INHIBITION OF MARINE BACTERIA AND MICRO-ALGAE
Antimicrobial assay using agar diffusion technique
Marine bacteria were isolated from biofilm associated with Balanus amphitrite and classified into five major groups, namely: Aeromonas sp, Alcaligenes sp., Flavobacterium sp., Pseudomonas sp and Vibrio sp. Over 40 species of marine bacteria has been isolated and maintained in continuous culture for the last 20 years.
METHOD. The agar diffusion technique follows the original method by Acar (1980). In sterilized petri dish 1 ml of 12 hour old nutrient broth culture, comprising of 3.7% of marine broth in distilled water, of each marine bacterial species was transferred along with 20 ml of antibiotic agar medium. Sterilized Whatman No. 1 paper discs (6.25 mm diameter) were loaded with the test solution at concentrations ranging from 10-2 to 10-6 mg/10ul. Control discs were loaded with only the vehicle. The discs were placed on the surface of the agar according to the standard scheme as shown in the example below, where A = 10-2, B = 10-3, C = 10-4, D = 10-5, E = 10-6 mg/10 µl.
After 24 hours of exposure to the test solution, the zone of inhibition, i.e., the area around the disc devoid of marine bacterial growth was measured by determining the distance from the edge of the disc to the edge of the area showing no bacterial growth.
Micro-algal Inhibition Assay
Dunaliella tertiolecta and Nitszchia sp were maintained in stock cultures in 20 ml test tubes at density of 2.5-5.0 x 10-4 cells per ml as seed cultures and maintained on seven day transfer cycle. The marine micro-algae were inoculated into 250 ml conical flasks containing 100 ml of filtered, sterilized seawater in F/2-Guillard-1975 growth medium. Test chemical, if not supplied in solution, was dissolved in distilled water and added at various concentrations to each algal culture for final concentrations ranging from 10-2 mg/ml to 10-6 mg/ml. Controls consisted of algal culture without the test solution. Each test group was comprised of 3 flasks.
The typical culture goes through four phases. Lag phase is characterized by little or no multiplication of the cells. Log phase or exponential phase is when there is a rapid increase in the rate of cell division. During the next phase, called stationary phase, the number of living cells remain constant and there is no further growth. In the decline phase, the cells begin the process of death or senescence. The growth of the algal cells in each flask was counted each day by haemocytometer. The entire growth and decline of the culture in the presence or absence of the test chemical was determined (Targett, 1988).
Acar JF (1980). The disc susceptibility test. In: Antibioteics in Laboratorymediciene, V Lorian (ed.), William and Wilkins, Baltimore, pp 24-54.
Targett NM (1988). Allelochemistry in marine organisms: Chemical fouling and antifouling strategies. In Marine Biodeterioration. MF Thompson, R Sarojini and R Nagabhushanam (eds). Oxford IBH Publishing Co. Pvt. Ltd., New Delhi, 609-617
For additional reading:
Avelin Mary, Sr., Sr. Vitalina Mary, Dan Rittschof and R. Nagabhushanam, Bacterial barnacle interaction: potential of using Juncellins and antibiotics to alter structure of bacterial communities. J. chem. Ecol. 19(10): 2155-2167.
Avelin Mary, Sr., Sr. Vitalina Mary R. Sarojini and R. Nagabhushanam, (1994 a) Bacteriostatic compounds in extracts of marine animals from the Indian Ocean. pp.229-239 in M. Thompson R. Nagabhushanam. R. Sarojini and M. Figerman (eds) Recent development in biofouling control. Oxford & IBW Publ.Co.Pvt. New. Delhi.
Avelin Mary, Sr., Sr. Vitalina Mary R. Sarojini and R. Nagabhushanam, (1994 b). Broad Spectrum natural products from the Indian ocean octocorals Euplexaura nuttingi. pp. 241-249 in M. Thompson R. Nagabhushanam. R. Sarojini and M. Figerman (eds) Recent development in biofouling control. Oxford & IBW Publ.Co.Pvt. New. Delhi.
Avelin Mary, Sr., C. Parasakthi and Sr. Vitalina Mary, 1995 a. Effect of extractsfrom marine red alga Gracilaria eucheumoides on the growth of Nitzschia sp. and Skeletonema costatum. Seaweed Res. Utilin. Vol. 17 Nos. 1 & 2 pp. 1-11.
Avelin Mary, Sr., J. Angel and Sr. Vitalina Mary, 1995b. Active compound from marine a alga: Hypnea muciformis as bacterostatic and immunomodulater. Sea weed Research and Utilization. 17: 69-77.