*4.3.2 Second enrichment culture*

For the second enrichment culture, 2 m1 of bacteria samples was transferred from the first enrichment culture into another 8 ml of MS media and 0.1% (w/v) substrate (Carbazole) as the sole carbon source. Next, the samples were incubated at 30 °C and shaken at 200 rpm for another two to four weeks until color change observed.

#### *4.3.3 Isolation of pure colony*

After second enrichment, the bacteria were inoculated on MS double layer agar plate. Heterocyclic hydrocarbon which is carbazole was added as the sole carbon source before incubating them for several days. Then the bacteria were sub cultured until pure colony is observed. Colonies that shows clear zone were picked by inoculating needles and purified by streaking several times on a fresh new plate. The growing bacteria colonies were observed as the pure cultures are obtained to be used in the next procedure.

#### **4.4 Glycerol stock preparation and test**

The pure colony of carbazole degrading bacteria isolated were inoculated into the MSM broth and incubated for 24 hours. Next, the bacteria culture was transferred into sterile 2 ml.

Eppendorf tube and sterile glycerol were added to make the final concentration 20% and 40%. Finally, the glycerol stocks were stored in 80 °C. After 3 weeks

stored, the bacteria from glycerol stock was streaked on MSM agar where growth were observed.
