biomass is determined from marine samples collected and freshly fixed at sea. For some procedures, chemical treatment and slide mounting may also be required in the field. Fixed samples are either counted at sea or analysed later in the laboratory by microscopy. Microscopic analysis involves counting and sizing of microzooplankton. Geometrical shapes are assigned to each microzooplankton taxon and organism volumes calculated. These are converted to organism biomass through appropriate volume to organic carbon ratios. Biomass of the microzooplankton community is the sum of biomass of individual organisms divided by the original water volume. The range of sizes of the microzooplankton (ca 2- 200 mm) requires two different
procedures for the quantification of microzooplankton biomass. The larger microzooplankton are quantified using settlement while the smaller cells are concentrated onto filters.

4.0 Apparatus
Research grade inverted/fluorescence microscope(s) and settlement chambers are essential for this research. An image-analysis system and an Apstein net are desirable but not essential. All other apparatus such as computers and spreadsheet software is assumed to be standard to a well-found oceanographic research laboratory.

5.0 Reagents
5.1 Lugol’s iodine. Acid Lugol’s is superior for preserving ciliates but it dissolves calcified  material. Separate samples should be preserved with buffered formaldehyde  where calcified microzooplankton are important.

5.2 Strontium sulphate. Used for preservation of Acantharians.

5.3 Glutaraldehyde: Use 25% Grade II (Sigma). Glutaraldehyde should be kept frozen  until preparation for sample preservation.

5.4 Proflavin

5.5 DAPI

5.6 Buffered formaldehyde: 37% formaldehyde solution saturated with sodium tetraborate  or hexamine.

5.7 Note that fixatives and preservatives are poisonous and some are probably carcinogenic.  Adequate care should be taken at all times.

6.0 Sampling
Vertical profile samples should be taken through the surface mixed layer by CTD/rosette  or Niskin bottle. Onboard ship, samples must be treated carefully as many protozoa are  delicate. The optimal approach is for samples to be siphoned into containers to which  fixative/preservatives have been added. Samples should be fixed as quickly as possible.  Drainage through small diameter valves in the bottom of the Niskin bottles my damage  some organisms.

7.0 Procedures
Two complementary techniques are required for the quantification of microzooplankton  biomass. The larger (ca 20 - 200 mm) organisms such as many ciliates and dinoflagellates  are quantified by settlement microscopy (as given in 7.1 below). The smaller (ca 2-20 mm)  organisms such as flagellates are enumerated by epifluorescence microscopic analysis of  stained samples held on microscope slides (as given in 7.2 below). These should be  processed immediately or stored frozen until analysis. Frozen slides should be stored once  only and analyzed, not thawed and refrozen.

Fluorescence microscopes should have filter sets for i) UV excitation and blue emission,  and ii) blue excitation and green and red emission. Analysis should be carried out with  either x63 or x100 objectives. Random fields or transects of filters should be examined,  and cells counted and sized either visually or by image analysis (Verity & Sieracki, 1993).  Exposure of cells to excitation light should be minimized.
7.1 Quantification of microzooplankton (ca 20-200 mm in size) abundance and biomass  by settlement microscopy. Take between 250 ml and 2L seawater depending on  microzooplankton concentrations, from a Niskin bottle, fix in 1-10% acid Lugol's  iodine. Add strontium sulphate solution to make 2 mg/l final soncentration. Store  samples in the dark. Take sub-sample of 50 to 100 ml and concentrate by sedimentation  for 24 hours. Identify, count and measure all microzooplankton using an  inverted microscope. Cells can be sized either by calibrated ocular micrometer or by  image analysis. This allows an estimate of cell volume to be made for the subsequent  calculation of carbon content.

7.2 Determination of pico- and nano-flagellates (ca 2 - 20 mm in size) by epifluorescence  microscopy. Take 50 ml (or more if concentrations are low) from a Niskin bottle, fix  in 0.3% final concentration fresh glutaraldehyde (previously stored chilled or fro-zen),  stain with 5 mg/ml DAPI for five minutes. Counterstain with proflavin which  allows the cell outline to be determined, also at a final concentration of 5 mg/ml.
Concentrate sample on a 0.8 mm black polycarbonate filter, using a backing filter to  enhance even distribution of cells. Mount filter onto a glass slide with a small drop of  immersion oil between the filter and cover-slip. Process slide immediately or freeze  until subsequent analysis.  Recent work (Stoecker et al., 1987) has shown that many protozoan microzooplankton  can be plastidic and may therefore be photosynthetic. This functional diversity  may be important and if so, the following procedure should be used to differentiate  plastidic from non-plastidic cells and autotrophic and mixotrophic from het-erotrophic
dinoflagellates.

7.3 Differentiation of plastidic and non-plastidic cells. Fix 250ml sea-water in 2% hex-amine  buffered formaldehyde as above. Store samples at 4°C in the dark until they can be enumerated by autofluorescence microscopy. Note that this technique is good  for ciliates and dinoflagellates.

7.4 Sampling microzooplankton using water bottles will produce a statistically inadequate  record of rare organisms. If quantitative information on rare microzooplankton  is required the following procedure should be used.

7.5 Enumeration of rarer microzooplankton. Gently filter 20 liters from water bottle  through a fine mesh (e.g. 20-30 mm) to a final volume of 200 ml. Fix for subsequent  settlement microscopy for sarcodines, tintinnids and metazoa. Alternatively for a  qualitative assessment of rarer microzooplankton species, tow an Apstein net fitted  with a 20 mm mesh vertically through the surface mixed layer. Samples collected can  be observed live and fixed, for later identification.

