|
|
|
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).
-
-
- Table I. Flask culture environmental
conditions.
|
Lighting |
Cycle: 12 hours light /
12 hours dark
Type: fluorescent bank:
2-F20T12 Westinghouse cool white
Intensity:
133-299 µEin/sec/cm2
Temperature:
20 oC |
|
Water |
Type: Aged, sterilized
Salinity:
30 ppt
Total volume:
250 ml
Flask size:
1 L Pyrex glass Erlenmeyer flask |
|
Concentration |
Seeding density:
2.5-5.0 x 104 cells per ml
Nutrients:
1 ml stock nutrients per liter of seawater
Time to bloom:
6-7 days
Bloom
density:
1- 3 x 106 cells per mal |
-
REFERENCES
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.
 Back
to top
|
