Preparation of the Culture Medium

The salt solution is prepared by dissolving the required salts one by one in the prescribed amount of water to which the agar is then added . The solution is double -cooked till all the agar is dissoved.

Erhlenmayer flasks of 250 ml capacity are the most convinient and popular of the culture bottles used. Some people, however, prefer to use milk bottles., which on being tilted at right angles give a larger surface for sowing of seeds than Erhlenmeyer flasks. The mouth of the flask is colsed with either cotton wool plugs or rubber -stoppers or screw caps. The latter two are now preferred to cotton plugs, because cotton -wool absorbs moisture and provide shelter for atmospheric spores whcih may later contaminate the medium . When rubber stoppers or screw-caps are used , bent tubes made of glass are passed through them in order to provide sufficient aeration for the growing seedlings. The mouth of this tube is then plugged with cotton wool . This arrangement will considerably reduce chances of contamination from the spores in the atmosphere. Thomasen (1964) suggests that , if at all cotton plugs are to be used , they should be previously soaked in 0.2 % picric acid and dried throughly before use.

Just before pouring into the culture bottles the pH of the culture medium is tested and adjusted to suit the requirement of the particular plant whose seeds are to be sown. In general orchids prefer a slightly acidic medium but this could vary slightly with species . Hotttum (1964) reports that a pH of 6 (7 is neutral) is deal for Spathoglottis , whereas ,Vanda and Phalaenopsis prefer a more acidic medium . He recommends addition of 5 cc of 5 % HCI to each litre of the solution to adjust the pH to the necessary level . Vacin (1950) reported that plants like Vanda and Dendrobium thrive well under a pH of 5.2 and suggested addition of citric acid at the rate of 0.2 gm to every litre of the medium.

Culture media suited to the requirements of the various genera are now available in powder form in many countries . Dissolve the power in the prescribed amount of distilled water and used directly , after autoclaving .

The prepared culture medium is poured into the flasks using a long-steammed funnel in order to avoid splashing of the liquid medium on the mouth and sides of the flask , which would invite contamination . The bottles are then stoppered tightly.

The culture bottles prepared thus are then autoclaved for 15-20 minutes at 15 lb pressure and allowed to cool inside the autoclave itself , from which the water has been removed.