CLASSIFICATION OF MICROORGANISMS USING IMAGE PROCESSING
TECHNIQUES
Teresa Alvarez, Yolanda Martin, Susana Pe'rez, Francisco Santos,
Fernando Tadeo, Susana Gonzalez
Department of Engineering Science and Automatic Control, University of Valladolid
Jose' Luis Arribas
Empresa Depuradora de Aguas Residuales, Avila
Pastora Vega
Department of Computer Science and Automatic Control, University of Salamanca
ABSTRACT
This paper presents an approach to the automatic
classification of microorganisms based on image-
processing techniques. A computer application that
processes and classifies the images has been developed.
The classification is carried out using a competitive
neural network. Its input pattern is evaluated from a
frequency analysis of images taken through microscope.
This paper focuses on the application of this technique
to the classification of living diatoms.
1. INTRODUCTION
As it is well known, image processing is a mature area,
which has successfully solved many problems in
different areas, such as Medicine, Astronomy or
Engineering ([21], [ l l ] , [20] and the references therein).
With this work, we propose to use these techniques to
solve a problem in Microbiology: the automatic
classification of microorganisms from their images
taken using light microscopes
The growth of some species of microorganisms is
directly related with the temperature, light intensity and
physiochemical characteristics of the environment (pH,
salinity, the presence of organic matter, etc.).
Environmental changes can be studied using these
microorganisms.
In particular, the identification of the type and
amount of diatoms in a sample eases the calculation of
+the Biological Diatom Index (BDI). The BDI is a
numerical value [ 11 that estimate the water quality based
' on the presence/absence and number of certain species
of diatoms. Diatoms or Bacillariophyceae (Figure 1) are
microscopic unicellular algae with silica shell that
inhabit all aquatic environments [13]. These
microorganisms are chosen as bioindicators because:
- They live in aquatic environments as different as
fresh, salt and brackish waters, running and stagnant
water, dripping and intermittent waters, etc.
0-7803-6725-1/011$10.00 02001 IEEE
- Changes in the quality of the water affect their
numbers and types.
- There are diatoms adapted to many different
pollution levels.
Figure 1: Diatom
TAX'IBD [6] and OMNIDIA [19] are two
examples of programs that can be used to calculate the
IBD, but the classification process is perfomed by the
user (choosing among different images the most
similar). ADIAC [ 5 ] is a European project that has
begun developing tools for automatically classifying
diatoms. Some of the results are presented in [24]. This
work is more a quantitative study than a qualitative one
[24], we focus on identifying and classifying the algae
instead of counting the number of them.
This paper summarizes our study of image
processing techniques applied to the automatic analysis
and recognition of diatom images. It has been developed
a computer application that performs the image
processing and the classification.
Next section describes the approaches followed for
the image processing and the generation of reference
patterns. Then, we justify the reason for choosing a
competitive neural net. Finally some results and
conclusions are discussed.
2. IMAGE ENHANCEMENT
At this stage, the goal is the enhancement of the images
of diatoms because there are more microorganisms and
impurities in the image. Diatoms can be discriminated
very well using their contour. The rest of the body is
transparent and the levels of intensity correspond with
those of the background of the image [ 131 (Figure 1).
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 We have implemented spatial domain methods
([20], [21]) because we consider they are a good
approach for our problem and they are not very
computationally or time demanding. The contrast
stretcing is done block by block. This way, memory
requirements variations in luminosity can be corrected
(from left to right or up-down).
2.1. 'Block' operation
The image is divided in areas of equal size (Figure 2, at
the right hand side, a magnified view of a block can be
observed) that will be called 'blocks'. Each region will
be processed independently. Some advantages are:
Figure 2: Block Obtention
- The decision parameters of the algorithm are
selected from the characteristics of each block. So,
there are no side effects from other areas of the
image that could worsen the result.
- An intensily compensation of the image is done.
- As the me1hod works with subimages, less memory
is needed t13 store the intermediate data generated by
the algorithm.
2.2. Contrast s.tretching
Compensation techniques increase the image contrast
and enhance the non transparent areas of the diatoms. In
particular, contrast stretching increases the dynamic
range of the gray levels in the image being processed.
We have applied a window-level contrast stretching
[20] (Figure 3), such that S = R", S is the final contrast
range, R is the initial contrast range and y i s a positive
parameter called gamma correction.
output 1 4 corpuscle from the other. We use information based on
Figure 3: Contrast Stretching
We have scaled the input range to the total range,
giving as output an image with the microorganisms
scaled towards the black.
2.3. Example
Figure 1 shows a diatom before the image processing
techniques are applied. The histogram (Figure 4) reveals
that the image contains relatively few low or high
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amplitude pixels and that the intensity is concentrated in
a small interval. The processed image (Figure 5) shows
a diatom that stands out the background.
3. DIATOM RECOGNITION
Image recognition ([9], [20], [15], [22]) implies the
existence of some characteristics or descriptors that
allow concluding which kind of object or situation is
represented in the image (in our case, the diatoms).
The ability to perform pattern recognition is
fundamental to image analysis. In our cases, we work
with quantitative descriptions that have been obtained
from the frequency analysis of the image. So, the
patterns we consider are vectors of real values.
