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An Image-Processing Based Automated

Bacteria Colony Counter

Conference Paper September 2009

DOI: 10.1109/ISCIS.2009.5291926 Source: DBLP

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 An Image-Processing Based Automated Bacteria Colony Counter
Hseyin Ate
e-mail: hates@anadolu.edu.tr
Department of Electrical and Electronics Engineering, Anadolu University. 26555 Eskisehir Turkey.
AbstractThis paper presents an image
processing based automated counting system to
detect the number of bacteria colonies that develop
in Petri dishes of microbiology laboratories. The
visible colonies represent the initial number of
bacteria present in the aqueous environment. The
counting system contains shape based segmentation
and classification algorithms. Colonies are
considered as (possibly overlapping with some
amount of amorphous deviations from) discs and
classified as a cluster of bacteria with respect to
their compactness ratio. The system is implemented
using Matlab, and tested using ground truth data
provided from Anadolu University, Dept. of
Environmental Engineering microbiology
laboratory. Results are presented.
1. Introduction
A colony counter is a tool used for
counting colonies of bacteria or other
microorganisms growing on an agar plate (also
called a Petri dish). Classically, the non-
automated counting process consists of
marking off with a felt-tipped pen on the outer
surface of the plate and keeping the marked
number [1]. This manual process is a long and
laborious process that depends on the visual
ability and care of human who counts the
colonies. In addition, especially for very high
number of colonies, manual counting results
can differ among different biologists. This is
due to the fact that, highly populated colony
counting methods mostly use estimation
methods, utilizing a small portion of the plate.
Bacteria colony is a group of bacteria
cells which develop from a single bacteria cell.
Different types of bacteria produce differently
shaped colonies with specific color, structure,
margin, and elevation. In general, colony
shapes are classified to four fundamental
groups; circular, irregular, filamentous and
rhizoid, according to their forms.
978-1-4244-5023-7/09/$25.00 2009 IEEE
18
mer Nezih Gerek
e-mail: ongerek@anadolu.edu.tr
Figure 1: Colony types according to their form.
Naturally, the most encountered type (by
experimenting E.coli) is the circular colony.
Consequently, circular colonies are examined
in this study. This selection reduces the
analysis to a worst case circle detection
problem. However, the problem is kept not to
assume any information about Petri dish image
such as image degradation and noise, size of
colonies, color, etc.
Some automated counters detect
colonies very sensitively by using special
growth medium contains fluorogenic substrates
[9]. The method is very useful for detecting
colonies but fluorogenic substrates are costly.
In recent studies regarding image processing
based colony counters (in fact, circular object
counters), transform based methods, such as
Hough transform based methods are mostly
used in order to extract circular shapes and
curves[2-4].
In this paper, we introduce a robust,
efficient, automated, software-centered and
low-cost counting system. Unlike the previous
methods, Hough transform based methods
were not adopted because of the indistinctness
and deviation of colony size, making the initial
estimate of the colony radius hard to
determine. Size of a colony is directly
proportional the amount of food in medium
and the distribution of the initial bacteria.
Typically, size of colony can vary in wide
range (see Figure 6-b).
As a result of deciding against shape
based transforms, a new two-pass iterative
method is developed. Initially, a simple
binarization is applied using histogram based
threshold determination (here, this step is
notated as Pattern Detector). Following the
September 14-16, 2009
METU
Northern Cyprus Campus
Figure 2: Colony counter flow graph.
binarization process, isolated binary objects are
analyzed according to their size and
compactness ratio to determine how close
the shapes are to the ideal circular colonies (the
operation is denoted by Pattern Classifier in
this work). If a binary shape fails the
compactness test, then the watershed algorithm
is applied to split the corresponding original
(gray) shape to possible colonies (the operation
is denoted by the term Cluster Splitter).
There is a possibility that the result of the
watershed splitting may still not correspond to
circular colonies. The compactness test is,
therefore, applied again. In this second pass, if
the test fails again, then the watershed
parameters are refined and splitting is applied
again. The proposed algorithm stops applying
watershed splitting at this point. However, if
the compactness ratio test continues to fail, an
alternative analysis tool is invoked where the
final (non circular) shapes are considered to
consist of combinations of colony shapes with
known areas ( AC ). Consequently, the number
of colonies inside a non-circular shape with
area ( A ) is assumed to be N A AC in the
module notated as Resulter. The average
colony area is assumed to be the arithmetic
mean of every other colony shape that has been
successfully recognized in the first pass.
Application of watershed algorithm twice
according to the compactness ratio test is found
to be more accurate than a single watershed
segmentation. The accuracy is tested in better
detail in Section 3.
Over 300 sample Petri images were
tagged to form the ground truth data in the
microbiology laboratory of the Environmental
19
Engineering Department of Anadolu
University. Results are compared to the tags
generated by an expert microbiologist and
results of freely available software colony-
counter [8].
2. Proposed System
The proposed system is illustrated in
Figure 2. The system has two main parts;
pattern detector and counter. Counter is kept as
a separate module to obtain a scalable and re-
usable system. Modifications can be applied to
the pattern detector part.
2.1 Pattern Detector
Pattern detector is the first step of the
system. It has three missions; Petri dish
boundary detection, noise removal, and colony
candidate pattern detection. Petri dish
boundary detection is not fully automated. The
program interface asks user for three points on
the boundary of the dish. The parameters of the
circle which encapsulates the dish can be found
by solving the circle equation which passes
through the three points (P1:(x1,y1); P2:(x2,y2) ;
P3:(x3,y3)), producing a circle center, (x0, y0),
and radius, r as the following equations:
E1: (x1-x0)2 + (y1-y0)2= r 2
E2: (x2-x0)2 + (y2-y0)2= r 2
E3: (x3-x0)2 + (y3-y0)2= r 2
Our data set is consist of about 2-3
megapixel photos taken by an amateur-grade
digital camera with a fair amount of mis-focus
and grain noise. Median filtering is used for
noise removal as pre-processing.
Colony candidate detection is a
thresholding process. Otsus threshold
algorithm [6] is used for this process.
 final compactness test is applied, but, this time,
its output is no further processed by the
watershed algorithm. Instead, if the
compactness test still fails, indicating non
circular clusters, they are marked as non-
Figure 3: Pattern detector steps separable, and they are sent to the module of
resulter, which calculates an estimate
2.2 Counter number of colonies using division of the area
Counter is the encapsulating part of the to a normal colony area. Due to personal
system which performs iterative splitting and communications with biologists, this kind of an
counting operations. The pattern detector estimation process was recommended as a
produces raw candidates and its output is far common practice for real life applications.
from being accurate. In fact, the main
detection process is embedded into the
counter part for better accuracy.
The critical and difficult problem is
caused by the fact that, the initial outputs
(clusters) may not always correspond to a
single colony (see Figure 5). The outputs of the
pattern detector (thresholding) are observed to
be fine initial points of the watershed algorithm
which is used to better segment clusters.
Despite this improved accuracy, many of the
relatively large clusters (with more than two
actual colonies) may not be topologically split
into the real colonies using watershed.
Besides, watershed algorithm does not Figure 4: 1-3: colony, 4-5: cluster
guarantee not to separate a colony into two or
more fake colonies, either. As seen in Figure 2, 2.2.2 Cluster Splitter
three states method is developed as a remedy Clusters are split into sub regions by
to such circumstances. Each block in figure is the cluster splitter module where sub regions
presented in the following sub sections. are assumed as colony. Here, the watershed
segmentation algorithm is adopted. A case of
2.2.1 Pattern Classifier watershed segmentation is illustrated in Figure
The examined bacteria colonies have 5. If the cluster consists of a few colonies,
circular shape. The pattern classifier tests this algorithm usually produces plausible
whether a binary shape corresponds to a colony results.
or a cluster according to its, so called,
compactness ratio, also known as the
circularity ratio. The definition of circularity
ratio is the ratio of the area of the closed shape
to the area of a circle (the most compact shape)
having the same perimeter, mathematically
given as;
CR = 4 (area) / (perimeter) 2.
As a shape deviates from circle to rather non-
convex shapes, the value decreases [7]. By
thresholding this ratio for each shape, the
pattern classifier module separates patterns into Figure 5: Watershed segmentation for two merged
2 group; colonies, and clusters of colonies. colonies
In order to improve efficiency, the
pattern classifier test is applied a second time 2.2.2 Resulter
after feeding to the watershed algorithm, which Resulter is the last module of the
splits the non-circular shaped cluster to finer counter. It gets colonies from the previous
colony shapes. Following the second pass, a modules and finds the number of colonies for

