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The following points highlight the six important methods of tissue culture. The methods are: 1. Hanging Drop Culture 2. Double Cover Slip Method 3. Culture in Stewards Auxophyton 4. Culture in Bottles, Conical Flasks etc. 5. Culture in a Micro-Chamber 6. Culture in Petridish, Watch Glass etc..
Method # 1. Hanging Drop Culture:
The simplest method of tissue culture is hanging drop culture (Fig. 2).
A portion of the tissue with 1 mm diameter is placed on a coverslip containing a drop of almost solidified medium. A 1″ × 3″ grooved slide is taken and the coverslip is placed over it. The rim region is sealed with wax. Harrison used this process for tissue culture. In such a culture the tissue remain viable only for few days.
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If the culture medium is changed from time to time the tissue remains viable for longer period. For subculture the tissue is divided into smaller parts and each part is sub-cultured in a new grooved slide with fresh medium. In 1955 de Ropp cultured single cells in hanging drop medium, but only few cells divided.
Method # 2. Double Cover Slip Method:
Maximov in 1928 first used this method. In this method (Fig. 3) a small round coverslip with the tissue and the medium is placed on a large square coverslip. A drop of water or salt solution attaches the small coverslip with the large coverslip. Double cover-slips are placed on a grooved slide and the rim of the coverslip is sealed with wax.
When the culture medium has to be changed, then the small coverslip is carefully detached from the larger coverslip and washed with nutrient solution. The small coverslip is attached to a new larger coverslip and placed on a new grooved slide and the edge is sealed. Torrey (’57) used Maximov’s double coverslip method for culture of pea root callus on agar medium.
Method # 3. Culture in Stewards Auxophyton:
Culture can be done in a tube (Fig. 4). The explant is placed in the culture tube and the nutrient medium is poured into it and the mouth of the tube is plugged. In 1952 Steward, Caplin and Miller designed a culture apparatus—called auxophyton for rapid proliferation of small explants (about 3 mg) from carrot root (conical tap root).
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They used a tube (12.5 cm long and 3.5 cm diameter) with a side neck at the middle. After introducing the explant and the medium (10 c.c.) the mouth is closed with a cotton plug, which helps gaseous exchange. About 24 such tubes can be mounted along the edge of a disc which is rotated at slight angle to the horizontal at a speed of 1-2 revolutions per minute.
The tissues are thus alternately exposed to air and the liquid medium, which flows along the tube from end to end. The apparatus is kept in a temperature controlled roam and with a fluorescent light the culture tubes are uniformly illuminated.
In 1956 Steward and Shantz designed nipple flasks to culture several explants for biochemical investigations. 1000 c.c. nipple flask has ten nipples and 250 c.c. nipple flask has eight nipples.
In these flasks explants are distributed in the nipples, and when placed in a auxophyton the explants are alternately exposed to air and culture medium. The medium generally becomes turbid due to the release of free cells and cell aggregates from the explants. This suspension itself may be used for subculture.
Method # 4. Culture in Bottles, Conical Flasks etc.:
When many cells are cultured together then culture is done in a conical flask, bottle or petridish. In culture vessel containing liquid medium inoculation is given.
Cells rapidly increase on the surface of the bottle or conical flask forming monolayer. When whole surface is utilised the cell growth is reduced. If the medium is changed at an interval of 3-4 days then the tissue remains intact for a longer period.
Subculture may be done by scraping the cells from the walls of the flasks or bottles or by shaking or cells are separated by chemicals (such as, trypsin). The cells are centrifuged and cultured in a new bottle or flask. If the flasks, tubes etc. are continuously shaked then the cells increase rapidly.
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In spinning culture 10 litre culture bottles each containing 4.5 litres of medium are placed in an apparatus at 45° angle to the horizontal and rotated at 80—120 .revolutions per minutes. The mouth of the culture bottle is closed by cotton plug. Adequate gaseous exchange occurs and the cells grow very rapidly.
Method # 5. Culture in a Micro-Chamber:
Jones, Hildebrandt, Riker and Wu (’60) cultured cells from a callus of the hybrid Nicotiana tabacum × N. glutinosa in a microchamber, which was sealed with inert mineral oil (Fig. 5). Cells can be minutely observed in such a culture. G.G. Rose (’54) used perfusion chamber for tissue culture of animal cells.
Method # 6. Culture in Petridish, Watch Glass etc.:
Organ may be cultured in a petridish or watch glass (Fig. 6). The petridish may be incubated in a humified incubator. For culture of small number of cells petridish is recommended.
