In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.
We are going to introduce our method to establish a string of algae especially unicellular conjugatophytes. For the establishment of a strain, collection of water sample, which contains algae through fieldwork, is necessary. The water sample contains foreign matters other than algae.
Only single cell is isolated from this sample. The THIL is transferred to a clean medium and cleansed repeatedly. Isolation is conducted through this process.
Sterilization is possible if it is conducted in a careful manner. Finally, the isolated cell is transferred to a test tube and a strain is established through cultivation. Here we focus on unicellular conjugatophytes in which varieties of forms are identified.
Unicellular conjugatophytes grow in stagnant, fresh water area such as lake, marsh, and paddy field. From these environments, algae containing water sample is collected to produce a culture strain. We will introduce a method to collect water sample from lake environment.
A plankton net is used for the lake environment. The upper net part is permeable to water. The algae caught by the net will be concentrated into the bottom container.
A cross mesh allows small algae to pass through. A fine mesh gets clogged easy. A proper net should be used depending on the purpose.
Bring the concentrated water sample to laboratory. Unicellular conjugatophytes also grow in marsh or paddy field. In the shallow water area, it is directly sampled with a dropper.
Water is carefully sampled from the soil surface, not to contain the soil. Confirm if the water sample contains algae. The algae, around 400 micrometer and over, is directly observed through loop.
As for small algae, a portable microscope is used. Water sample is mounted on the lid of a Petri dish and covered with the body. This method keeps the sample from moving and makes observation easier.
On this water sample, isolation of algae is conducted. We will introduce the method to make a pipette, used for the isolation of single cell algae from water sample. How to make a pipette for isolation.
Ignite an alcohol burner. Heat a sterile Pasteur pipette on the flame. Take the pipette off the flame and stretch it.
Cut the tip of the pipette to the proper length. Be sure to discard the capillary of the melting off. Be sure to check if the flame is put out.
To produce a fine pipette for isolation, you should make sure the flame is not flickering. Sharpen the flame and melt the pipette at the tip. When stretching, you should make sure the pipette is off the flame.
Behold the bowing part is the finest. It is ideal for the tip. The bubbles from the pipette indicate the size of the tip.
Select proper size for the cell to be isolated. Shown is for the cell width of 10 to 20 micrometers. Adequate number of watch glasses needed for cradling is prepared.
Put the medium on a watch glass. Isolate a target cell from water sample collected in the field. A very fine glass tube takes up water due to capillary reaction.
Put the glass tube beside the cell to take it up. Isolate the cell from the medium on the watch glass and transfer it to a new watch glass. Use a new pipette to transfer the cell from the first watch glass to the second.
And another pipette to the third. Finally, the isolated cell is transferred to a test tube. Through cultivation of this test tube, cell multiplication is promoted and a strain is established.
The culture may turn green in two weeks if successful. In this study, we isolated 15 cells from the water sample collected at lake in Biwa, Japan. Two weeks later, green sediment was observed in nine test tubes marked with a red frame, indicating that cell purification of algae was ongoing.
The sediment was observed only for the isolated cells, showing that cell isolation was successful. This is our method to establish a unique algae culture strain. It is possible to establish an axenic culture strain if you conduct an isolation under accepted conditions, such as, in a food.
