5- Monitoring algal populations

Transcription

1 5- Monitoring algal populations A regular check of microalgae cultures is essential to prevent crashes and to keep high quality standards. The main parameters to be monitored are: color, density, ph and contaminant levels. As an example, a change in color to opaque grey and a ph level lower than 7.5 may indicate a high degree of bacterial contamination. A lighter color than normal may reveal insufficient nutrients or poor lighting. The algal culture growth can be also followed daily by counting the number of cells per ml with a haemocytometer (see below) Algal cell count in cultures is useful for a number of other reasons as it is possible: to determine the growth curve for each species of algae under local conditions; to devise criteria for quick identification of possible troubles (eg.; presence of foam, sedimentation pattern, changes in colour, etc.); to determine optimal utilization time, i.e. the age at which the algal population reaches the peak of the log-phase; to adjust environmental conditions to maximize production; to control possible contaminants and try countermeasures. Cell counting using improved Neubauer Haemacytometer Haemacytometer As the name suggests these counting chambers have been developed for counting blood cells but they can be used to calculate the cell density of an algal culture providing the cells are relatively small (~ 5-50µm and either single cells or short chains. Larger cells or long chains of cells are more appropriately counted using a Sedgewick Rafter cell or settling chamber. A haemacytometer is used for cell densities up to 500 million cells/ml. The size of these chambers can vary with manufacturer but we use a Neubauer brand. This device consists of a thick rectangular slide with two counting areas. With its special cover slip in place, each area forms a chamber 0.1 mm deep. The total area of each chamber is 9 mm 2. Each chamber is divided into 9 large squares, each 1mm x 1mm (1 mm 2 ). The four corner squares are subdivided into 16 medium squares, whereas the central one is subdivided into 25 medium squares, 0.2 x 0.2 mm, each with an area of 0.04 mm 2. The medium squares are separated by triple lines, the middle one of which is the boundary, and each square is further divided into 16 small squares. 19

2 1 mm Method 1. Algal Sample Non-motile cells which do not need fixing can be counted as soon as the sample is collected. However, if there will be a delay between sample collection and counting, or if the cells are motile then the sample will need to be preserved. The most common fixative used for marine microalgae is Lugol s Solution (alternatively, use formalin 4% to fixate moving algal cells). The recipe for the acidic form is given at the end of this sheet. 20

3 For cultures add 1 drop of Lugols solution to 1 ml sample, for field samples 10 drops per 200 ml of sample or until the colour of weak tea. Overuse of Lugol s will cause some delicate flagellate species to overstain, lose flagella or blow up entirely. Note: Sample dilution or concentration: The haemacytometer can be used where cell densities are in the range 5 x cells / ml. It is more likely that cultures will be less rather than more dense than this range but occasionally very dense cultures may need to be counted. It is both inefficient and very difficult to accurately count these cultures without first diluting the sample. Therefore dilute with a known volume of culture media and then fix. Alternatively a Lugols Dilution Solution (LDS) may be used where 100 ml of 0.2µm filtered seawater is prestained with concentrated Lugols until it is a weak tea colour. Then a known volume of culture can be added to a known volume of the LDS. Using LDS also means that all cells are exposed to the optimum concentration of Lugol s whereas adding concentrated Lugol s could destroy some cell types when it mixes into the sample. If the culture is dilute, concentrate by centrifuging or settling in a flat bottom measuring cylinder (allow 1 hour of sinking for each 10mm of cylinder height; therefore overnight is a practical solution). For either method once concentrated, remove and discard up to 90% of the clear supernatant (upper portion of the liquid) without disturbing the settled biomass. Homogenize the remaining sample and count, bearing in mind the need to integrate the concentration factor = final count x (settled volume / initial volume). 2. Prepare clean Neubauer slides and covers; clean slide and cover-glass with Kleenexpaper. 3. Attach the coverglass to the counting chamber, pressing it carefully into place. When the coverglass is properly attached, coloured bands, called Newton s rings, appear between the two glass surfaces. 4. Mix well the sample. 5. Take a Pasteur pipette and fill its tip by capillary action with sample. Hold the pipette at an angle of ~45 0 (higher or lower to control flow rate) and place the tip at the leading edge of the coverslip. With very gentle pressure, allow the sample to flow quickly and evenly into the chamber, exactly filling it. The chamber surface in the Neubauer brand is a flat mirror-like rectangle and the sample must cover this rectangle. Take care not to overfill beyond the ruled area. It is useful to rest your hand on a bench and steady the pipette tip with a finger. 6. Check under low magnification that the algal cells are evenly distributed: avoid the presence of air bubbles, over flowing, underfilling and uneven distribution of cells. If flooding occurs, rinse haemacytometer and coverslip with distilled water, and repeat procedure. Refill the pipette for each chamber. The time taken to fill the chamber should be short, to minimize setting of cells in the pipette. 7. Leave the counting chamber on the bench for 1-3 minutes before counting to allow the cells to settle. Check grid under the microscope (x 10 objective) for satisfactory distribution of cells, i.e. evenly spread. 21

