The following points highlight the five important methods employed for culturing single cells.
The methods are: (1) The Paper Raft Nurse Technique (2) The Petri Dish Plating Technique (3) The Micro-chamber Technique (4) The Nurse Callus Technique and (5) The Micro-droplet Technique.
Method # 1. The Paper Raft Nurse Technique:
1. Single cells are isolated from suspension cultures or a friable callus with the help of a micropipette or micro-spatula.
2. Few days before cell isolation, sterile 8 mm x 8 mm squares of filter paper are placed aseptically on the upper surface of the actively growing callus tissue of the same or different species.
3. The filter paper will be wetted by soaking the water and nutrient from the callus tissue.
4. The isolated single cell is placed aseptically on the wet filter paper raft (Fig 9.1).
5. The whole culture system is incubated under 16 hrs. cool white light (3,000 lux) or under continuous darkness at 25° C.
6. The single cell divides and re-divides and ultimately forms a small cell colony. When the cell colony reaches a suitable size, it is transferred to fresh medium where it gives rise to the callus tissue.
The callus tissue, on which the single cell is growing, is called the nurse tissue. Actually the callus tissue supplies the cell with not only the nutrients from the culture medium but something more that is critical for cell division. The single cell absorbs nutrients through filter paper. The nutrients actually diffuse upward from culture medium through callus tissue and filter paper to the single cell. A callus tissue originating from a single cell is known as a single cell clone.
Method # 2. The Petri Dish Plating Technique:
1. A suspension of purely single cells is prepared aseptically from the stock cell suspension culture by filtering and centrifugation requisite cell density in the single cell suspension is adjusted by adding or reducing the liquid medium.
2. The solid medium (1.6% ‘Difco’ agar added) is melted in water bath.
3. In front of laminar air flow, the tight lid of falcon plastic petri dish is opened With the help of sterilized Pasteur pipette 1 5 ml of single cell suspension is put an equal amount of melted agar medium when it cools down at 35°C, is added in the single cell suspension (Fig 9.2).
4. The lid is quickly replaced and the whole dish is swirled gently to disperse the cell and medium mixture uniformly throughout the lower half of the petri dish.
5. The medium is allowed to solidify and the petri dish is kept at the inverted position.
6. The cultures are incubated under 16hrs light (3,000 lux, cool white) or under continuous dark at 25°C.
7. The petri dishes are observed at regular intervals under inverted microscope to see whether the cells have divided or not.
8. After certain days of incubation, when the cells start to divide, a grid is drawn on the undersurface of the petri dish to facilitate counting the number of dividing cells.
9. The dividing cells ultimately form pin-head shaped cell colonies within 21 days of incubation.
10. The plating efficiency (PE) can be calculated from the counting of cell colonies by the following formula:
PE = Number of colonies per plate/Number of total cell per plate x 100
11. Pin-head shaped colonies, when they reach a suitable size, are transferred to fresh medium for further growth.
Method # 3. The Micro-chamber Technique:
1. A drop of liquid nutrient medium containing single cell is first isolated aseptically from stock suspension culture with the help of long fine Pasteur pipette.
2. The culture drop is placed on the centre of a ‘ sterile microscopic slide (25 x 75 mm) and ringed with sterile paraffin oil.
3. A drop of paraffin oil is placed on either side of the culture drop and a cover-glass (called raiser) is placed on each oil drop (Fig 9.3).
4. A third cover-glass is then placed on the ‘ culture drop bridging the two raiser cover-glasses and forming a micro-chamber to enclose the single cell aseptically within the paraffin oil. The oil prevents the water loss from the culture drop but permits gaseous exchange.
5 The whole micro-chamber slide is placed in a petri-dish and is incubated under 16 hrs. white cool illumination (3,000 lux) at 25 C.
6. Cell colony derived from the single cell gives rise to single cell clone.
7. When the cell colony becomes sufficiently ‘ large, the cover-glass is removed and the tissue is transferred to fresh solid or semisolid medium.
The micro-chamber technique permits regular observation of the growing and dividing cell.
Method # 4. The Nurse Callus Technique:
This method is actually a modification of petridish plating method and the paper raft nurse culture method. In this method, single cells are plated on to agar medium in a petridish as described earlier Two or three callus masses (Nurse tissue) derived from the same plant tissue are also embedded directly along with the single cells in the same medium (Fig 9.4).
Here the paper barrier between single cells and the nurse tissue is removed. Cells first begin to divide in the regions near the nurse callus indicating that the single cells closer to nurse callus in the solid medium gets the essential growth factors that are liberated from the callus mass. The developing colonies growing near to nurse callus also stimulate the division and colony formation of other cells.
Method # 5. The Micro-droplet Technique:
1. In this method, single cells are cultured in special Cuprak dishes which have two chambers—a small outer chamber and a large inner chamber. The large chamber carries numerous numbered wells each with a capacity of 0.25-25µl of nutrient medium.
2. Each well of the inner chamber is filled with a micro-drop of liquid medium containing isolated single cell. The outer chamber is filled with sterile distilled water to maintain the humidity inside the dish (Fig 9.5).
3. After covering the dish with lid, the dish is sealed with paraffin.
4. The dish is incubated under 16hrs cool light (3,000 lux) at 25°C.
5. The cell colony derived from the single cell is transferred on to fresh solid or semisolid medium in a culture tube for further growth.