I am actually trying to determine and compare the protease activity of bacterial biofilms biofilm and planktonic cells using 9 mL of the 3% azocasein solution. The specific method was given below:

The proteolysis assay was performed using the azocasein method of Bussamara et al., (2009). A 3% azocasein solution (Sigma-Aldrich, St. Louis, USA) was prepared in 5 mM phosphate buffer solution, pH 7.5 with 0.1% sodium azide (Sigma-Aldrich, St. Louis, USA) and 0.1 mg chloramphenicol/mL (Sigma-Aldrich, St. Louis, USA). After the incubation of the biofilm and planktonic cultures as given above, the stainless-steel coupons were transferred into 9 mL of the 3% azocasein solution while the planktonic cultures were centrifuged at 10,000 g for 5 mins. .0.1 mL of the supernatant was transferred into 0.9 mL of the 3% azocasein solution. For the controls, an un-inoculated coupon and RSM were used. The azocasein solutions were incubated at 40 °C for a period of 24 h. After the incubation, 0.4 mL of the azocasein solution was mixed with 0.8 mL of 20% trichloroacetic acid (Merck, Darmstadt, Germany) to halt the reaction process. The mixture was centrifuged at 10,000 g for 5 min. A volume of 0.15 mL of the supernatant was transferred into microtitre plate wells (Thermo Scientific, Massachusetts, US) in six replicates and the absorbance was read at 405 nm (Thermo Scientific, MA, USA). The proteolysis was measured by comparing the absorbance value of the samples with the absorbance value of the standard curve of the proteolysis by Streptomyces griseus (3.5 units/mg; Sigma-Aldrich, Auckland, New Zealand). One unit of proteolysis was defined as 1 mg of proteolysis produced by S. griseus under the assay conditions. The estimated concentration of the proteolysis produced was divided by the number of bacterial cells colonizing the stainless-steel surfaces or in the planktonic cultures. The data were expressed as picoUnits of proteolysis per CFU (ρU/CFU) for proteolysis and nanoUnit per CFU for the lipolysis (nU/CFU).