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Applied Microbiology Group, Craneld Health, Craneld University, Craneld MK43 0AL, UK
School of Biomedical Sciences, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK
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This study describes a method to obtain parameter condence intervals from the tting of
non-linear functions to experimental data, using the SOLVER and Analysis ToolPaK Add-In
of the Microsoft Excel spreadsheet. Previously we have shown that Excel can t complex
26 May 2011
multiple functions to biological data, obtaining values equivalent to those returned by more
Keywords:
correlations between them. Using a simple Monte-Carlo procedure within the Excel spread-
method was the inability to return condence intervals for the computed parameters or the
Monte Carlo simulation
sheet (without recourse to programming), SOLVER can provide parameter estimates (up to
SOLVER
200 at a time) for multiple virtual data sets, from which the required condence intervals
Excel
and correlation coefcients can be obtained. The general utility of the method is exemplied
by applying it to the analysis of the growth of Listeria monocytogenes, the growth inhibition of
Pseudomonas aeruginosa by chlorhexidine and the further analysis of the electrophysiological
data from the compound action potential of the rodent optic nerve.
2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
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2.
For a given data set and a particular model (yt ), the sum of
squares of the errors is given by
SSE =
n
(yi yt )
i=1
n
(Yi yt )
i=1
Another set of virtual data can be generated and the NLR tting
repeated. In the procedure outlined here the sum of squares of
the errors from multiple data sets are summed and the tting
of m-sets of data are conducted simultaneously
SSEtotal =
n
m
(Yi yt )
j=1 i=1
2.1.
Fitting the modied Gompertz equation to
microbial growth data
The modied Gompertz equation is a standard empirical
model for the tting of microbial growth data [5].
(1)
(2)
1
B
(3)
2.2.
Fitting the LambertPearson model to microbial
growth inhibition data
The time taken for a microbial culture to reach a specic optical density (also known as the time to detection, TTD) in the
presence of an inhibitor is dependent on the concentration
and doseresponse of that inhibitor. The LambertPearson
model (LPM, Eq. (4)) [4] describes the visual growth of a culture
as an exponential decay function of the concentration of the
applied inhibitor. A plot of the log concentration against the
relative rate to detection (RRTD, the ratio of the time to detection of the uninhibited culture, or positive control, to the time
to detection of the test culture) gives a characteristic sinusoid,
with inexion at RRTD = 1/exp(1). A linear extrapolation from
this point to the log concentration axis allows the estimation
of the minimum inhibitory concentration (MIC, Eq. (5)), and a
linear extrapolation to the RRTD = 1 axis allows the estimation
of the non-inhibitory concentration (NIC, Eq. (6)), the concen-
c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e 1 0 7 ( 2 0 1 2 ) 155163
if
[x] = 0, 1
else if
then
[x] P2
RRTD =
exp
P1
else if
(1 P2 (ln[x] ln P1 )) < 0, 0
else
(1 P (ln[x] ln P ))
1
1
(5)
P2
1 e
(6)
P2
2.3.
Further analysis of compound action potential of
the rodent optic nerve
The compound action potential from the rodent optic nerve
typically has three peaks (indicating the presence of three populations of axons with different conduction velocities) with a
rapidly decaying transient or artefact from the initial stimulus. Originally this phenomenon was modelled using the sum
of four Gaussian functions, one for each feature of the CAP
(Brown [3]).
CAP =
4
i=1
wi
/2
exp
t c 2
wi
(7)
i=1
Ai
wi
/2
exp
3.
(4)
t c 2
wi
(8)
157
3.1.
NLR: Excel analysis of the growth of L.
monocytogenes at 30 C in high salt media
3.1.1.
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10.00
9.00
8.00
7.00
6.00
5.00
4.00
3.00
0
20
40
60
80
100
Time (mins)
Fig. 2 Plot of the growth data of L. monocytogenes at 30 C
in 9% salt (symbols) with the tted modied-Gompertz
model (line).
3.1.2.
the modelled data was calculated per data set (Cells C115 to
AZ115). The SSE from each data set was summed and this total
value placed in cell C117; SOLVER was used to minimise this
total SSE by changing all the 200 parameters concurrently. This
procedure gave 50 sets of modelled parameters per run. A target SSE of 50 times the SSE obtained from the initial t (cell
B118) was used to monitor the progress of the tting.
