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VOL. 75 (4), 883-899, 2009 A FASCINATING EXAMPLE OF MICROALGAL ADAPTATION...

In each point physicochemical characterization of the water were
carried out using an YSI- 6820 Multi-parameter Water Quality
Monitors Sonde (1700/1725 Brannum Lane. Yellow Springs
Ohio 45387, USA). In addition water samples of 1 L (+ crude oil)
were collected in dark glass (amber type) for chemical analysis.
Phytoplankton was identified in situ directly after collection using a
McArthur portable microscope (Kirk Technology, England), as well as
in laboratory on fresh and a 4% PBS-buffered formalin samples using
a Zeiss microscope with phase contrast and Nomarski (Oberkochen,
Germany). Identification of algae was carried out in accordance with
Bourrelly (21), Cox (22), Zalocar et al. (23), Mirande & Tracanna (24,
25), Mirande et al. (26). Cell densities of phytoplankton were estimated
on 4% PBS-buffered formalin fixed samples in settling chambers using
an inverted microscope (Axiovert 35, Zeiss, Oberkochen, Germany).

In addition, we isolated microalgae from the crude spill area of
Arroyo Minero to be cultured in laboratory. Sampling was performed
using 13 ml polystyrene culture sterile tubes (Sarstedt, Aktien-
gesellschaft & Co., D-51588 Nümbrecht, Germany). The samples were
maintained at 20 ºC until we arrive to laboratory.

Two methods were used for isolation: i) direct isolation using
micropipettes with a Zeiss-Eppendorf micromanipulator connected
to inverted microscope (Axiovert 35, Zeiss, Oberkochen, Germany),
and procedures of successive dilutions in polystyrene culture sterile
tubes [as previously described in Costas et al. (27) and Brand (28)].

Once isolated the strains were re-cloned by isolating a single cell.
The strains were grown in 100 ml cell culture flasks with aerator cap
(Greiner Bio-One Inc Longwood, NJ, USA), in 20 ml of culture
medium BG-11 (Sigma Aldrich Chemist, Taufkirchen, Germany),
under continuous light of 60-mmol m–2 s–1 over the waveband 400-
700 nm provided by daylight fluorescent tubes (Phillips TLD 36W/
33, France), at 20 °C in growth chambers (Cámaras de Crecimiento,
Modelo AGP, Ing. Climas, C/ Industria 498-500, Badalona 08918,
Barcelona, Spain). BG-11 culture medium was prepared with distilled
water from the distiller (Elix 3uv Millipore) and filtered through
sterile-cup (Express Plus membrane, 250 ml) with filter of 0.22 mm.
Cultures were maintained axenically in mid-log exponential growth
phase by serial transfers of subcultures of a small inoculum (1-3 ml)

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