8.0 Calculation and expression of results
An example of the complete computation for procedures 7.1.1 and 7.1.2 above is shown  below:

8.1 Assign number to each microzooplankton organism to be counted, starting from 1  and work sequentially upwards.

8.2 Identify microzooplankton organism to appropriate level of taxonomic resolution.

8.3 Determine dimensions including length of organism (mm) from microscopic measurements and /or image analysis.

8.4 Calculate volume of organism using appropriate geometric formula. Ciliate sp a is an  ellipsoid volume for which the appropriate volume (in mm³ ) is (8/3p) * area² /length  (image analysis) or (1/6) p length*breadth*depth (measurement by eye).

8.5 Calculate organism carbon content (pgC) using appropriate volume to carbon conversion factor. In this case, for ciliates, this is 0.19 pg C mm -3 (Putt & Stoecker, 1989) and for dinoflagellates 0.14 pg C mm -3 (Lessard, 1991). Note that conversion factors can vary depending on type of fixative and concentration.

8.6 Calculate organism biomass concentration (pg C ml^-1 ) by dividing carbon content by  volume of sample settled (mls). In this case, 50 mls were settled.

8.7 Convert concentration to mgC l ^-1 , multiply by 10³ .

8.8 Sum biomass for each taxonomic group (e.g. Ciliate sp A) to obtain total biomass of that taxon and then sum all taxa to get microzooplankton biomass in sample.

8.9 Calculations on the standing stock per unit sea surface may be made by integrating  microzooplankton biomass with depth.

9.0 Quality control and assessment
There is no standard for this measurement and the accuracy is unknown.

As many cells as is practically possible should be counted; this is likely to be 50-200 cells  of each of the common taxonomic groups. If possible subsamples should be taken for a  few of the water-bottles to check sample replication.

10.0 Notes
10.1 The iodine present in Lugol's samples is volatile and photosensitive. The concentrations  may therefore decrease with time. Samples should ideally be stored in colored  glass bottles in the dark and inspected yearly. Readdition of Lugol's solution may be  required. The initial concentration of Lugol’s used has been found to vary from 1 to  10% depending on the scientist. There is no evidence to date, as to which concentration  is preferable, although some scientists believe that cell loss occurs at 1%.

10.2 A general discussion of biomass conversion factors among various planktonic  trophic and size groups is given in Verity et al., (1992).

10.3 It should be remembered that many microzooplankton organisms are fragile; water  samples should be treated with care prior to fixation and are best fixed as soon as  possible after collection.

11.0 Intercomparison
No intercomparisons have been carried out in JGOFS, although this is a recommendation  for the future.

12.0 References & JGOFS papers published using these techniques
Burkill, P.H., Edwards, E.S., John, A.W.G, & Sleigh, M.A. 1993. Microzooplankton and their herbivorous activity in the north-east Atlantic Ocean. Deep-Sea Research II. 40: 479-494.
Dussart, B.M. 1963. Les differentes categories de plancton. Hydrobiologia 26: 72-74.
Harrison, W.G., Head, E.J.H., Horne, E.P.W., Irwin B., Li, W.K.W, Longhurst, A.R., Paranjape, M.A. & Platt, T. 1993. The Western North Atlantic Bloom Experiment. Deep-Sea Research II 40:279-305.
Lessard, E.J. 1991. The trophic role of heterotrophic dinoflagellates in diverse marine environments. Marine Microbial Food-Webs 5: 49-58.
Putt, M. & Stoecker, D.K. 1989. An experimentally determined carbon:volume ratio for marine oligotrichous ciliates from estuarine and coastal waters. Limnology & Oceanography 34: 1097-1103.
Sieburth, J.McN., Smetacek, V. and Lenz, J. 1978. Pelagic ecosystem structure: Heterotrophic compartments of the plankton and their relationships to planktonic size fractions. Limnology and Oceanography 23: 1256-1263.
Sieracki, M.E., Verity, P.G.and Stoecker, D.K. 1993. Planktonic community response to sequential silicate and nitrate depletion during the 1989 North Atlantic spring bloom. Deep-Sea Research II 40: 213-226.
Stoecker, D.K., Michaels, A.E. & Davies, L.H. 1987. Large proportion of marine planktonic ciliates found to contain functional chloroplasts. Nature 326: 79-792.
Verity, P.G., and Siercacki, M.E. 1993. Use of color-image analysis and epifluorescence microscopy to measure planktonic biomass. In: Handbook of Methods in Aquatic Microbial Ecology (Kemp, P.F., Sherr, B.F. & Cole, J.J. Eds) Lewis, Boca Raton.
Verity, P.G., Robertson, C.Y., Tronzo, C.R., Andrews, M.G., Nelson, J.R. and Sieracki. 1992. Relationships between cell volume and the carbon and nitrogen content of marine photosynthetic nanoplankton. Limnology and Oceanography 37: 1434-1446.
Verity, P.G., Stoecker, D.S., Sieracki, M.E., Burkill, P.H., Edwards E.S. & Tronzo, C.R. 1993a. Abundance, biomass and distribution of heterotrophic dinoflagellates during the North Atlantic Spring Bloom. Deep-Sea Research II. 40: 227-244. 1993
Verity, P.G., Stoecker, D.K., Sieracki, M.E. and Nelson, J.R. 1993b. Grazing, growth and mortality of microzooplankton during the 1989 North Atlantic spring bloom at 47°N, 18°W. Deep-Sea Research II 40: 1793-1814.