International Journal of Advances in Engineering & Technology, Sept 2012.

IJAET
ISSN: 2231-1963

COMPUTATIONAL APPROACH TO COUNT BACTERIAL

COLONIES
Navneet Kaur Uppal1, Raman Goyal2
1

M.Tech Scholar & 2Assistant Professor,

Department of CSE, at LLRIET, Moga, Punjab, India

ABSTRACT
Research involving bacterial pathogens often requires enumeration of bacteria colonies. Bacterial
colony enumeration is an essential tool for many widely used biomedical assays. Bacterial colony enumerating
is a low throughput, time consuming and labor intensive process. Since there might exist hundreds or
thousands of colonies on a Petri dish, and the counting process is often manually performed by well-trained
technicians .There are several methods for enumeration of bacterial colonies. An increased area of focus in
Microbiology is the automation of counting methods. An increased area of focus in Microbiology is the
automation of counting methods. Several obstacles need to be addressed for methods that count colonies
present. These obstacles include: how to handle confluent growth or growth of colonies that touch or overlap
other colonies, how to identify each colony as a unit in spite of differing shapes, sizes, textures, colors, light
intensities ,etc. This method is designed to provide a degree of accuracy in counting. A colony counter is
used to count colonies of bacteria or other microorganisms growing on an agar plate. This method is used to
overcome these obstacles are thresholding, segmentation, time domain frequency, Watersheding, edge
detection and morphology operator, regional descriptors etc.. This method provides high degree of accuracy.
Our proposed counter has a promising performance in terms of both precision and recall, and is flexible and
efficient in terms of labor- and time-savings.

KEYWORDS: Bacteria, Bacterial colony, Thresholding, Morphology, Watersheding.

I. INTRODUCTION
Colony counting of bacteria is crucial for quantitative, precise assessment of pathogens in clinical
research and diagnosis. Manually counting of bacteria colony is very difficult. An automated colony
counting is time saving & less labour intensive process. To provide the fast & accurate result & to
reduce the labour workload two colony counting approaches are developed. These approaches may
utilize both direct and indirect methods of counting colonies. The traditional plate count method is
considered an indirect method, desirable due to its low cost. Some examples of technologies that may
be used for quantification of microbial growth include: ATP Bioluminescence, Spiral Plating,
Membrane Filtration, Direct Epi-fluorescent Filter Microscopy, and Membrane Laser Scanning
Fluorescence Cytometry. Several obstacles need to be addressed for methods that count colonies
present. This method is designed to provide a degree of accuracy in counting that could be correlated
to the counts that would be obtained using a well-trained operator. In the automated approach ,
Bacteria are grown onto filter for 24 to 48 hours to check the contamination level of the sample. To
count these bacterial colonies microbiologist uses some dyes so that bacterial colonies appear as
colored spots and our problem is to count the number of these bacterial colonies. Further in an
Industry thousands of such samples are formed per day and colonies on each sample are counted
manually, then this becomes a time consuming hectic and error prone job. The goal of our proposed
project is to develop software to save time with accurate results and fast delivery to customers. This

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ISSN: 2231-1963
project will be extended in such a manner that it will count the colonies after 6 to 8 hours prior, saving
a lot more time. In this paper the proposed approaches differentiates the processing of colony images
with color information filtering is used for denoising.

II. BACTERIA
Bacteria are unicellular microorganisms. They are typically a few micrometers long and have many
shapes including curved rods, spheres, rods, and spirals. The study of bacteria is bacteriology, a
branch of microbiology. Bacteria are ubiquitous in every habitat on earth, growing in soil, acidic hot
springs, radioactive waste, seawater, and deep in the earth's crust. A group or cluster of bacteria
derived from one common Bacterium. A bacterial cell is a unit of bacterial colony. In colony of
bacteria there can be thousands to millions of cells can be present. The morphology of the colony of
bacteria can be seen with naked eyes without the help of any gear.
A colony counter is an instrument used to count colonies of bacteria or other microorganisms growing
on an agar plate. Bacterial colony is a group of bacteria growing on a plate that is derived from one
original starting cell. An agar plate is a sterile Petri dish that contains a growth medium used to
culture microorganisms. Bacterial colony counting process is usually performed by well-trained
technicians manually.

