In order to obtain axenic cultures, the unialgal cultures
were submitted to a mixture of antibiotics containing Penicillin
G (16,250 U), Streptomycin (5 mg ml–1) and
Chloranphenicol (2 mg l–1) (Guillard’s Antibiotic
Concentrate—Sigma G-5535) at different concentrations
(corresponding to dilutions of the original antibiotic mixture
between 1:10 and 1:30) and at different exposure times
(12-60 h). Cultures were grown in borosilicate test tubes with
polypropylene screw caps containing f/2 medium at the
above described temperature and light conditions. At the end
of each exposure time, viability tests and axenic tests were
performed, in order to determine the success of the treatment.
The viability tests consisted of the inoculation of 1 ml of the
cultures that had been exposed to the antibiotic mixture in 9
ml of antibiotic-free sterile culture medium, and incubated
under the same conditions. To check for the presence of
microorganisms other than the microalgae, 100 μl of the
axenic assay were inoculated on enriched organic solid medium
TSA (Alcântara et al., 1996).