66
(ADAVANTECH) AD/DA interface. The FIA system, for sulfate signals, is shown in Figure
1. For the best comparison the sign for cells suspensions were obtained with the same FIA
system used for barium sulfate (Figure 1), but, a physiological solution was introduced into
the carrier stream, R1 and R2 channels.
Cell count in Neubauer chamber:Aliquots of 10 until 15 µL of homogenized
suspension were introduced into a Neubauer Chamber5. The count was made through the
stereoscopic microscope (OLYMPUS BX41 and CX40).
Interference studies:The selectivity of the proposed method was tested considering the
composition of studied samples (two ferments and one probiotic samples). Thus, the studied
interferents were: ascorbic acid, folic acid, amide, calcium, sodium chloride, iron,
phosphorous, glucose, hystidine, magnesium, proteins, pyridoxine, potassium and thiamine).
Aliquots of solutions of those interferents were mixed with Saccharomyces cerevisiae
solution (3.83 x 10
9
cells L
-1
). Three interferent concentrations were examinated: the first one
was the same found in the sample and the others were ten times and a hundred times higher
than the first one.
Application in real samples:The samples were prepared adding 2.0 g of the samples to
1000 mL of physiologic solution containing thimerosal and chloramphenicol, under agitation
and constant temperature. After the period of one hour, the samples were determined in the
FIA system. To check the accuracy, the determination by the optical microscope was also
done, leaving an aliquot of 200 µL diluted in 800 µL of the physiologic solution. Soon after,
an aliquot of 20 µL was removed and placed in the camera of Neubauer.
Cells growth studies: Saccharomyces cerevisiae strains had been inoculated into an
assay tube containing 10 mL of Sabouraud broth with chloramphenicol. After 48 hours 1 mL
of the Sabouraud broth was transferred to an erlenmeyer containing a sterile new broth,
totalizing a final volume equal to 50 mL. Samples were kept at 25º C under continuous
shaking throughout the procedure. The microbial growth was followed by the proposed
method, and for this, measurements were done each hour, until the microbial population
reached the steady state of growth phase.
Results and discussion
After optimization, the obtained sulfate analytical curve was: (S = 0.0087C
sulfate
+
0.0005) and the S. cerevisiae analytical curve: (S = 4.01 x 10
-11
C
S.cerevisiae -
0.0018).
The linear equations of both analytical curves have been combined in order to
establish proportionality between the sulfate concentration and the S. cerevisiae
concentration. Thus, the obtained linear equation (C
S. cerevisiae
= 1.088 C
sulfate
+ 0.0023) can be
used to determine the microorganisms concentrations in the unknown samples.
The interference studies: There is no significant interference, based on reduced
increase of the analytical signal (in percentage) of the Saccharomyces cerevisiae solution
(3.83 x 10
9
cells L
-1
). For the concentration two and three the main interference is caused by
protein, nevertheless, these concentrations are not present in real sample, so these results
indicate that analyst should be attentive with this interfering. The accuracy test, presented
below, confirm the no occurrence of interference for the studied samples.
Precision and Accuracy studies: In order to check the accuracy of the proposed
method samples of Saccharomyces cerevisiae were prepared and the results obtained with the
FIA method were compared with the Neubauer Chamber results. According to results there
was no significant difference at 95% of confidence level (paired t-test). In the determination
through FIA system advantageous characteristics were obtained such as high sample
throughput (120 h
-1
), besides the excellent precision (Figure 1a). The limit of detection was
2.53x10
8
cell L
-1
.