Symbiotic Fungi: Principles and Practice (Soil Biology)

22 Co-Culture of Linum album Cells and Piriformospora indica 365
3. Subculture the fungus on the same medium every month for maintenance of
the culture.
22.3.2 Initiation of Fungal Culture
22.3.2.1 Inoculum Preparation
1. Transfer 5-day-old fungal inoculum (3 1 cm squares of grown fungi) from
solid MYPG media plates into a 250 ml Erlenmeyer flask containing 50 ml Hill
and Kaefer’s medium (Hill and Kaefer 2001).
2. Place inoculated flasks on incubator shaker rotating at 200 rpm and maintain at
30 2 C.
3. Allow cells to grow in suspension culture for 5 days.
4. Centrifuge the grown cells in sterile centrifuge tubes at 5,000 rpm for 10 min.
5. Decant the spent media and use cells to initiate suspension cultures of P. indica.
Preparation of Hill and Kaefer Medium
1. The components of Hill and Kaefer medium are divided into two parts: A and B
(Compositions are given in Tables 22.1 and 22.2).
(a) The concentrations of various components mentioned in part A and part B
are the final concentrations of components after mixing part A and part B.
(b) Prepare FeCl3 and FeSO4·7H2O solutions in 1 N H2SO4.
(c) Separately prepare the stock solutions of all vitamins in distilled water.
Bring the pH of solution to 12.5 in order to solubilize them except Nicotinamide.
2. Autoclave both stock solutions separately. After adding both the solutions, bring
pH to 6.5 by adding required amount of autoclaved 2 M NaOH under aseptic
conditions.
3. Store all the stocks at 4 C except vitamins.
4. Store stock solution of vitamins at 20 C.
22.3.2.2 Development of Fungal Culture for Co-Culture Setup
1. Transfer these fungal cells (0.6 g l 1 on dry cell weight basis) to a 250 ml
Erlenmeyer flask containing 50 ml Hill and Kaefer’s medium aseptically with
the help of a sterile spatula over flame.