Journal of Molecular Structure 606 (2002) 181188

www.elsevier.com/locate/molstruc

Differentiation and detection of microorganisms using Fourier

transform infrared photoacoustic spectroscopy
Joseph Irudayaraj*, Hong Yang, Sivakesava Sakhamuri
Department of Agricultural and Biological Engineering, The Pennsylvania State University, 227 Agricultural Engineering Building,
University Park, Pennsylvania, PA 16802, USA
Received 19 March 2001; accepted 10 August 2001

Abstract
Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) was used to differentiate and identify microorganisms on
a food (apple) surface. Microorganisms considered include bacteria (Lactobacillus casei, Bacillus cereus, and Escherichia
coli), yeast (Saccharomyces cerevisiae), and fungi (Aspergillus niger and Fusarium verticilliodes). Discriminant analysis was
used to differentiate apples contaminated with the different microorganisms from uncontaminated apple. Mahalanobis distances
were calculated to quantify the differences. The higher the value of the Mahalanobis distance metric between different
microorganisms, the greater is their difference. Additionally, pathogenic (O157:H7) E. coli was successfully differentiated
from non-pathogenic strains. Results demonstrate that FTIR-PAS spectroscopy has the potential to become a non-destructive
analysis tool in food safety related research. q 2002 Elsevier Science B.V. All rights reserved.
Keywords: Fourier transform infrared photoacoustic spectroscopy; Chemometrics; Microorganisms; Pathogen; Apple surface

1. Introduction
Microbial determination is a primary consideration
with respect to food safety and quality in a processing,
retail, or production environment. Conventional
methods for differentiation and identication of
microorganisms depend on biochemical and serological tests that usually involve incubating the culture
in selective agar media or broth for up to several days
and then performing a specic test to determine the
presence of a certain species of bacteria. The risk of
such a lag in determination could potentially deter
timely intervention and appropriate remedial
measures to counteract food contamination. There* Corresponding author. Tel.: 11-814-865-2807; fax: 11-814863-1031.
E-mail address: josephi@psu.edu (J. Irudayaraj).

fore, rapid and direct screening of microorganisms

on food products could be an essential and a valuable
tool to the food industry.
With the improvement in analytical instrumentation, Fourier transform infrared (FTIR) spectroscopy
has been applied to chemically differentiate intact
microbial cells nondestructively, by producing
complex, yet distinct and reproducible biochemical
ngerprints of different bacteria [1]. FTIR spectroscopy has been used to classify microorganisms by
Naumann and coworkers [212]. The authors have
demonstrated its capability to discriminate and
classify intact microbial cells at the strain level [6].
Naumann et al. [9] characterized the infrared spectra
of bacteria into ve spectral areas or windows (W1
W5), which are potentially informative for bacteria
identication. These spectral windows are associated
with specic chemical groups of the different bacterial

0022-2860/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.
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Table 1
Composition of the medium used for different microorganisms
Microorganisms

Medium components for 1 l of water and

incubation temperature

A. niger

Yeast extract 3 g, malt extract 3 g, peptone

5 g, dextrose 10 g, 258C
Potato dextrose agar, 258C
Glucose 20 g, yeast extract 6 g, CaCl22H2O
0.23 g, (NH4)2SO4 4 g, MgSO47H2O 1 g,
and KH2PO4 1.5 g, 308C
Dextrose 20 g, yeast extract 5 g, peptone
5 g, NaCl 5 g, K2HPO4 5 g, 258C
Glucose 20 g, yeast extract 8 g, K2HPO4
0.5 g, CH3COON 1 g, MgSO4 0.6 g,
MnSO47H2O 0.03 g 258C
Yeast extract 10 g, tryptone 16 g, and NaCl
5 g, 258C