After the minimisation procedure, from the tted parameters the mean and standard deviations were found. The
95% condence intervals were calculated from the 95% percentiles using the syntax =PERCENTILE(array, 0.025), and
=PERCENTILE(array, 0.975). The correlation coefcient was
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Table 1a Excel MC (100 iterations), JMP (non-linear regression) and Mathematica MC (10,000 iterations) analyses for the
tting of the modied Gompertz equation to Listeria monocytogenes growth data.
Method
Parameter
Estimate
StdErr
LCL
Excel
MC
A
C
B
M
A
C
B
M
A
C
B
M
3.948
4.896
0.095
30.318
3.951
4.900
0.095
30.341
3.95
4.9003
0.095
30.340
0.0278
0.0520
0.0033
0.2617
0.0279
0.0486
0.0033
0.2858
0.0279
0.0481
0.0033
0.2833
3.906
4.801
0.090
29.766
3.892
4.802
0.089
29.738
3.895
4.807
0.089
29.776
JMP
NLR
Mathematica
MC
UCL
3.999
4.977
0.101
30.792
4.008
5.000
0.102
30.937
4.0048
4.995
0.102
30.882
Table 1b Parameter correlation table obtained using Excel MC and JMP NLR analysis (brackets).
A
A
C
B
M
1
0.697 (0.675)
0.347 (0.357)
0.546 (0.499)
1
0.658 (0.650)
0.135 (0.088)
can be estimated from the parameter data of the MC analysis. For each parameter set the particular biological parameter
was calculated, giving 101 values (including the original tting parameters). From these values the required percentiles
were calculated. In this particular case a re-parameterized version of the modied Gompertz [5] was used to show that the
ranges obtained by running a non-linear analysis using JMP
were equivalent to those obtained directly from the MC analysis (Table 2).
4.
4.1.
1
0.0122 (0.161)
4.2.
Further analysis of compound action potential of
the rodent optic nerve
The LPM (Eq. (4)) can be written in Excel as a nested series of IFstatements (e.g. see Fig. 6). Initial estimates for the regression
parameters can be obtained from an analysis of a plot of the
chlorhexidine concentration against the RRTD (Fig. 5). Using
SOLVER estimates for parameters P1 and P2 and the RMSE were
Table 2 Calculation of biological parameters from the modied Gompertz using Excel MC, compared to the JMP NLR
tting of the re-parameterized modied Gompertz equation for the growth of Listeria monocytogenes at 30 C in high salt
(9%).
Method
Parameter
Estimate
StdErr
Excel
MC
Growth rate
Lag
MPD
Growth rate
Lag
MPD
0.172
19.806
8.845
0.171
19.829
8.850
0.005
0.470
0.039
0.005
0.499
0.036
JMP
NLR
Units. Growth rate: log10 cfu ml1 h1 ; lag: h; MPD: log10 cfu ml1 .
LCL
0.162
18.733
8.765
0.162
18.765
8.778
UCL
0.182
20.589
8.918
0.182
20.878
8.924
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RRTD
1.2
0.8
0.6
0.4
0.2
0
0.01
0.1
10
Chlorhexidine (mg
100
l -1)
the brief stimulus artefact (Eq. (7)). This model was set up in
Excel and the 16 parameters regressed. The sum of squares
obtained was 0.09987; the parameter values for Peaks 1, 2 and 3
were essentially identical to those published (nb, a typographical error in the publication gave the area of Peak 1 as 7.663,
whereas it should have read 0.633). The estimated parameter values for the artefact were, however, different from those
published. The peak area found in this analysis was 5.577 vs.
7.180 found previously and a calculated amplitude of 30.8 vs.
38.042.
The standard error of the t (0.02986) was used to produce
an array (96 124) of normally distributed random numbers.
These values were added to the modelled data (on a separate Excel sheet) to produce 96 virtual data sets (Cells DH5 to
GY128), Fig. 7 (nb, columns and rows have been hidden to
show the full sheet). The model was added to each cell M5 to
Table 3 Excel MC (100 iterations), JMP (non-linear regression) and Mathematica MC (10,000 iterations) analyses for the
tting of the LPM to the inhibition of Pseudomonas aeruginosa in the presence of chlorhexidine.