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two cases: If the input shape is small and
circular, then the shape is assigned to a single
colony. For larger and non-circular shapes
(non-separable clusters), the number of actual a b
colonies is estimated as the ratio of cluster area
to an average colony area. The average colony
area was automatically recognized by
obtaining the size magnitude corresponding to
the most populated colony sizes, as
suggested by the microbiologists. Integer
division is applied one by one to all
c d
inseparable clusters to minimize error.

3. Experimental Results
In our experiments, we used over 300
images which were taken in different times
using completely different consumer grade
compact digital cameras at resolutions of
3264x2448 and 2048x1536. Dataset is e f
provided from Department of Environmental
Engineering Microbiology Laboratory,
Anadolu University in Eskisehir. All Petri
dishes included colonies grown from the E.coli
bacteria (samples are shown in Figure 6).
The results are compared with (i)
ground truth data, (ii) freely available colony Figure 6: Some sample colony images
counter (clono-counter), and (iii) one-shot corresponding to six different colony formations.
watershed segmentation based counting
method. In order to better visualize the
performance of the methods under different Table I: Colony numbers found by various
circumstances, the data set is split into six sub methods over colony formations indicated in Fig.6.
categories with different colony shape Clono- One-shot Proposed Ground
characteristics. The categories are exemplified counter watershed method truth
by the six sample images in Figure 6, notated
from (a) to (f). a 172 29 32 37
Colony number results are presented in
Table I. A quick inspection of this table b 222 194 282 225
indicates that the proposed two-pass method
c 211 119 149 141
gives plausible results as compared to the
single pass output. Surprisingly, the popular d 347 1018 1375 1825
clono-counter gives unacceptable results in
e 932 527 626 660
several of the cases, making the search for
alternative image processing based methods a f 191 423 431 513
necessary work.

