Bio-Rad Model 550, Ultramark, and Benchmark Microplate Reader Accessories - Manual (Page 11)

Notes:
1. If one of the positive or negative control wells has an out-of-range absorbance
value, the values reported for positive and negative mean and standard devi-
ation will be asterisks. If the out-ot-range value is below -3.500, then the aster-
isks will be preceded by a minus sign (-). For example, the report may show
Pos. Mean *.***
Pos. Dev. *.***
Neg. Mean *.***
Neg. Dev. *.***
Cutoff 1.234
2. If the calculated cutoff value is greater than 3.500 or less than
-3.500,then the report will show
Cutoff *.***
or
Cutoff -*.***
3. If the number of standards is zero for the formula method, then the report
will show the positive and negative means and standard deviations as 0.000.
4. If the number of standards is one in the formula method, then the positive
control mean and the negative control mean will be the absorbance val
ues of the one positive control and the one negative control, respectively,
and the reported standard deviations will be 0.000.
5. If the calculated cutoff value is 0.000, then all readings greater than 0.000
are reported as ‘+’, all negative readings are reported as ‘-’, and all 0.000
readings reported as ‘+/-’.
F. The CONCENTRATION REPORT lists the absorbances of the samples and thencal-
culates concentrations of the samples based on the absorbances of a series of standards.
The on-board software calculates the best fit straight line between each set of two con-
secutive data points in the standard curve of absorbance vs. concen-tration. When there
is only one standard, a line is drawn between the data point and the origin to create a stan-
dard curve. When there is more than one standard, the ori-gin is not considered a data
point. Because of the way in which standard curves are calculated, standard data must be
input in either ascending or descending order (see Section 4.3, Set Concentration Mode).
Consider the case when there are four standards. The software makes a plot of
absorbance vs. concentration and then calculates the three equations (Eq1, Eq2 and
Eq3) which describe the straight lines that join (1) the first (Conc1, Abs1) and second
(Conc2, Abs2) standard data points, (2) the second and third (Conc3, Abs3) standard
data points, and (3) the third and fourth (Conc4, Abs4) standard data points. The
equation used by the software to calculate the sample concentration depends on the
absorbance of the sample. If the sample absorbance is less than Abs1, the software
extrapolates Eq1, the line determined by standard data points 1 and 2. The same
equation is used to determine the unknown concentration when the absorbance of
the unknown is between Abs1 and Abs2. However, Eq2 is used to determine the
concentration of unknowns with absorbances between Abs2 and Abs3, and Eq3 is
used to determine the concentration of all samples with absorbances greater than Abs3.
There are six pre-determined microplate formats associated with the concentration report.
The assignment of the template is made by the software based on (1) the maximum
number of samples, which may be set at 40, 44, 80 or 88; (2) the number of standards,
which range from zero to seven; (3) the number of sample replicates, one or two and (4)
the number of standard replicates, also one or two. The six different formats are shown
in the figures below. Format information is summarized in the following table.
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