Date:

   Name:

   The Topic:       Culture Indexing for Bacterial and Fungal Contaminants

   The presence of latent microbial contaminants in tissue cultures affects growth and
   development, multiplication rate and acclimatization. Indexing is usually accomplished by
   inoculating segments or intact shoots into selection medium. For indexing of latent bacterial
   contaminants of tissue cultures is recommended a broad-spectrum Leifert and Waites Sterility
   Test Medium (Leifert et al. 1989) or B523 (Viss et al. 1991).

   

   Material: Nodal segments of potato, carnation, raspberry and tobacco long-term cultures

   media:

   Agar solidified Leifert and Waites Medium (M-S basal salts medium 2,2g.l^-1, Meat extract
   7g.l^-1, Glucose 5g.l^-1, Peptone 4 g.l^-1, NaCl[2] 2 g.l^-1, Sucrose 15 g.l^-1, Yeast Extract
   10 g.l^-1)

   Agar solidified B523 medium (Caseinhydrolyzate 8 g.l^-1, MgSO[4] . 7 H[2]O 0,15 g.l^-1,
   K[2]HPO[4] 2 g.l^-1, Yeast Extract 4 g.l^-1, Sucrose 10 g.l^-1, agar 10 g.l^-1)

   Maintenance M-S medium (Murashige and Skoog)

   

   Procedure:

   1.      Remove shoots from culture and place in a sterile Petri dish in a laminar flow hood.

   2.      Cut the shoots into single node segments or thin cross-sectional disks leaving the
   shoot tips intact.

   3.      Transfer the shoot tips onto maintenance medium.

   4.      Inoculate the B523 agar medium by digging the segment into the medium, slowly lift out
   of the medium and place onto the medium. This procedure inoculates lower oxygen levels of the
   medium that may promote the growth of some microbes. Do the same type of inoculation to M-S
   medium.

   5.      Label corresponding segments, disks and shoot tips cultures.

   6.      Incubate inoculated Petri dishes in the dark at 22-30˚C for 3 weeks.

   

   Evaluation

   Screen the cultures for visible contamination in the following period of cultivation.

   Clouding of the liquid media or colonies on the surface of the medium and/or halo in the
   medium indicate the presence of cultivable contaminants in the segments or disks. The
   contaminated cultures should be autoclaved before disposing.

   Compare contamination of the corresponding segments, disks and shoot tips cultures cultivated
   on both agar media.

   Literature:

   Leifert et al. (1989): J. Appl. Bacter. 67: 351 – 361.

   Viss et al. (1991): In Vitro Cell Dev. Biol.-Pl. 27P, 42.