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VICTORIA LÓPEZ-RODAS Y COLS. AN. R. ACAD. NAC. FARM.

PAM fluorimeter (Walz, Effeltrich, Germany) at seven different time
points (0.1, 1, 10, 12, 24, 48 and 72 h). Effective quantum yield was
calculated as follows:

F PSII = (F’m - Ft) / F’m (22)

F’m, Ft, maximum and steady-state fluorescence of light-adapted cells,
respectively.

2.4. Fluctuation analysis of TBT-sensitive ? TBT-resistant
2.4. transformation

A modified Luria-Delbruck fluctuation analysis (23) was
performed to distinguish resistant cells that had their origin in
random spontaneous pre-selective mutations (mainly prior to TBT
exposure) from those arising through acquired post-selective
adaptation (during the exposure to TBT) (Figure 1). Two different
sets of experimental cultures were prepared. In the set 1 experiment,
96 culture flasks were inoculated with N0 = 102 cells (a number small
enough to reasonably ensure the absence of pre-existing mutants in
the strain). Cultures were grown in BG-11 medium until Nt = 1 × 105
cells and afterwards exposed to TBT. For the set 2 controls, 26
aliquots of 1 × 105 cells from the same parental population growing
in BG-11 medium were separately transferred to culture flasks
containing TBT. Cultures were observed for 60 d (thereby insuring
that one mutant cell could generate enough progeny to be detected),
and the resistant cells in each culture (in set 1 and set 2) were
counted. The cell count was performed by at least two independent
observers.

Two different results can be found in the set 1 experiment, each
of them being interpreted as the independent consequence of two
different phenomena of adaptation. In the first case (Figure 1, set
1A), the variance in the number of cells per culture could be found
to be low if resistant cells arose by physiological adaptation or
specific post-selective mutations. Because every cell is likely to have
the same chance of developing resistance, inter-culture (flask-to-
flask) variation would be consistent with the Poisson model
(variance/mean = 1). On the contrary, if high variation in the inter-

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