PLOS ONE

RESEARCH ARTICLE

Experimental setup and image processing

method for automatic enumeration of
bacterial colonies on agar plates
Lola Hogekamp1, Stefan H. Hogekamp2, Mario R. Stahl ID1*

1 Institut für Lebensmittel- und Bioverfahrenstechnik, Max Rubner-Institut, Karlsruhe, Germany, 2 PALAS
GmbH, Karlsruhe, Germany

* [email protected]

a1111111111
a1111111111
a1111111111 Abstract
a1111111111
a1111111111 Automated colony counting methods have long been known in Microbiology. Numerous
methods for automated image analysis have been described and a wide range of commer-
cial products exists. Known advantages are saving cost by reducing enumeration time, auto-
matic documentation, reproducibility, and operator independence. Still, even today the
OPEN ACCESS
realization of all advantages of automated image analysis makes it necessary to either
invest in an expensive, high performance commercial system, or to acquire expert knowl-
Citation: Hogekamp L, Hogekamp SH, Stahl MR
(2020) Experimental setup and image processing edge in image processing. This is a considerable obstacle for many laboratories, and the
method for automatic enumeration of bacterial reason why manual colony counting is still done frequently. This article describes an easy to
colonies on agar plates. PLoS ONE 15(6): apply automatic colony counting system–including suggestions for sample preparation–that
e0232869. https://doi.org/10.1371/journal.
can be put into operation with basic knowledge of image processing and low budget.
pone.0232869

Editor: Abdelwahab Omri, Laurentian, CANADA

Received: May 3, 2019

Accepted: April 23, 2020

Introduction
Published: June 24, 2020
Reliable and exact quantitative analysis of microorganisms in liquids is an important part of
Copyright: © 2020 Hogekamp et al. This is an open
access article distributed under the terms of the
the daily work in the authors’ laboratory. Plate counting of, e.g., Escherichia coli is a recurring
Creative Commons Attribution License, which task; the strain E. coli DH5α in particular is used as biological indicator for the efficacy of
permits unrestricted use, distribution, and UV-C treatment of liquids. The results play an important role in the assessment and design of
reproduction in any medium, provided the original processing steps.
author and source are credited. Plate counting is carried out on samples prepared by surface inoculation according to DIN
Data Availability Statement: All relevant data are standard [1]. Samples from a dilution series (e.g., 1:103, 1:104, 1:105) are inoculated on broth
within the manuscript and its Supporting plates. The original concentration is calculated from the count of colony forming units (CFU)
Information files. carried out after a given incubation period. Manual CFU counting is still standard. This, for
Funding: The authors received no specific funding once, cannot be easily repeated since the colonies will continue to grow unless the plates are
for this work. The purchase of equipment for this kept in cool storage, where space is limited. Also, there is usually no photographic documenta-
study was funded by Max Rubner Institut. Author tion but only the CFU number noted down by the counting person.
SH received support in the form of salary from the
Exact counting is easily done for high dilution, at the cost of statistical significance [2, 3].
private, commercial company PALAS GmbH
(https://www.palas.de/en/). PALAS GmbH had no
Also, dilution errors take influence. Assessing at lower dilution provides better statistical
role in study design, data collection and analysis, safety, but the colonies can be so numerous that they become indistinguishable and the count
decision to publish, or preparation of the value becomes subjective [2]. Ideally a sample is assessed at a dilution that leads to a colony

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

manuscript. The specific roles of this author are count per plate in the order of magnitude of 102. According to Breed and Dotterrer [4], satis-
articulated in the ‘author contributions’ section factory results can be achieved at up to 400 colonies per plate.
Competing interests: The authors have read the Manual counting gets even more difficult if, for saving cost, samples from several different
journal’s policy and have the following competing dilution levels are inoculated on segments of a single nutrient plate. Fig 1 shows a plate used
interests: author SH is employed by the private, for enumeration at three different dilution levels. At the lowest dilution the available space is
commercial company PALAS GmbH. There are no
already densely covered by colonies. In such a case it is particularly difficult to determine CFU
patents, products in development, or marketed
products to declare. This does not alter the number without subjective influence and with sufficient statistical reliability. Due to the
authors’ adherence to all PLOS ONE policies on reduced area per dilution stage colonies must be counted after a short incubation period;
sharing data and materials. counting a large number of small objects, however, is time consuming [5] and tires the assess-
ing person quickly.
Based on the recommendation of no more than 400 colonies per full plate [4], any segment
of a plate split in six segments as shown in Fig 1 should contain no more than 70 colonies. This
is, however, difficult to achieve, as the initial microbial load is unknown. Laboratory practice
therefore often requires enumeration of non-ideal plates like the one shown in Fig 2 with a
count of approximately 300 in the segment.
In principle automated colony counting, as various publications suggest [1, 6–9], increases
laboratory productivity considerably. Ideally there will be fewer faults, improved reproducibil-
ity, and the results become independent of the training level of the person who carries out the
assessment. An image showing the distribution of the colonies is created and stored digitally.
If required, this image can be assessed again automatically or manually at any time, which is a
significant advantage. Image processing is challenged by variations in colony size, shape, color,
contrast, and density as well as by confluence, so some experience in image processing as well
as some effort for developing a suitable procedure are required. The effort for finding a solu-
tion for the well defined, repetitive task presented here appeared to be, however, manageable.
Initial tests with the public domain image processing software package ImageJ showed that
recording a digital image and CFU enumeration in software, executing all steps manually,
required less time compared to manual counting for CFU numbers as low as 50 per plate pro-
vided that predefined regions of interest (defining the area to be processed) could be used–
experienced persons count approximately 2–3 colonies per second by marking & taking notes
(see "Results" section below) [8]. At this stage it was estimated that fully automated processing