III.

METHODOLOGY

3.1. Image Capturing

Bacterial Colonies are grown onto filter for 16 to 24 hours. Some colored dyes are spread over each
filter so that bacterial colonies appear as colored spots. Now this filter is kept on a Petri plate.
Background is made of black or white intensity so, it becomes easier to separate the filter from its
surroundings while processing the image. Petri plate is kept in a box containing a digital camera and
light arrangement. Images are then captured using this arrangement. The collected images are
digitized on a computer utilizing a image processing software package that has programming
capabilities. The digitized picture is processed using the various procedures described to separate and
detect the colonies present.

3.2. Wavelet Transform

A discrete wavelet transform (DWT) is any wavelet transform for which the wavelets are discretely
sampled. As with other wavelet transforms, a key advantage it has over Fourier transforms is temporal
resolution: it captures both frequency and location information. The Haar wavelet is a sequence of
rescaled "square-shaped" functions which together form a wavelet family or basis. Wavelet analysis is
similar to Fourier analysis in that it allows a target function over an interval to be represented in terms
of an orthonormal function basis.

3.3. Extracting Image Contents

Extraction of Bacteria present on petri dish is done using various procedures. Extraction procedure
consists of following steps. Each extraction step has its own significance
3.3.1. Conversion of RGB to HSV
When it comes to computer vision, RGB values will vary a lot depending on strong or dim lighting
conditions and shadows, etc. In comparison, HSV is much better at handling lighting differences, and
it gives an easy to use color value. The advantage of HSV is that it provides a way of selecting colors
that is more intuitive to most artists.
3.3.2. Gray Scaling of Image
Grayscale digital image is an image in which the value of each pixel is a single sample, that is, it
carries only intensity information. Images of this sort, also known as blackened-white, are composed
exclusively of shades of gray, varying from black at the weakentsst intensity to white at the strongest.
Grayscale images are distinct from one-bit bi-tonal black-and-white images, which in the context of
computer imaging are images with only the two colors, black, and white (also called bi level or binary

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images). Grayscale images have many shades of gray in between. Grayscale images are also called
monochromatic, denoting the presence of only one (mono) color (chrome).
Image capturing

Wavelet transform

Gray scaling

Applying filter

Thresholding

Thinning

Removing distortion

Eroding

Subtracting from original image

Removal of spurs

Dilation

Edge detection

Watersheding

Segregating overlapped and non overlapped

Non overlapped

Overlapped

Total=overlapped +non overlapped

Fig.1. Flow Work

3.4. Applying Filter for Noise Removal

Adaptive filters adapt or learn the characteristics of the signal. Therefore the adaptive median filtering
has been applied widely as an advanced method compared with standard median filtering. The
Adaptive Median Filter performs spatial processing to determine which pixels in an image have been
affected by impulse noise. The Adaptive Median Filter classifies pixels as noise by comparing each
pixel in the image to its surrounding neighbor pixels. The size of the neighbourhood is adjustable, as
well as the threshold for the comparison. A pixel that is different from a majority of its neighbors, as
well as being not structurally aligned with those pixels to which it is similar, is labeled as impulse
noise. These noise pixels are then replaced by the median pixel value of the pixels in the
neighbourhood that have passed the noise labeling test.
3.4.1. Weiner Filter
The most important technique for removal of blur in images due to linear motion or unfocussed optics
is the Wiener filter. From a signal processing standpoint, blurring due to linear motion in a

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photograph is the result of poor sampling. The inverse filtering is a restoration technique for deconvolution, i.e., when the image is blurred by a known low-pass filter, it is possible to recover the
image by inverse filtering or generalized inverse filtering. However, inverse filtering is very sensitive
to additive noise. The approach of reducing one degradation at a time allows developing a restoration
algorithm for each type of degradation and simply combining them. The Wiener filtering executes an
optimal tradeoff between inverse filtering and noise smoothing. It removes the additive noise and
inverts the blurring simultaneously. The Wiener filtering is optimal in terms of the mean square error.
In other words, it minimizes the overall mean square error in the process of inverse filtering and noise
smoothing.
W ( f1 , f 2 ) =

H ( f1 , f 2 ) S xx ( f1 , f 2 )
2

H ( f1 , f 2 ) S xx ( f1 , f 2 ) + S ( f1 , f 2 )

Where, Sxx(f1,f2), S(f1,f2) are respectively power spectra of the original image and the additive
noise, and H (f1,f2) is the blurring filter.

