Isolation, stock culture, mass culture and mixed algal culture can be done in the following manner.
For the isolation of the required species of phytoflagellates, the serial dilution culture technique is employed. In this method mainly 5 dilution steps (the inocula corresponding to 1, 10-1, 10-2, 10-3 and 10-4 ml) are employed. After filtering the sea water through 10 micron sieve, the filtrate has to be inoculated to 5 series of culture tubes in various concentrations. These are kept under sufficient light and with uniform temperature (25°C) conditions. After 15–20 days, discoloration of the tubes can be observed. On examination, the growth of unialgal species can be observed. Purification of these organisms can be done by sub-culturing the same in 250 ml, 500 ml, 1 /. and finally 3 or 4 7. Haufkin culture flasks as stock culture.
Conway medium is used for the stock culture of the above algal. Cultures are maintained in 5 / Haufkin flasks. Nutrients are added along with 10 ml of inoculum and this is kept under illumination (1000 lux) in an air-conditioned room. When the exponential growth phase is reached in 8–10 days, the light intensity is reduced to half. The flagellates will enter into stationary phase after 15 days and it can be maintained for about 2 months with or without aeration. Before it enters into death phase, these cultures should be used as inoculum for carrying out mass production.
Solution A-Chemicals
| Potassium nitrate | 100 g. |
| Sodium orthophosphate | 20 g. |
| Sodium EDTA | 45 g. |
| Boric acid | 33.4 g. |
| Ferric chloride | 1.3 g. |
| Manganese chloride | 0.36 g. |
| Distilled water | 1000 ml. |
Solution B-Trace metals
| Zinc chloride | 4.2 g. |
| Cobalt chloride | 4.0 g. |
| Copper sulphate | 4.0 g. |
| Ammonium molybdate | 1.8 g. |
| Distilled water | 1000 ml. |
| Acidify with HC1 to obtain a clear solution. |
Solution C-Vitamins
| Vitamin B (Thiamin) | 200 mg. |
| Vitamin B12 (cyanocobalamine) | 10 mg. |
Each vitamin is dissolved separately in 100 ml. distilled water and stored in a refrigerator.
Solutions A, B and C are prepared in different re-agent bottles. 1 ml. of A., 0.5 ml. of B, and 0.1 ml. of C each are added to 1000 ml. of filtered and sterilized sea water.
Persepex tanks of 100 /. capacity are used for mass culture. Two litre inoculum is added per 100 /. of sea water Maximum algal bloom is obtained in 5–6 days. Vigorous aeration is provided.
Mixed algae are produced in 1t. FRP tanks in the sunlight using filtered sea water. Algale from the indoor mass culture is used as inoculum. The mixed algae in the open may bloom in 3–4 days. This may contain a mixture of diatoms along with the Isochrysis. Chemicals used for open culture in 1000 /. of sea water are given below:
| Potassium nitrate | 13.2 g. |
| EDTA | 6.6 g. |
| Sodium orthophosphate | 6.6 g. |
| Sodium silicate | 6.6 g. |
Initially the stocks were maintained in test tubes. When we have sufficient test tubes of pure culture we have to sub-culture them in 500 ml. and in one litre conical flask. Then we have to sub-culture them into four-litre Haufkin's culture flasks. Twenty litres of sterilized sea water is taken in glass carbouys and enrich them with Walne's medium and to this 500 ml. of inoculum is added from Haufkins culture flask. Within four days a good bloom will be developed. This can be used as inoculum for the presepex tanks. For mass culture we take 60 litres of sterilized sea water and enrich the same with Walney's Conway's medium. For one litre of sterilized sea water 1 ml. of A and 0.5 ml of B are taken. We can harvest micro-algale within four days. When golden yellow colour develops, it indicates that it is a good bloom. Normally, cell concentration is around 1.2 to 2 million cells per ml. The cell concentration can be estimated with the help of haemocytometer.