Fig 1. Sample Petri dish. Petri dish split in six segments for inoculation with sample liquids of three different dilution
levels (2 repetitions per dilution). Regions of interest for CFU counting outlined.
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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Fig 2. One segment enumerated on a segmented plate. Sample inoculated with E. coli DH5α, enumerated by point-
and-click on screen using ImageJ.
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of plate images might lead to a reduction in processing time by one order of magnitude com-
pared to manual counting, with the additional benefit of simultaneous digital documentation.
This prospective increase in efficiency was the main motivation for further evaluation of auto-
mated counting methods.
At the Max Rubner-Institut automated counting methods had been investigated previously;
one major challenge at the time had been satisfactory processing of segmented plates, contain-
ing multiple subsamples from different dilution levels. Commercially available colony counters
in the midrange price segment, which perform digital imaging and automated counting, had
been rated at the time to be unsuitable due to lack of flexibility.
Presently the purchase cost of components for industrial image processing has fallen due to
the widespread use of this technology, down to a level which allows the design of high quality
systems even with a budget as small as €2,000 –the amount available to us for this project. Soft-
ware that allows processing of images in seconds even on economically priced personal com-
puters is available commercially as well as in the public domain (on which we focused due to
budget restraints).
We thus aimed at developing an image analysis process for counting E. coli DH5α colonies,
which should be simple and robust so that non-expert operators could apply it, preferably
without parameter adjustment, even to segmented plates. Part of the project was determining
optimized steps for plate preparation that would simplify the subsequent image processing.

Materials and methods

A review of commercially available products for plate enumeration (optical counting aids,
compact automatic counters, and fully programmable image analysis systems) led to the con-
clusion that commercially available solutions would not meet our budget. While even mid-

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

priced products (order of magnitude 10k €) seemed to be not sufficiently flexible, fully config-
urable systems appeared to be suitable but far too expensive for our task. We therefore
designed a system of our own using individually selected components, as described below.
A review of the features of commercial as well as public domain software for colony count-
ing like NICE (NIST’s Integrated Colony Enumerator) [10, 11] and OpenCFU [12] showed
that a universally configurable software package would be required. We decided to use ImageJ
(version 1.51 64bit for Microsoft Windows1), a popular scientific image-processing tool. Ima-
geJ allows step by step manual processing as well as scripting for automation and linking of
external modules, and is therefore suitable for experts as well as for beginners who can easily
comprehend the effect of a processing step.

Petri dish preparation

Simplicity of enumeration by image analysis greatly benefits from careful preparation of the
Petri dishes. Effects caused by bubbles and foreign particles in the nutrient can, if at all, only be
compensated with substantial effort in the processing of the image. One should also aim at
constant plate quality, i.e., identical amounts of nutrient and constant layer thickness by pre-
cise horizontal alignment when pouring the dishes.
Colony clusters on the rim of the Petri dish are a substantial obstacle for automatic count-
ing (true for manual counting as well). Various, in some cases quite complex algorithms have
been published for treating clusters. Since we intended to develop a simple method it appeared
to be more worthwhile to try and avoid clusters in the first place by choosing an appropriate
inoculation method.
For this purpose lines drawn on the backside of each Petri dish (see Figs 1 and 2) indicate
borders, which are not to be crossed during inoculation. If this is duly observed (e.g., with the
help of a jig), colonies only grow within the outlined areas. These can subsequently be pro-
cessed as separate enumeration areas by the image analysis software by defining respective
regions of interest (ROIs). The lines facilitate alignment of the dish prior to image recording,
making sure that inoculated areas are located within the ROIs of the processing software. In
radial direction these regions end a few millimeters short of the rim of the Petri dish, prevent-
ing the formation of colony clusters in this area. Avoiding the dish edges also means avoiding
the possibly thicker broth layer around the rim of the plate, thus achieving a more uniform
background brightness of the image.