4. Packaging of the algorithm. Despite the difference in the running

In this study, the project is
implemented and tested on Matlab
environment because of its flexibility and
rather intensive library. Obviously, such an all
purpose platform is not suitable to develop a
distributable, fast and user-friendly program.
Therefore, an executable version of the
program will eventually be developed.
environment, a detailed comparison is made
over image processing and/or machine vision
libraries which are under a freely distributable
license like GPL, LGPL, MIT etc. The
platform capability, efficiency, usability and
documentation are listed as follows:

21
AForge:
Compatible Language: CLR capable
languages (Visual c++, C# VB.net etc.)
Platform: Windows (Common Language
Runtime)
Documentation: Complete API documentation
with few example project and article.
Usability: Easy to use and learn.
Efficiency: too slow.
IPL98:
Compatible Language: C, c++
Platform: Cross-platform
Documentation: Complete API documentation
only
Usability: Hard to use and learn.
Efficiency: Not tested.
CVIPTools:
Compatible Language: C, c++
Platform: Cross-platform
Documentation: Complete API documentation
with example projects and a book.
Usability: Hard to use and learn.
Efficiency: Not tested.
OpenCV:
Compatible Language: C, c++
Platform: Cross-platform
Documentation: Complete API documentation
with several projects and pages on internet.
Usability: Hard to use
Efficiency: Very efficient and extensible with
available modules.
EmguCV: EmguCV is .net wrapper of
openCV so it has nearly all advantages of
openCV.
Compatible Language: CLR capable
languages (Visual c++, C# VB.net etc.)
Platform: Windows (Common Language
Runtime), cross-platform capable using
mono-project.
Documentation: Complete API documentation
Usability: Easy to use and learn.
Efficiency: Similar to openCV.
It can be noticed that the algorithms
used in our method (such as Otsus
thresholding, watershed segmentation, median
filter, edge detection etc.) are also available in
several free license libraries. Therefore the
transformation of our method to executable
form is clearly possible. Initial compilation
22
work has started using .net based and Qt based
application structures with openCV libraries.
5. Conclusions and Future Work
In this paper, a new image processing
based method is proposed for robust, efficient,
and automated counting of colonies in Petri
dishes. The system is purely software-centered
and depends on the digital images produced by
consumer grade (cheap) digital cameras. The
proposed system can handle colonies in fairly
noisy images with occasional focus problems.
The main problem of concern was the
existence of clusters of colonies which cannot
be visually split into individual colonies.
Application of more than one method over the
same image produced reasonable performance
in such circumstances.
As a future work, full automation of
the overall process (including detection and
extraction of the plate region) will be
developed. It is also desired to generalize and
develop methods that are not solely specific to
circular colony shapes, in order to handle more
than one type of bacteria colony.
6. References
[1] M. Goyal Machine Vision Based
Bacteria-Colony Counter, Thesis, Electrical
and Instrumentation Department, Thapar
University.
[2] J. M. Bewes ,N. Suchowerska , D. R.
Mckenzie Automated cell colony counting
and analysis using the circular Hough image
transform algorithm (CHiTA), Physics in
Medicine and Biology 2008, vol. 53, no. 21,
pp. 5991-6008.
[3] P. R. Barber, B. Vojnovic, J. Kelly, C. R.
Mayes, P. Boulton, M. Woodcock, M. C.
Joiner, Automated counting of mammalian
cell colonies, Physics in Medicine and
Biology, 2001, vol. 46, no.1, pp. 63-76.
[4] Shih-Hsuan Chiu , Jiun-Jian Liaw, An
effective voting method for circle detection,
Pattern Recognition Letters, vol.26 no.2,
pp.121-133.
[5] Meyer, Fernand, Topographic distance
and watershed lines, Signal Processing, Vol.
38, July 1994,pp. 113-125.
 [6] N. Otsu, A Threshold Selection Method
from Gray-Level Histograms, IEEE
Transactions on Systems, Man, and
Cybernetics, Vol. 9, No. 1, 1979, pp. 62-66.

[7] Compactness measure of a shape, online

http://en.wikipedia.org/wiki/Compactness_mea
sure_of_a_shape.

[8] M. Niyazi , I. Niyazi and C. Belka,

Counting colonies of clonogenic assays by
using densitometric software, Radiation
Oncology 2007, 2:4 doi:10.1186/1748-717X-2-
4.

[9] Colifast, online at http://www.colifast.no/

[10] AForge.Net Framework, online at

http://www.aforgenet.com.

[11] Image Processing Library 98, online at:

www.mip.sdu.dk/ipl98/.

[12]CVIPTools, online at:

www.ee.siue.edu/CVIPtools/

[13] OpenCV Wiki, online at:

http://opencv.willowgarage.com/.

[14] Emgu CV, online at:

http://www.emgu.com/.

[15] Qt software, online at:

http://www.qtsoftware.com/.

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