1. aeration
1-If the plant part is grown on the surface of a semi solid medium it acquires sufficient aeration without a special device.
2-But if liquid medium is used, submerged plant part requires some special device for aeration.
3-One such device is a "filter paper bridge" whose two legs remain dipped in the medium and the horizontal part carrying the plant part is raised above the level of the medium.
4-Common method - shaking the cultural flasks/tubes on an automatic shaker.

2. 5-The culture vials containing medium are plugged with non-absorbing cotton wrapped in cheese cloth; such a closure allows the exchange of gases but does not permit the entry of micro-organisms into the culture vials.

3. Steps of tissue culture
1-Inoculation and callus formation
2-Multiplication or sub-culturing callus
3-Development and differentiation of sub-culture
4-Acclimatization

4. Inoculation and callus formation
1-explant - vegetative parts or reproductive part, young or old.
2-Old parts have accumulation of phenols, terpenoids, coumarins, steroids (secondary metabolites) - pre-soaked in an anti-oxidant solution to remove the substances
3-desirable to take explant from young plant especially from meristem and cortical parenchyma

5. 4-Explant is extracted with a sharp scalpels - tissues remain undamaged.
5-Damaged tissues release various compounds which are air oxidized.
6-explant surface is sterilized with chlorine water or HgClz (0.1 %),
7-then it is washed with distilled water to remove adherent chemical particles.
8-explant is transferred to sterilised culture medium kept in test tubes or flasks in culture chamber.
9-This chamber is also pre-sterilized with ultra violet ray emitting tubes.

6. 10-all inoculated tubes/flasks are kept at 25 ± 2°C temperature in culture chamber with light exposure intensity of 2000 lux for 16 hours
11-light is not essential for callus formation in tissue culture experiment.
12-It is essential for plant regeneration only.
13-Callus formation is observed after two weeks of inoculation, i.e. incubation period is of two weeks.