Camera setup for image acquisition

Results of preliminary tests (on digital images recorded in grayscale and RGB color at resolu-
tions between 300 and 800 dpi using a Canon EOS 500D 15 MP SLR camera) suggested gray-
scale digital imaging of the Petri dishes at a detail resolution of approximately 40 μm in order
to analyze colonies down to 0.5 mm diameter. This corresponds to an image of approximately
2,500 by 2,500 pixels for a 100 mm dish. A digital camera with 10 MP sensor (format: 3,840 by
2,748 pixels) of type Basler acA3800-14um with USB3 interface was chosen. This camera is a
true grayscale camera, i. e., the sensor is not covered by a RGB filter pattern. Though RGB
cameras are frequently used as an image source for grayscale processing, the typically used
Bayer filter pattern reduces spatial resolution for the R and B channels to one quarter and
halves it for the green channel. Image analysis tasks which do not require differentiation by
color are thus preferably solved using grayscale cameras; should filtering by color become nec-
essary again one can still attach a suitable high, low, or band pass lens filter.
Effective use of the resolution offered by a camera’s sensor requires a lens that matches the
sensor regarding image circle diameter and contrast performance. The usually applied limit is

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

50% contrast at a line pairs per mm value corresponding to the sensor’s pixel pitch (a pixel
pitch of, e.g., 1.6 μm equals 300 lp/mm). The setup shown here employs a Basler C125 lens of
16 mm fixed focal length and f/1.8 maximum aperture. Nominally, contrast performance and
image circle of this "5MP" lens are slightly below the optimum for the selected camera. This,
however, would mainly affect the corners of the image, which were partially cropped in the
first place (the sensor’s 3,840 by 2,748 pixels were cropped to 2,748 x 2,748 pixels), and also are
not part of the regions of interest since the Petri dish is circular. Furthermore, stopping the
lens down to at least f/2.8 improves the contrast performance of the lens and equalizes back-
ground brightness as well [13]. This lens-camera combination is therefore well suited for
recording circular Petri dishes and, at approximately €500 purchase cost, economically priced.
A common LED powered photographer’s backlight for film and slide viewing is used for
creating a transmissive illumination, which follows the suggestions published, e.g., by Chiang
et al. [14]. The panel we used (M.WAY size A4 LED light panel, EAN 4710956777647) emits
flicker free light at constant brightness over an area much larger than a Petri dish. The dish is
kept in a fixed position at the center of the light panel by use of a jig and is illuminated uni-
formly. Fig 3 shows the final setup used.
The chosen camera comes complete with software (Basler pylon for Windows1), which
allows parameter configuration and saving of images to disk. Recording parameters can be
saved permanently in the camera allowing constant operating conditions without need of
reconfiguration after startup. Optionally, direct camera control and image acquisition from
within ImageJ is possible by using a software plugin made by Phase GmbH, Lübeck. This
allows processing the recorded image immediately without previous saving to disk. Software
was installed on a standard office PC equipped with 8 GB RAM and an Intel1 CoreTM i3 CPU
running Windows1 10, which was purchased for approximately €500.

Image processing and colony enumeration

The camera used in our setup allows recording images with a brightness resolution of 8 or 16
bits per pixel. Both data formats can be processed in ImageJ. Changes of brightness and con-
trast in an 8-bit image will produce gaps in the brightness histogram, which can be avoided by
performing all image manipulations on a 16-bit image. However, working in 8 bit returned
results equivalent to the gold standard method (see below in “Results”) and thus all subsequent
experiments were carried out on images of 8-bit grayscale depth.
Two assumptions were made in order to simplify processing of the recorded images:
• The size of the detected / counted objects is irrelevant, only their number needs to be deter-
mined exactly. This makes isolating the objects from the background of the image consider-
ably easier. Instead of thresholding and segmentation, which is difficult for low contrast,
confluent objects and proved unsatisfactory in preliminary tests, image processing focuses
on identifying objects as features that cause a local brightness extreme. This approach can
also detect multiple extremes in a neighborhood. Compared to complex strategies like fuzzy
logic based detection [15] the solution suggested here is quite simple.
• Objects of interest (the CFUs) appear generally darker than the (local) background and each
CFU possesses one darkest spot, which marks the center. This is in principle already
achieved by the design of the illumination, although some post-processing of the image is
required for noise suppression.
Taking the digital image and saving it to disk typically required less than ten seconds during
our preliminary tests. Counting the colonies by image processing (sequential execution of one
command after another) produced the result within less than ten seconds as well. For all