Method
Parameter
Estimate
StdErr
Excel
MC
JMP
NLR
P1
P2
P1
P2
P1
P2
MIC
NIC
MIC
NIC
7.265
1.150
7.257
1.149
7.257
1.149
17.34
1.630
17.333
1.626
0.093
0.022
0.085
0.019
0.085
0.019
0.428
0.044
0.372
0.037
Mathematica
MC
Excel
MC
Mathematica
MC
LCL
7.109
1.107
7.092
1.114
7.095
1.113
16.61
1.550
16.62
1.554
UCL
7.455
1.187
7.430
1.185
7.427
1.186
18.18
1.709
18.09
1.698
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161
Fig. 6 Spreadsheet used for the Excel MC analysis of the tting of the LPM to the data for the growth inhibition of P.
aeruginosa by chlorhexidine.
Fig. 7 Spreadsheet used for the Excel MC analysis of the tting of a multiple Gaussian function (Eq. (7)) to CAP data.
DD128, the parameters were placed below each set of modelled data: cells M132 to DD143. The calculated SSE between
the modelled data and their respective virtual data set was
placed in cells M145 to DD145. The sum of these SSE was calculated in Cell L146. Due to the SOLVER limit of 200 parameters,
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Parameter
Estimate
Stdev
LCL
Pk1
A1
w1
c1
A2
w2
c2
A3
w3
c3
A4
w4
c4
0.633
0.243
1.397
1.552
0.418
1.875
1.288
0.609
2.566
8.214
0.152
0.883
0.007
0.002
0.001
0.017
0.004
0.001
0.013
0.006
0.004
2.675
0.009
0.021
0.620
0.238
1.394
1.517
0.410
1.872
1.262
0.598
2.559
4.002
0.135
0.863
Pk2
Pk3
Artefact
UCL
0.648
0.247
1.398
1.579
0.423
1.877
1.313
0.621
2.574
10.697
0.162
0.920
Parameter
Estimate
Stdev
LCL
Pk1
A1
w1
c1
A2
w2
c2
A3
w3
c3
D1
D2
0.621
0.238
1.397
1.564
0.421
1.874
1.283
0.607
2.568
4.996
30.906
0.008
0.002
0.001
0.019
0.005
0.002
0.015
0.007
0.004
0.034
0.194
0.606
0.234
1.394
1.522
0.411
1.871
1.254
0.596
2.559
4.928
30.447
Pk2
Pk3
Artefact
UCL
0.636
0.243
1.399
1.595
0.427
1.877
1.313
0.621
2.577
5.058
31.455
4.3.
4.4.
Comparison of the Excel NLR and MC analysis
with Mathematica
Parameter
Estimate
Approx StdErr
Pk1
A1
w1
c1
A2
w2
c2
A3
w3
c3
D1
D2
0.620
0.238
1.396
1.566
0.421
1.874
1.281
0.607
2.568
4.997
30.907
0.009
0.003
0.001
0.019
0.004
0.002
0.015
0.007
0.004
0.034
0.414
Pk2
Pk3
Artefact
LCL
0.603
0.233
1.394
1.529
0.413
1.871
1.252
0.593
2.560
4.931
30.124
UCL
0.637
0.243
1.399
1.603
0.430
1.877
1.312
0.621
2.576
5.063
31.712
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Parameter
Estimate
Approx StdErr
Pk1
A1
w1
c1
A2
w2
c2
A3
w3
c3
D1
D2
0.621
0.238
1.397
1.564
0.421
1.874
1.282
0.607
2.568
4.997
30.925
0.008
0.002
0.001
0.018
0.004
0.016
0.015
0.007
0.004
0.034
0.413
Pk2
Pk3
Artefact
5.
6.
LCL
0.603
0.233
1.394
1.530
0.413
1.871
1.253
0.594
2.560
4.931
30.141
UCL
0.636
0.242
1.399
1.601
0.430
1.877
1.312
0.621
2.576
5.063
31.752
Program availability
Acknowledgment
We would like to acknowledge the nancial support given by
the Food Standards Agency (UK) to Mr. Ioannis Mytilinaios
(post graduate scholarship PG 1025).
references