10 20 30 40 53 M
Figure 4: Histogram
Figure 5: Processed image
3.1. Generating patterns for diatoms images
In order to recognize the microorganism that appears in
the image, it will be necessary to analyze certain spectral
properties or characteristics that distinguish one
the frequency components of the images, generated from
the power spectrum (or spectral density) of the images.
The power spectrum of bidimensional images
corresponds to a real bidimensional matrix where each
component is defined by:
where
M-I N-l
x=oy=o
the Fqurier transform of the digital image f(x,y) and
F(u,v) is its conjugate [17].
The image input to the frequency analysis is the image
output of the methods presented in 2.1. The 2-D
spectrum is (diagonal inverse) symmetric even if the
component at frequency0 has or has not been moved to
the centre of the frequency plane. So, it is only
necessary to consider one of the spectrum quadrants.
Even so, the features vector is too big and the
classification cannot be done fast enough. For instance,
one of this microscopic images can generate an intensity
vector of 100 to 400 values. We need to obtain some
parameters from the spectrum of the images in such a
way that the features vector decreases its size
dramatically and contains the information necessary to
classify the images. So, we decided to use as parameters
the spectral maximum, the total energy and the
bandwidth where the 95% of the intensity lies (in
horizontal and vertical directions). We found
experimentally that these values summarizes rather well
the features that discriminate one images from other.
Once the features vector has been calculated, the
classification can be done.
4. MICROORGANISMS CLASSIFICATION
4.1. LVQ neural nets
We have chosen neural nets [12], [14] as the
classification tool because they have been applied
successfully to many different problems in pattern
recognition. Moreover, there are promising results in the
analysis of microscopic images in industry: sugar
factories (growth of crystal sugars [lo]), food industry
[7], ....
Some years ago, people that worked on Artificial
Vision preferred "feed-forward'' nets (such as
Perceptron). Then, the self-organized nets [2] spread and
the ART nets [I71 were used as visual patterns
classifiers. During the last years, [2], [3], [23] Learning
Vector Quantification (LVQ) neural nets (Figure 6) have
been used because as it has been reported in the
literature, they give very good results in image
classificationconsidering frequency information.
Competitive
layer
Inputs
Figure 6: LVQ Neural Net
We have used competitive learnig to train the neural
net. In competitive learning only a single output neuron
is active at any one time. This method of learning
divides the input set in groups or classes according to
their properties.
Our net uses a non-supervised algorithm in the
hidden layer (so, the classes can be grouped) and a
supervised algorithm in the output layer (so the user sees
at which class belongs the image).
In a LVQ neural net, there connections among all
the output units and all the input units. The weights of
33 1
these connections and the input vectors belong to the
intervals [0, 11 and [-1, 11. And they are normalized to
unity.
4.2. LVQ nets and diatom classification
There are four values in the pattern, so the input vector
has four elements. The number of neurons in the
competitive layer (hidden layer) and the number of
linear neurons in the output layer are chosen following
the classification goals. For instance, if there are five
types of microorganisms, then there are five output
neurons. The links between the competitive layer and
the output layer establish which and how many neurons
of the first layer belongs to a certain class, the other
links are kept out because they represent links with non
adequate classes.
5. COMPUTER APPLICATION
As it was summarized in the abstract, we have
developed a computer application (Figure 7) that
processes and classifies images of microorganisms. The
tool recognizes successfully the types of diatoms for
which it has been trained.
The user interface has been programmed in C++.
The neural net was trained using the Neural Nets
toolbox of Matlab. We have used Mideva to compile the
Matlab code into C++.
This program performs typical operations such as
rotating, selecting an area or zooming the image, among
others. It is possible to write and print informs and many
more things. But what it is really interesting is that
images of microorganisms are processed via a simple
and not wide used technique (block operation) and
contrast strectched giving as result images where the
diatoms are clearly distinguished by their contour.
The application has been evaluated with several
images. The results have been satisfactory: the diatoms
are correctly classified when they belong to the trained
sets or rejected otherwise.
6. CONCLUSIONS AND FUTURE WORK
Neural nets have proved to be a valuable tool for
classifying water images using their frequency
components. It has also been stated the importance of
the preprocessing stage prior to the feature extraction.
Future works considers the inclusion of the
presence/absence of certain colors in the feature pattern.
Moreover, it is now required that the user rotates the
image until the main axis of the microorganism lies
horizontally. This is cumbersome. A possible approach
is to detect automatically the direction using the Hough
transform. Other solution would be to substitute the
feature pattern with a pattern generated using the radial
frequency vectors (that are independent of the direction)
and the variance along the points at the same distance of
the origin. Other hture work could be the classification
of this type of images considering frequency and
morphologic properties.
 Figure 7 : Computer application
8. ACKNOWLEDGMENTS [ 121, Haykin. Neural Networks. A comprehensive
Foundation. MacMillan College. Publishing Company,
This work has been funded by the Junta de Castilla y 1994.
Leon, project refe:rence C01/200F. A special thanks to
[13] Krammer, K., Lange-Bertalot H.,
Prof CCsar de Prada (University of Valladolid, Spain)
Bacillariophyceae I., 2., 3. and 4. G Fischer Verlag,
for his knowledge and encouragement.
Stuttgart, 1986-91
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