Methods of Single Cell Culture
There are five important methods which are widely employed for culturing single cells. The methods are:
1) The filter – paper raft nurse technique.
2) The Petri dish planting technique.
3) The micro-chamber technique.
4) The nurse callus technique.
5) The micro droplet technique.
1. The Filter-paper Raft Nurse Technique:
i) Single cell are isolated from suspension cultures or a friable callus with the help of a micropipette or microspatuala.
ii) Few days before cell isolation, sterile 8X8 mm square of filter paper are placed aseptically on the upper surface of the actively growing callus tissue of the same or different species.
iii) The filter paper will be wetted by soaking the water and nutrient from the callus tissue.
iv) The isolated single cell is placed aseptically on the wet filter paper raft.
v) The whole culture system is incubated under 16 hrs cool white light (3000 lux) or under continuous darkness at 25 0C.
vi) The single cell divides and redivides and ultimately forms a small cell colony. When a 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 cultures medium but sometimes more that is critical for cell division. The single cell absorbs nutrient 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 the single cell is known as a single cell clone.
2. Petri Dish Plating Technique:
The technique developed by Bergmann (1960) is the most popular one for plating of single cells. The techniques are as follows:
i) A suspension of purely single cell is prepared aseptically from the stock cell suspension culture by filtering and centrifugation. The requisite cell density in the single cell suspension is adjusted by adding or reducing the liquid medium.
ii) The solid medium (1.6 % Difco agar added is melted in water both).
iii) In front of laminar air flow, the tight lid of Falcon plastic petri dish is opened. With the help of sterilized pasteurpipette, 1.5 ml of single cell suspension is put and equal amount of melted agar medium when it cools down at 35 0C is added in the single cell suspension.
iv) The single cell divides and redivides and ultimately forms a small cell colony. When a cell colony reaches a suitable size, it is transferred to fresh medium where it gives rise to the callus tissue.
v) The medium is allowed to solidity and petri dish is kept the inverted position.
vi) The cultures are incubated under 16 hrs light (3000 lux cool white) or under continuous dark at 25 0C.
vii) The petri dishes are observed at regular interval under the inverted microscope to see whether the cells have divided or not.
viii) After certain days of inoculation, when cells start to divide, a grid is drawn on the under surface of the petri dish to facilitate counting the number of dividing cells.
ix) The dividing cells ultimately form pin-head to facilitate counting the number of dividing cells.
x) The plating efficiency (PE) can be calculated from the counting of cell colonies by the following formula:
PE= Number of colonies per plate X 100
_________________________
Number of total cells per plate
xi) Pin-head shaped colonies when they reach a suitable size are transferred to fresh medium for further growth.
3. The Micro-chamber Technique:
i) A drop of liquid nutrient medium containing single cell is isolated aseptically from stock suspension culture with the help of long fine Pasteur pipette.
ii) The culture drop is placed on the centre of a sterile microscopic slid (25X75 mm ) and rinsed with sterile paraffin oil.
iii) A drop of paraffin is placed on either side of the cultural drop and a cover glass is placed on each oil drop.
iv) A third cover glass is 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 permit gaseous exchange.
v) The whole micro-chamber slide is placed in a petri-dish and is incubated under 16 hrs white cool illumination (3000 lux) at 25 0C.
vi) Cell colony derived from single cell gives rise to single cell clone.
vii) When the cell colony becomes sufficiently large, the cover glass is removed and tissue is transferred to fresh solid or semisolid medium.
The micro – chamber technique permits the regular observation of the growing and dividing cell.
4. The Micro-droplet Technique:
i) In this method single cells are cultured in a special Cuprak dishes which have two chambers- a small outer chamber and a large inner c
Chambers. The large chamber carries numerous numbered wells each with a capacity of 0.25-25 of nutrient medium.
ii) Each well of 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.
iii) After covering the dish with lid, the dish is sealed with paraffin.
iv) The dish incubated under 16 hrs white cool light at 25 0C.
v) The cell colony derived from the single cell is transferred on to a fresh solid or semisolid medium in a culture tube for further growth.
5. The Nurse Callus Technique:
This method is actually a modification of petri-dish plating method and paper raft nurse culture method.
In these methods, single cells are plated on to a agar medium in a petri-dish as described earlier. Two to three callus masses derived from the same plant tissue are also embedded directly along with the single cells in the same medium. 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 stimulates the division and colony formation of the other cells.