3.5. Thresholding
It is one of the methods of image segmentation. This method is based on a clip-level (or a threshold
value) to turn a gray-scale image into a binary image. Thresholding can be defined as mapping of the
gray scale into the binary set {0, 1} that is thresholding essentially involves turning a color or
grayscale image into a 1-bit binary image. Individual pixels in a gray scale image are marked as
object pixels if their value is greater than some threshold value and as background pixels
otherwise. Generally the object pixels are black and the background is white. After thresholding a
binary image is formed where an object pixel is given a value of 1 while a background pixel is given
a value of 0. The key of this method is to select the threshold value (or values when multiple-levels
are selected). Several popular methods are used in industry including the maximum entropy method,
Otsu's method (maximum variance), and et al. k-means clustering can also be used. & the main
purpose of thresholding is to discard irrelevant data and keep only the important segments of data
which lie above threshold curve to Classify pixels on the basis of color or a local property. To provide
a bi-level picture that distinguishes objects from background. To represent the text and diagrams in
binary formats.

3.6. Thinning
It is a morphological operation that is used to remove selected foreground pixels from binary images,
somewhat like erosion or opening. It can be used for several applications, but is particularly useful for
skeletonization. In this mode it is commonly used to tidy up the output of edge detectors by reducing
all lines to single pixel thickness. Thinning is normally only applied to binary images, and produces
another binary image as output.

3.7. Morphology
Morphological image processing (or morphology) describes a range of image processing techniques
that deal with the shape of features in an image..The language of mathematical morphology is set
theory. The mathematical morphology simplifies image data, preserves essential shape characteristics
and eliminates noise. Morphological operations are based on simple expanding and shrinking
operations. The primary application of mathematical morphology occurs in binary images, though it is
also used in grey scale images. Morphological operations are typically applied to remove
imperfections introduced during segmentation. Mathematical morphology is a foundation of
morphological image processing, which consist of a set of operators that transform images according
to the above characterizations. The morphological operator dilation acts like a local maximum
operator. Dilation generally increases the sizes of objects, filling in holes and broken areas, and
connecting areas that are separated by spaces smaller than the size of the structuring element. Erosion
acts like a local minimum operator. Erosion generally decreases the sizes of objects and removes
small anomalies by subtracting objects with a radius smaller than the structuring element.

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3.8. Edge Detector
It is a process that detects the presence and location of edges constituted by sharp changes in color
intensity of an image. . For a good edge detection, the edge line should be thin and with few speckles.
The purpose of edge detection in general is to significantly reduce the amount of data in an image,
while preserving the structural properties to be used for further image processing.
Cannys aim was to discover the optimal edge detection algorithm. Sobel and Prewitt which uses first
derivative have very simple calculation to detect the edges and their orientations but have inaccurate
detection sensitivity in case of noise.

