MATERIALS AND METHODS

The diatom, R. alata was isolated from the plankton sampled from Vellar estuary (11° 29’ N; 79° 46’ E) in January 2006, southeast coast of India by using plankton net (Cloth No. 25 with mesh size of 42 μm) and cultured in F/2 medium (Guillard, 1983) for 9 days under optimal laboratory conditions. The cells were harvested at the end of 9th day (cell density 2.6x10⁵ mL^-1) and then freeze dried. Dried samples were extracted with different solvents viz., acetone, chloroform, chloroform: methanol (1:1), methanol: distilled water (4:1) and distilled water. Fifty milligram of dried algal powder was added with 1 mL of solvent, shaken well, kept for one hour at room temperature and centrifuged in the cold temperature for 20 min at 3000 rpm. The clear supernatant was pipetted out and stored under -4°C until use.

In vitro antibacterial activity of diatom extracts were determined against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, Salmonella typhi and Vibrio cholerae (Source: Jawaharlal Nehru Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry) using the paper disk assay method (El-Masry et al., 2000). Whatman No.1 filter paper disk of 6-mm diameter was sterilized by autoclaving for 15 min at 121°C. The sterile disks were impregnated with different extracts (500 μg mL^-1). Agar plates were surface inoculated uniformly from the both culture of the tested microorganisms. In all cases, the concentration was approximately 1.2x10⁸ CFU mL^-1. The impregnated disks were placed on the medium suitably spaced apart and the plates were incubated at 37°C for 24 h disk of penicillin-G (P) (Himedia No. SD 028) was used as a positive control and discs with solvents (without extract) as control. The Antibacterial activity was read by noting the presence or absence of clear zones of inhibition surrounding the discs and was measured as the radial extent of the clear zone (diameter mm). All assays were carried out in duplicate.

RESULTS AND DISCUSSION

The results (Fig. 1) obtained in the present study revealed that R. alata has produced some antibacterial compounds against human pathogenic bacteria tested. Among the solvent extracts of R. alata tested, chloroform extract showed growth inhibition activity against all the seven pathogenic bacteria tested and maximum inhibitory activity was found against P. vulgaris with zone of clearance of 17 mm and less activity against S. aureus (5 mm). There was no inhibition activity found with distilled water extracts against all pathogenic bacteria tested. The overall observation inferred that maximum growth inhibition activity was found with chloroform extract against P. vulgaris and minimum with chloroform + methanol extract against E. coli (2 mm). As earlier reports of Walter and Mahesh (2000), in the present study acetone and chloroform extracts showed considerable activity against most of the test organisms. However, chloroform + methanol extracts showed only moderate activity, methanol + water fraction showed negligible activity and the water fraction revealed no activity.

The antibacterial activity of marine diatom shown in the present study was supported by the observation of Manivasagam et al. (1989). Their observation on antibacterial activity of the diatom Pleurosigma elongatem showed its growth inhibition activity against B. subtilis, S. aureus, E. coli, S. typhi, V. cholerae in acetone, chloroform, chloroform + methanol, methanol + water, as the observations of present study. There was no antibacterial activity was found in distilled water extracts. The difference in the antibacterial activity may be due to species specific characteristics and the efficiency of the solvents used in the extraction of antibacterial substances (Walter and Mahesh, 2000).

The results of the present study inferred that the antibacterial activity of R. alata may be to prevent the colonization of epiphytic bacteria on the surface of the algae. Such control and regulation of the epibiota by antibiotics diffusing outwards from the thallus of some marine planktonic algae was indicated by McN Sieburth (1964). The less activity found with methanol + water extract may be due to the inefficiency of the method and further research on the characterization of the antibacterial compounds from R. alata are under process. Disc diffusion tests for antibiotic activity have been reported as of doubtful quantitative significance (Schneierson and Amsterdam (1959). According to Jorgensen and Nielson (1961), it was found that antibiotics in charged dried discs underwent a progressive loss in potency when kept at room temperature. We have found that freshly prepared discs when refrigerated in dark showed negligible loss in activity up to one month storage.

The present study is a preliminary attempt to screen marine microalgae for bioactive compounds. It appears from the present investigation that, a marine microalga, R. alata is potential source of novel antibacterial compounds. Characterization of the antibacterial compound and evaluation of methods employed in antibiotic assay deserve further research to give a clear picture of the antibacterial activity of the algae.

Fig. 1:	Antibacterial activity of different grade solvent systems of Rhizosolenia alata

ACKNOWLEDGMENTS

The authors are grateful to the authorities of Annamalai University for the facilities and Department of Ocean Development (DOD), Government of India for providing research fellowship to R. Venkatesan, first author of this manuscript.