Page 263 - Algae Anatomy, Biochemistry, and Biotechnology
P. 263
246 Algae: Anatomy, Biochemistry, and Biotechnology
2
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