2.8. Worksheets

Worksheet 2.1.: Isolation of pure algal strains by the agar plating technique
Worksheet 2.2.: Determination of cell concentrations using haematocytometer according to Fuchs-Rosenthal and Burker.
Worksheet 2.3.: Cellular dry weight estimation of micro-algae.

Worksheet 2.1.: Isolation of pure algal strains by the agar plating technique

The following agar plating technique can be used to isolate algal strains from raw seawater and for the maintenance of existing algal strains. · prepare a 0.9% agar medium by weighing out 9 g of agar powder and placing it into a 2 l conical flask to which 1 l of sea water is added · heat the flask on a Bunsen flame and let it boil twice, i.e. heat until it boils, let it cool and let it boil a second time. · add nutrients (see Tables 2.3 & 2.4) before autoclaving · cover the flask with aluminium foil · autoclave at 125°C for 30 minutes at 1 atm · sterilise Petridishes by incubation for 30 minutes at 150°C · agar plates are prepared aseptically by pouring the warm autoclaved agar into the sterile Petridishes near a Bunsen flame or in a laminar flow, cover up the Petridishes and leave them to cool for about 2 h · streak the algal sample onto the agar surface with a sterile platinum loop (previously heated to red-hot and cooled) · place the Petri dishes upside-down on an illuminated glass rack · depending on the density of the inoculum, cell colonies can be observed to grow on the surface after 5 - 21 days · select the best colonies and transfer them with a sterile platinum loop into a test tube filled with 5-10 ml of culture medium and shake it regularly during incubation on an illuminated glass rack. · when a colour change is observed in the tube, check under the microscope the isolated algal strain

Worksheet 2.2.: Determination of cell concentrations using haematocytometer according to Fuchs-Rosenthal and Burker.

A variety of counting chambers (normally used for blood cell counts) can be used for cell counts (algae, yeast) (see Table 2.9.). Two types are most common: Fuchs-Rosenthal and Bürker (Fig. 2.17.). Both types have 2 rafters allowing for 2 subsamples to be examined. They have the following characteristics:
	Fuchs - Rosenthal	Bürker
Depth (in mm)	0.200	0.100
Surface of smallest square (in mm2)	0.0625	0.0400
minimal cell concentration (in cells.ml-1)	103	106
· dilute sample if needed (use formalin 4% to fixate moving algal cells) clean slide and cover-glass with Kleenex-paper · press cover glass onto the slide until the Newton diffraction rings appear · fill both slides of the counting chamber under the cover-glass with a single smooth flow of suspension using a Pasteur pipet (avoid air bubbles) · count cells in, respectively, 80 (Fuchs-Rosenthal) and 20 (Bürker) small squares under a microscope (objective 40 x). Count cells which touch the upper and left border but not those which touch the lower and right borders (see schematic diagram) · the subsample on the other side of the chamber is counted in the same way · calculation for Fuchs-Rosenthal numbers of cells.ml-1 = (n1 + n2)/(2×80) ×80×103×d = (n1 + n2)/2×103×d · calculation for Bürker numbers of cells.ml-1 = (n1 + n2)/(2×20) ×250×103×d = (n1 + n2)/160×106×d with: n1 = number of cells counted in upper rafter n2 = number of cells counted in lower rafter d = dilution factor · For greater accurary make 3 duplicate counts (3 separate dilutions each counted in two rafters).

Figure 2.17. Counting directions (follow arrow) for Fuchs-Rosenthal chamber (upper diagram) and for Bürker (down left corner). For both, count the cells in the square and those which touch the top and left border (·). Do not count the ones touching the right and lower border (o) (see down right corner).

Worksheet 2.3.: Cellular dry weight estimation of micro-algae.

Dry weight of algal cells can be determined by filtering and drying algae from aliquots of culture of known concentration · determine accurately (3 duplicate counts, see Worksheet 2.2.) the concentration of the algal culture to be sampled for dry weight analysis · filter an exact volume of culture on pretared glass-fiber filters (1 µm pore size) using a Büchner setup connected to a vacuum pump (triplicate). Wash the filter with a solution of ammonium formate (0.5 M) to remove salts · follow the same procedure with control filters on which an equal volume of 0.22- µm filtered seawater is filtered (triplicate). The strength of the applied vacuum will determine the amount of salts retained on the control filters. · dry the filters at 100°C for 4 h to volatilize the ammonium formate · weigh on an analytical balance · calculate the dry weight per algal cell according to the formula: DW (g.cell-1) = (DWA - DWC).(N.V)-1 with DWA = average dry weight retained on algal filter (g) DWC = average dry weight retained on control filter (g) N = algal concentration (cells.ml-1) V = volume of algal culture and filtered seawater filtered on algal and control filter, respectively (ml) In order to improve the correction for salt residues and the variation among samples, cellular dry weight can be determined from regression analysis of DW retained on the filter versus number of algal cells filtered (Fig. 2.18).

Figure 2.18. Dry weight analysis of algae by means of linear regression of dry weight retained on the filter versus number of algae cells filtered. Each data set represents the dry weight determination for algae obtained from one culture of, respectively Isochrysis sp. (T-iso) and Chaetoceros neogracile (Chg). Linear regression equators are: T-iso: y = 1.83 × + 107.7 (r² = 0.99); Chg: y = 2.61 × +130.1 (r² = 0.95)