F. verticilliodes
S. cerevisiae
B. cereus
L. casei
E.coli HB101,
DH5a, JM107,
JM101, K12
E. coli (O157:H7)

Veal infusion broth: Lean Veal infusion

500 g, bacto proteose peptone 10 g, sodium
chloride 5 g, 258C

components. The authors concluded that the selection

of appropriate spectral windows along with the corresponding weights is essential for spectroscopic
comparison of strains.
Gram-positive and -negative bacteria based on rst
derivatives of W1 region were used for differentiation
using cluster analysis. The authors [11] attributed the
discrimination due to the cell wall constituents of the
two-types of bacteria studied. Within gram-negative
bacteria, the regions of 30502800 and 1500
700 cm 21 were used to differentiate Legionella
pneumophila, Aeromonades, E. coli, Pseudomonas
aeruginosa, and Salmonella typhimurium. This
analysis also demonstrated that closely related strains
of Staphylococcus, Streptococcus, and Clostridium
could be distinguished [6] using the different regions
of the spectra. Legionella produces poly-b-hydroxyfatty acid that has a special effect on the infrared
spectra in the region 1800800 cm 21 while E. coli
has an O-antigen that has a distinct peak in the polysaccharide region from 1200 to 700 cm 21 [6]. Articial neural network (ANN) was applied to build a
hierarchical classication model from the FTIR
spectra of microorganisms [2]. Using this approach,
42 different strains of Pseudomonacae, 33 strains of
Bacillus, 46 strains of Staphylococcus, and six species
and 24 strains of yeast genera Candida were classi-

ed. The critical factors in the analysis of microbial

FTIR spectra according to the authors [2] were data
pretreatment, wavelength selection by a feature
extraction algorithm, type of network, and the
learning function used for the training of ANN.
Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) has been used to obtain chemical
`nger-prints' of the food surface and successive
layers close to the surface with minimal or no sample
preparation [13]. FTIR-PAS measurement is based on
the photoacoustic effect caused when a modulated
infrared beam impinges on a sample surface in a
sealed environment [14]. Photoacoustic signals
proportional to the pressure oscillation due to a corresponding non-radiative de-excitation process of the
modulated infrared beam can then be detected by a
sensitive microphone and transferred into a suitable
spectrum by A/D conversion. FTIR-PAS spectroscopy has been used to directly determine fungi
contamination on the surface of corn [1521]. The
FTIR-PAS spectra of corn with and without the
fungi were divided into 12 distinct areas for identication [19]. The regions in the spectrum were
identied and compared with the relative chemical
compositions of fungi and corn. An ANN model was
trained to distinguish contaminated from uncontaminated corn by pattern recognition. Results demonstrated that FTIR-PAS with its multi-component
analysis capability was more reliable than the bright
greenish yellow uorescence (BGYF) method [20]. In
another experiment, the protein biomass in Saccharomyces cerevisiae and E. coli was measured by FTIRPAS, from the dried microorganisms deposited on a
lter [21]. The increase of protein biomass in the
microorganism during the growth period was
conrmed by FTIR-PAS spectra.
The proposed work is one of the rst applications of
FTIR-PAS to distinguish and characterize microorganism on food surfaces. The overall focus of the
research was to explore the concept of direct examination of apple skin for microorganism contamination
using FTIR-PAS. The specic objectives were (1) to
characterize and discriminate apple skin surfaces with
different microorganism contamination, (2) to characterize fruit surfaces with different E. coli strains, and
(3) to discriminate pathogenic (OH157:H7) from nonpathogenic E. coli strains (JM101, JM107, HB101,
K12, and DH5a).

J. Irudayaraj et al. / Journal of Molecular Structure 606 (2002) 181188

183

2. Experimental

to provide CO2 and H2O free dry air to the interferometer, while helium was used to purge the PAS
detector. The interferometer in the spectrometer can
operate in the rapid and step scans modes. A carbon
black reference was used to collect the corresponding
reference spectra for spectra-intensity normalization.
Aliquots of resuspended microbial suspensions were
evenly placed at the surface of apple, kept at room
temperature to equilibrate, and then used for spectroscopic analysis. The stainless steel PA cell with a
small section of the fruit sample (, 3 3 mm) was
purged for 5 min by helium to provide a CO2 and
moisture free environment. The spectra were
collected by rapid-scan at a speed of 5 kHz scan, a
resolution of 16 cm 21, at 256 scans/sample. Each
experiment was replicated 10 times.

2.1. Preparation of samples

2.3. Chemometric analysis

The microorganisms used in this study were S.

cerevisiae (ATCC 24859), Lactobacillus casei
(ATCC 11443), Bacillus cereus(NRRLB-3711), E.
coli (K-12, DH5a, JM101, HB101, JM107, and
O157:H7), Aspergillus niger(NRRL 326) and
Fusarium verticilliodes(NRRL 13586).
Frozen cultures were thawed and the organisms
were grown in the respective media (Table 1). The
grown cultures were centrifuged at 5000 rpm for
15 min, and washed two times with distilled water.
F. verticilliodes samples were harvested from 10day old slant cultures on potato dextrose agar plates
that has been incubated at 258C, suspended in sterile
distilled water. The concentration of microorganisms
was presented as optical cell density measured by a
Bausch and Lomb Spectronic 20 spectrophotometer at
620 nm (Table 2). Apple skin was sectioned from an
apple at room temperature (,258C) and microorganisms were gently smeared and used for measurements.

