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246                                   Algae: Anatomy, Biochemistry, and Biotechnology

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                  where C is the number of cells counted, A is the area of field in mm , D is the depth of a field
                  (Sedgewick-Rafter chamber depth) in mm, and F is the number of fields counted.
                     For colonial taxa multiply the count of units by the average number of cells per unit and use the
                  resulting value as C in Equation (6.1). To adjust for sample concentration of dilution the result is
                  divided or multiplied by the appropriate factor. To obtain total cell density per milliliter, sum all
                  counts of individual taxa.
                     If cell density is low (,10 units per field) counting of long transects to cover a large proportion
                  of the chamber floor is more appropriate. Several transects with a width of a chamber field are
                  counted. The number of strips depends on the required precision and the phytoplankton density.
                  The number of cells per millimeter is calculated according to the following formula:


                                                               C   1000 mm 3
                                                           1
                                        Number of cells   mL  ¼                              (6:3)
                                                               L   D   W   S
                  where C is the number of cells counted, L is the length of strip in mm, D is the depth of a field
                  (Sedgewick-Rafter chamber depth) in mm, W is the width of strip in mm, and S is the number
                  of strips counted. To adjust for sample concentration or dilution the result is divided or multiplied
                  by the appropriate factor.
                     A “high-tech” alternative to counting algae in mixed assemblages with the Sedgewick-Rafter
                  cell is the inverted microscope method. The expensive component here is the inverted microscope,
                  whose great advantage is that settling chamber depth does not preclude the use of high magnifi-
                  cation objective lenses. In 1931, Utermo ¨hl solved the problem of concentrating and enumerating
                  algae in mixed populations when he described a one step settling and enumeration technique
                  using the inverted microscope. The procedure involved the gravitational sedimentation of preserved
                  phytoplankton into a counting chamber. This counting technique correctly assumed that phyto-
                  plankton would fall randomly to the bottom of the chamber and that counts would then be made
                  on random fields or transects. The inverted microscope counting technique has gained broad popu-
                  larity for phytoplankton enumeration. One of the advantages of this randomized counting technique
                  is the capability of calculating error estimates to verify the accuracy of the enumeration. Through
                  the years, many modified chambers have been designed and used with the inverted microscope.
                     Special and expensive “Utermo ¨hl” chambers can be purchased, but cheaper ones can be con-
                  structed from large cover slips, and plastic syringe barrels. If a long focal-length lens is available,
                  chambers may be constructed from glass slides.
                     A measured volume of preserved sample is added to the settling chamber and allowed to settle
                  for at least an hour. Time periods as long as 24–48 h are preferred, especially if small algae are
                  present in the sample (these will settle only very slowly). Upon settling, the upper portion of the
                  chamber is removed and replaced with a glass plate. The sample is then transferred to an inverted
                  microscope (condenser numerical aperture 0.70; objectives 25  and 40 ; oculars 12.5 ) with
                  phase contrast optics.
                     The sample is initially enumerated at 500  using a random fields technique. A minimum of 20
                  random fields and 200 individual cells are enumerated. Additional fields are counted until the
                  minimum count is attained. When there is a large number of cells of a particular taxon in a
                  sample, fewer than 20 random fields are enumerated with a minimum of five random fields exam-
                  ined for this taxon. Individual cells are enumerated, whether in chains, filaments, or colonies. This
                  allows for a more accurate estimate of biomass which is determined from the cell densities. Upon
                  achieving 20 random fields and 200 individual cells, a low magnification scan (25 ) of 20 random
                  fields is used to estimate the rarer, larger forms within the sample.
                     In the case of unialgal samples (unicells, small colonies, or relatively short filaments) chambers
                  such as the haemacytometer, the Thoma chamber (Figure 6.3), the Fuchs-Rosenthal or the Burker
                  chambers are effective and commonly used for estimating the densities of cultures. The
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