4 8. Reduce the amount of light entering the microscope s condenser by adjusting the iris diaphragm. Focus the rulings of the chamber and the algal cells. Do not mistake pieces of dust for algae. 9. Counting of Algae For all haemacytometers, the fundamental measurement is the average number of cells per 1mm square, so the centre large square is usually counted. To obtain the total number of cells in this large square, the number of cells in each of the 25 medium squares are counted, recorded then added (see sample cell count) 10. Start counting at the top left square and count only those cells bordering on triple rulings, the convention is to count only those cells touching the top and left-hand side rulings of each square. 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. 11. Usually the procedure is repeated twice more to give a total of 6 counts. 12. Record your results in a table like that shown in the example below 13. After counting each of the two haemacytometer chambers, the haemacytometer and coverslip are rinsed with distilled water. 14. To obtain the cell density, calculate the average cell count and multiply by the conversion factor (for Neubauer = x10 4 ) Explanation of Neubauer conversion factor The volume of the central large square = length width depth = 1 mm 1 mm 0.1 mm = 0.1 mm 3 Multiplication by 10 will give the number of cells in 1 mm 3. Since there are 1000 mm 3 (= 1 cm 3 ) in 1 ml, so multiplication by 1000 will give the number of cells per ml. Accordingly, the cell density per milliliter or the number of cells/ml = average cell count 10 4 which is called the chamber conversion factor for Neubauer. 22

5 Example Isochrysis sp. (Tahitian) CS-177 Age; 8 days Growth conditions; 25 0 C, light intensity 50µmol. photons m -2 s 1, 12:12 light: dark cycle Total no. = 175 Total no. = Total no. = 181 Total no. = Total no. = 174 Total no. = 165 Each block 1-6 represents the total number of cells in the large centre square. Average = = Cell Count = Average X (chamber conversion factor for Neubauer = x10 4 ) = X 10 4 cells/ml = 1.74 X 10 6 cells/ml Impoant Notes: If the sample was diluted; multiply the above value by the dilution factor, where the dilution factor = total volume/sample volume. Dilution of 5 ml sample in 95 ml of water will give a dilution factor = 100/5 = 20. If the sample was concentrated; multiply the above value by the concentration factor, where the concentration factor = settled volume/initial volume. Settling of 1 liter sample to 100 ml will give concentration factor of 100/1000 = 1/10. Population dynamics Microalgae culture systems are highly dynamic. The populations show a typical pattern of growth that has been distinguished a succession of six different phases. These phases are characterized by variations in growth rate, and are summerized in the next Table. 23

6 Table. Description and interpretation of the different phases of batch culture growth curves. phase growth interpretation 1 lag zero physiological adaptation of the innoculum to changing conditions 2 acceleration increasing trivial 3 exponential constant population growth changes the environment of the cells 4 retardation decreasing effects of changing conditions appear 5 stationary zero one or more nutrients (or light) are exhausted down to the threshold level of the cells 6 decline negative the duration of stationary phase and the rate of decline are strongly dependent o the kind of organisms It is advisable to harvest phytoplanktonic organisms during their log phase, since in the new culture they will grow more rapidly and will yield a more viable population. Plotting of the Growth Curve. 1. Determined the cell number each day of your experiment, 2. Plot the cells number against time in days. PREPARATION OF LUGOL SOLUTION Lugol is a fixative staining solution for easier counting of organisms. Reagents: Solution A: dissolve 50 g potassium iodide (KI) and 25 g iodine (I2) in 100 ml boiling water Solution B: 25 g sodium acetate (NaOAc) dissolved in 250 ml water Procedure: when solution A cools, mix the two solutions and store in a cool, dark place as the iodine is light sensitive and will degrade. It should also be stored with a tight fitting lid and kept away from the general culture environment. Use: Add a few drops to each 1-ml sample Alternatively, Lugol s is made by dissolving 100 g Potassium Iodide (KI) in 1L of distilled water, then 50 g crystalline iodine (I 2 ) is dissolved in this solution and then100 ml glacial acetic acid is added. 24