As the fungal growth suppression by biocontrol agents (BCA) in solid media using dual plate assay has some issues regarding nutrient limitation. A protocol for fungal suppression in liquid medium containing bacterial supernatant in different ratios is being designed in the name of "Hydrolytic Activity of bacterial supernatants for fungal suppression."

Procedure

 

 

Grow bacterial cells in 100 ml Waksman broth at 110 rpm at 28°C till the O.D reaches 0.590 at 650 nm. Centrifuge the suspension and discard the pellet. Take the supernatant in a separately autoclaved flask with Waksman broth in different ratios (1:1, 1:2, and 1:3). Inoculate the dual liquid medium with a fungal (plant pathogen) block of 5mm diameter. Keep the flask at 110rpm at 28°C for 4 days. Keep the positive control having fungal culture in Waksman broth. Observe the medium for fungal mycelium comparing with control. Instead nutrients were supplied to the fungus in the medium; bacterial supernatant will inhibit the fungal growth, if having some antifungal metabolites.

Reagents

 

Waksman broth
Constituents	(g/lit)
Protease peptone	5.0
Glucose	10.0
Beef Extract	3.0
NaCl	5.0
pH 6.8