Benson Lab

Carla's CcI3 Solid "CB" Medium


 

 

Component
Per liter final volume
1.
NaPyruvate
5 g 
2.
Proteose Peptone #3 (Difco)
1.6 g
3.
NH4Cl 
0.5 g
4.
10X stock salts*  
100 ml
5.
1000X Iron stock**  
1 ml
6.
10X Buffer stock***
100 ml (pH 6.5; after autoclaving)
7.
Phytagel - Gellan Gum (Sigma)
2.5 g (0.25% final conc.)


 

*10X Stock Salts: CaCl2.2H2O, 6.0 g; MgSO4.7H2O, 2.0 g; dH2O, 1000 ml

**1000X Stock Iron: Dissolve 0.75 g Na2EDTA.2H2O, 0.56 g FeSO4.7H2O and 0.02 g of Na2MoO4.2H2O in 100 ml dH2O

***10X Buffer: KH2PO4 6.8 g/l; MOPS (3-(N-morpholino) propanesulfonic acid) 11.56 g/l.  Adjust pH to 6.5 with HCl. The buffer should be prewarmed to about 50 C and added to the medium before plating to avoid clumping of the gellan gum.

Notes: 

- The medium is made by adding the first five ingredients plus gellan gum to make 900 ml. The buffer stock is added seperately after autoclaving. Phytagel is a polysaccharide gum produced by "Pseudomonas elodea". It is solubilized during autoclaving but resolidifies at a higher temperature than agar. Therefore plates are poured at about 70 C using a glove to protect the hand. Before spread plating Frankia, allow the surface to "harden" by cooling the plates with the lid ajar for an hour or two in the clean hood.