Principal component (PC) and canonical variate

(CV) analysis were applied to the spectra obtained
from microbial characterization experiments. windas (Wiley, Chichester, UK) software was used for
discriminant analysis. In general, weighted linear
combinations of the spectra are structured to maximize the differences among the group means, relative
to their variances. The linear combinations of the
variances, known as canonical variates, are plotted
to cluster the data into different groups based on
spectral similarity. Mahalanobis distance (DM)
dened by the matrix Eq. 1 was used for discriminant
analysis [22]. In vector notation, the Mahalanobis
distance, DM between the `jth' observation `xj' and
the `kth' group center `mk' is given by:

Table 2
The concentration of different microorganisms
E. coli strains

Concentration (OD620nm)

A. niger
F. verticilliodes
S. cerevisiae
B. cereus
L. case
O157:H7
K-12
DH5a
JM101
HB101
JM107

0.45
0.30
0.39
0.75
0.29
0.45
0.30
0.30
0.31
0.30
0.32

2.2. FTIR-PAS measurement

A Bio-Rad FTS 6000 FTIR spectrometer
(Cambridge, MA) with a Bio-Rad demodulator and
a helium-purged photoacoustic cell (MTEC Model
100 PA Ames, IA) was used. The FTIR spectrometer
contains a cooled ceramic mid-IR source and a KBr
beamsplitter. A model 75-52 FTIR purge-gas
generator (Whatman, Inc., Haverhill, MA) was used

DM {xj 2 mk }W 21 {xj 2 mk }T 0:5

Where `xj' and `mk' are both 1 r row vectors, and

`W' is the average `within group' covariance matrix,
calculated separately for each group, and `T' is the
transpose. Hence, if there are `g' groups, then `W' is
given by [22]:
g
X

k1

nk 2 1Sk
n2g

Here `nk' is the number of observations and `Sk' is the

covariance matrix of kth group.

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J. Irudayaraj et al. / Journal of Molecular Structure 606 (2002) 181188

Fig. 1. FTIR-PAS spectra of uncontaminated and contaminated apples with different microorganisms.

Fig. 2. Discriminant canonical variate analysis based on the rst two canonical variates of apple with/without microorganisms.

J. Irudayaraj et al. / Journal of Molecular Structure 606 (2002) 181188

185

Table 3
Mahalanobis distances for bacteria and fungi obtained from discriminant analysis of the FTIR-PAS spectra

A. niger
B. cereus
E. coli HB101
F. verticillioides
L. casei
S. cerevisiae
Apple skin

A. niger

B. cereus

E. coli HB101

F. verticillioides

L. casei

S. cerevisiae

Apple skin

1.82

5.89
2.00

7.43
7.00
2 2.18

5.41
6.59
8.11
2.44

8.18
9.01
4.69
8.98
1.87

6.49
6.77
4.36
7.60
4.31
2.61

7.01
5.55
9.15
8.31
11.25
8.22
2.11

3. Results and discussion

In the rst step, apple surfaces smeared with the
different bacteria and fungi (E. coli HB101, A.
niger, F. verticilliodes, B. cereus, L. casei, and S.
cerevisiae) were analyzed, to demonstrate whether
FTIR-PAS could differentiate the different types of
microorganisms. FTIR-PAS spectra of pure apple
skin (or surface) together with the different types of
microorganisms are shown in Fig. 1. The major peaks
that represent an apple skin could be observed around
3372 cm 21 (OH stretching), 2928 and 2856 cm 21
(CH stretching), 1732 cm 21 (CyO stretching),
1557 cm 21 (CyO stretching and CC ring stretching),
1466 cm 21 (CH bending), 1258 cm 21 (COH
bending), 1047 cm 21 (COC stretching), and
723 cm 21 (CH bending). The assignments of peaks
in the FTIR-PAS spectra were consistent with the
assignment given by Silverstein and Webster [23].
Visual appearance of the spectra of the fruit surfaces
with microorganisms are similar to the spectra of pure
apple skin, but on closer inspection slight differences
may be found in the spectra of the different types of
microorganisms. Hence, further interpretation and
examination of such complex, yet, usually similar
spectra requires the application of multivariate statistical techniques.
When discriminant analysis was applied to analyze
the FTIR-PAS spectra of different microorganisms,
the ellipses of clusters in Fig. 2 show the 95% condence regions for each group. In this study, 100%
correct classication was achieved without considering overlap regions. Fig. 2 plots the results obtained
by discriminant analysis based on the rst two canonical variate (CV) scores while the Mahalanobis
distances (DM) between the means of the different