3.9. Watersheding
This is method of image processing segmentation. There is a need to split the colony to get correct
colony counts. To separate the connected colonies intensity are considered. Intensity gradient image
as a topological surface, thus Watershed algorithm can be applied to divide the clustered colonies in
the image just as water fold in the topological surface. Think of the grey-level image as a landscape.
Let water rise from the bottom of each valley (the water from each valley is given its own label). As
soon as the water from two valleys meets, build a dam, or watershed. These watersheds will then
define the borders between different regions. Watershed can be used directly on the image, on an edge
enhanced image or on a distance transformed image. A grey-level image may be seen as a topographic
relief, where the grey level of a pixel is interpreted as its altitude in the relief. A drop of water falling
on a topographic relief flows along a path to finally reach a local minimum. Intuitively, the watershed
of a relief corresponds to the limits of the adjacent catchment basins of the drops of water The
gradient magnitude of an image is considered as a topographic surface for the watershed
transformation. Watershed lines can be found by different ways. The complete division of the image
through watershed transformation relies mostly on a good estimation of image gradients. The result of
the watershed transform is de-graded by the background noise and produces the over-segmentation.
Also, under segmentation is produced by low-contrast edges generate small magnitude gradients,
causing distinct regions to be erroneously merged.
Our proposed method can recognize chromatic and achromatic images and thus can deal with both
color and clear medium. In addition, our proposed method can also accept general digital camera
images as its input. The whole process includes detecting dish/plate regions, identifying colonies,
separating aggregated colonies, and finally reporting consistent and accurate counting results.

IV.

RESULTS

TEST 1(a) (Processed image of Non Overlapped Bacterial Colonies)

Figure 1.1 Processed image of Non Overlapped Bacterial Colonies

Figure 1.2 Total number of Overlapped Bacterial Colonies found

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ISSN: 2231-1963
TEST 2(a) (Processed image of Non Overlapped Bacterial Colonies)

Fig. 2.1: Processed image of Overlapped Bacterial Colonies

Fig. 2.2: Total number of Overlapped Bacterial Colonies found

Table 1. Result Image 2. jpg
Sr No.

Fig Name

Overlapped

Total

2.jpg

20

100

TEST 2(b) (Processed image of Overlapped Bacterial Colonies)

Fig. 3.1: Processed image of Overlapped Bacterial Colonies

Fig. 3.2: Total number of Overlapped Bacterial Colonies found

TEST 2(c) (Processed image of Overlapped Bacterial Colonies)

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Fig. 4.1: Processed image of Overlapped Bacterial Colonies

Fig. 4.2: Total number of Overlapped Bacterial Colonies found

TEST 2(d) (Processed image of Overlapped Bacterial Colonies)

Fig. 5.1: Processed image of Overlapped Bacterial Colonies

Fig. 5.2: Total number of Overlapped Bacterial Colonies found

Table 2. Manually Count & Proposed Method Count
SR.NO.

Fig.1.1

TOTAL
COUNT
MANNUALY
64

Fig.2.1

102

370

FIG.
NAME

OVERLAPPED

NONOVERLAPPED

64

PROPOSED
COUNT
METHOD
64

20

80

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ISSN: 2231-1963
3

Fig.3.1

10

10

Fig.4.1

17

14

17

Fig.5.1

59

11

46

57

V. CONCLUSION
From the results and comparison of the different methods of edge detection, it is concluded that the
Computational Bacterial Colony Counter is better than the traditional method of manual
counting of bacterial colonies on petri dish.

120
100
80
Manual Method

60

Proposed Method

40
20
0
1

Figure 6. Proposed count and Manual count

We can design an automated bacterial colony counter which used many image processing algorithms
such as grayscaling, thresholding, filtering, Watersheding etc. to count these colonies efficiently.
Hence, Images with high contrast and low or medium density give accurate or near to accurate count
(99-100%). Images with low contrast or high density give less accurate count (95-98%) as given in
graph. The reason thereof is that in case of low contrast after thresholding shape of colony/colonies
gets distorted which leads to appearance of high curvature points along the boundary. These high
curvature points (corners) get accumulated in count result.

VI.

FUTURE SCOPE

Image processing is very advanced and extensive field which needs extensive research and hard work.
1. This work can be extended to run on different types of micro biological bodies in order to
count and evaluate their different parameters like shape, density etc..
2. Parallel Computing can also be done in order to speed up the process.
3. Process the most complex samples and give accurate count.

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AUTHORS BIOGRAPHY
Navneet Kaur Uppal was born in Punjab, India, in 1988. She received her Bachelor degree
in Computer Science Engg. degree from Amritsar College of Engineering and Technology
in 2009 and persuing the Masters from the Lala Lajpat Rai Institute of Engineering and
Technology .

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