1. BY
D.SABARIANNAI

2. AIM OF THE STUDY
To screen the cyanobacteria which tolerate high salinity.
To study what are the Antioxidant enzymes produced in this cyanobacteria.
To use this cyanobacteria for reclamation of saline soil.

3. We selected 10 organism for screening.
Oscillatoria salina
Oscillatoria boryana
Oscillatoria willei
Chrococcus minor
Synechococcus elongatous
Spirulina subsalsa
Phormidium valderianum 20041
Phormidium valderianum
Phormidium corium 91921
Gleocapsa crepidium

4. Out of 10 organisms Oscillatoria willei Mo711 tolerate high salinity.
This organism is treated in 3 different conditions 0,25,100 ppt.
The treatment is done for 24 , 48 hours.
Then it is centrifuged & protein is extracted.
protein is estimated and loaded in native gel for anti-oxidative enzyme study.

5. Anti oxidative Enzyme SOD staining: Gel is washed in Tris HCl PH 8.
Riboflavin - 4mg, EDTA - 2mg, NBT – 20mg are added to Tris HCl PH 8.
Incubate gel for 30 min in dark.
Illuminate the gel under light for min at RT.
Achromatic regions are visualised dark blue background.

6. Total SOD
24 Hrs 48 Hrs

7. Types of SOD 24 Hrs 48 Hrs 24 Hrs 48 Hrs 0 25 100 0 25 100

8. Peroxidase 3,3‘-diaminobenzidine(DAB) oxidised DAB.
Gel is washed with sodium acetate buffer PH 4.5.
DAB – 30mg, H µl are added to sodium acetate buffer & incubate gel in this solution until brown bands are visualised.

9. Peroxidase
24 Hrs 48 Hrs

10. Catalase
H2O2 H2O+ O2
Gel is washed in potassium phosphate buffer PH 7.0.
Gel is incubated in potassium phosphate buffer containing H2O2 →3µl, 2% potassium ferric cyanide, 2% ferric chloride.
The yellow colour bands are appeared on a dark green background.

11. Esterase Gel is washed in phosphate buffer pH 7.5.
Gel is incubated in phosphate buffer PH 7.5 containig α- napthyl acetate → 50mg, β -napthyl acetate → 50mg,Fast blue RR → 100mg .
Incubate the gel in this solution until dark gray colour band appeared.

12. 24 Hrs 48 Hrs

13. SDS – PAGE still in process.
We are standardizing the SDS-PAGE.
Yet to be done:
2D gel

14. SDS-PAGE
24 Hrs 48 Hrs