types of microorganisms are listed in Table 3. The

higher the value of the Mahalanobis distance, the
greater is the difference between these groups. The
Mahalanobis distance metric indicates the extent of
similarity or dissimilarity of the different microbial
group. Consequently smaller the distance greater the
similarity, the greater the distance greater the dissimilarity. Comparing Fig. 2 and Table 3, it can be noted
that pure apple skin and L. casei are the farthest apart
and consequently has the highest Mahalanobis
distance value of 11.25. The second highest distance
of 9.15 is between E. coli HB101 and apple skin. The
S. cerevisiae and E. coli HB101 clusters are very close
with the lowest Mahalanobis distance measure of 4.36
and hence overlaps each other signicantly (Fig. 2).
Mahalanobis distance of L. casei and E. coli HB101 is
4.69.
The clusters from the analysis demonstrate that
pure or uncontaminated apple skin could be differentiated from the microorganism-contaminated surface
(Fig. 2 and Table 3). Such classication of the FTIRPAS data using clusters with the corresponding
distance can be a useful tool to quantify not only the
differences but also the similarities of biological
materials. The FTIR-PAS technique with an appropriate analysis procedure can be successfully applied
to characterize a wide variety of microorganisms and
contamination of food surface when properly
calibrated. However, a wide array of tests with
different food surfaces and microorganisms need to
be conducted.
In the second step, FTIR-PAS was used to examine
different E. coli strains, including the pathogenic E.
coli O157:H7 and non-pathogenic E. coli JM107,
JM101, DH5a, HB101, and K12. Fig. 3 shows the
spectra of the different E. coli strains on apple skin

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Fig. 3. FTIR-PAS spectra of contaminated apples with different strains of E. coli.

Fig. 4. Discriminant canonical variate analysis of non-pathogenic strains of E. coli on apple surfaces.

J. Irudayaraj et al. / Journal of Molecular Structure 606 (2002) 181188

187

Fig. 5. Discriminant canonical variate analysis of apple surfaces with pathogenic and non-pathogenic strains of E. coli.

along with a pure apple skin and the key peaks identied are discussed in Fig. 1.
Fig. 4 shows the classication of the different nonpathogenic E. coli strains and Fig. 5 shows the classication of pathogenic from the non-pathogenic
strains. The peaks in Fig. 4 are similar to Fig. 1,
however, a slight shift was observed at the indicated
locations. Further evaluation requires the application
of statistical methods. The Mahalanobis distance
metric in Table 4 quanties the differences between
the different bacterial strains. It should be noted that
the greatest separation was obtained between the E.
coli O157:H7 pathogen and the non-pathogenic

strains considered (indicated by high Mahalanobis

distance values in Column 6 of Table 4). Fig. 5 clearly
shows the separation of the clusters. The similarity of
the non-pathogenic E. coli strains (JM101, JM107,
HB101, K12, and DH5a) appears in the clusters in
Fig. 4. The Mahalanobis distances between the
different non-pathogenic E. coli strains (Column 1
5 of Table 4) are much lower compared to its distance
from the E. coli O157:H7 pathogen (Column 6 of
Table 4). The results shown, present a unique and a
novel approach to discriminate different strains of
microorganisms including pathogenic and non-pathogenic bacteria on food surfaces by direct probing

Table 4
Mahalanobis distances for E.coli strains obtained from discriminant analysis of the FTIR-PAS spectra

E. coli JM101
E. coli JM107
E. coli HB101
E. coli K12
E. coli DH5a
E. coli O157:H7

E. coli JM101

E. coli JM107

E. coli HB101

E. coli K12

E. coli DH5a

E. coli O157:H7

2.44

5.17
2.43

5.78
6.81
2.29

5.98
5.86
6.40
2.44

5.71
5.19
5.54
5.02
2.89

9.58
9.44
10.14
7.59
8.34
2.61

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using FTIR-PAS spectroscopy. The results show

excellent promise and potential in food safety and
quality studies.
4. Conclusion
We have demonstrated that FTIR-PAS with multivariate analysis can be used to discriminate apple
surface contaminated with different microorganisms.
Classication of pathogenic from non-pathogenic
E. coli by direct non-destructive probing was successfully demonstrated using FTIR-PAS and chemometrics. With extensive testing and analysis, the
procedure has the potential to become a rapid
non-destructive tool for bacteria classication and
identication on a food system.
Acknowledgements
The assistance of Dr Chitrita Debroy in supplying
microorganisms and preparing microbial cultures for
analysis is greatly appreciated.
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