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Fig 3. Camera setup. Grayscale digital camera for industrial image processing and LED backlight installed in a protective housing. PC screen displays predefined regions
of interest for a Petri dish split into six segments.
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comparisons discussed subsequently we thus generally assume that complete digital processing
of a Petri dish by an operator with some routine requires about 20 s.
The processing steps that lead from the digital image to the CFU count are described one
by one below. Fig 4 illustrates how the various steps affect the content of the region of interest.
As a first step the area to be processed and enumerated was selected in the recorded image.
This is particularly important for the subsequent automatic contrast adjustment, which would
be hampered by the presence of, e.g., marks or text written in black ink. Since the inoculation
areas as well as the position of the Petri dish for imaging were fixed we could predefine regions
of interest (ROIs), and no readjustment by the operator was required. In Fig 1 such regions of
interest are shown, outlining the six segments of the plate.
In the following step the background of the selected region(s) of interest was smoothed by
using ImageJ‘s Subtract Background. . . command (compare Fig 4A and 4B to see the effect).
This function applies the Rolling Ball algorithm with a fixed radius, which should be larger
than the largest object to be detected. The E. coli DH5α samples investigated here had been

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Fig 4. Sequence of processing steps. From top left to bottom right: a) original image, b) after background removal, c)
after automatic contrast expansion, d) median filtered, e) after increasing image brightness (additional background
removal step), f) with indicator spots for detected objects.
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incubated for 24 h at 37˚C, forming colonies smaller than 2 millimeters in diameter. We used
a radius of 50 pixels for background subtraction, equivalent to 4 mm maximum object diame-
ter (preservation of larger objects may require that the radius value is increased accordingly),
to accommodate for different colony growth rates. Uniform results were achieved for varying
background brightness (caused, e.g., by variable thickness of broth layer).

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

After background elimination we expanded image contrast by using ImageJ‘s Brightness/

Contrast command in automatic mode. Applying this function spreads the gray values of the
image across the available dynamic range, independent of the dynamic range in the original
image (compare Fig 4B and 4C to see the effect). Background removal plus contrast expansion
thus equalize images that may have initially differed in brightness. Proper contrast expansion
requires that any gray value in the image represents either a part of the remaining background
or, respectively, of the objects to be detected–no dark spots caused by impurities, smudging or
writing must be present.
At this stage an image still contains residual noise in the background and within the objects
to be detected and counted. In the next step thus noise was removed by median filtering.
Median filtering suppresses noise in digital images much more efficiently than application of
the Gauss or the average value filter while preserving edges and removing outliers (compare
Fig 4C and 4D to see the effect). In our preliminary experiments we also tested other noise
removal methods but, as expected, median filtering turned out to be the most suitable. Below
the approximate size of the work area of the median filter objects get eliminated, which is
sometimes regarded as a disadvantage [16]. This behavior, however, can also be used inten-
tionally to exclude microcolonies. The chosen operating range (working radius of 6 pixels,
equal to approximately 0.25 mm) corresponded to the minimum size of objects that would
have been considered in manual counting.
Finally, the remaining background features had to be removed from the filtered image.
Since the final counting step required a grayscale image thresholding could not be applied.
Therefore a constant brightness value was added to all pixels of the image in order to lift the
background, still containing low contrast artifacts, above the numerical brightness maximum
(255 at 8 bit depth). Good results were achieved by raising image brightness by 135, so this
value was generally applied. Compare Fig 4D and 4E to see the effect; the background area is
uniformly white and the colonies appear lighter, but still with a grayscale structure.
After these processing steps the images showed, in front of a uniform white background,
objects which had originally been larger than the minimal size defined by the median filter.
The CFU number was then determined by counting local brightness minima in the image
using ImageJ’s Find Maxima. . . command (using the Light background option). Fig 4F shows
the result: In this example, the detected center points were copied into Fig 4E.
The image processing method described above eliminated background artifacts and
ensured that a single local density maximum represented the center of a CFU. Still, clustered
colonies show multiple local density maxima, i.e., multiple CFUs may be recognized up to a
certain degree of overlapping.
Fig 5 shows two examples of non-segmented plates from a series of images recorded during
the test phase of the image processing sequence at approximately 600 dpi resolution, using a
digital SLR camera. The images were first enumerated manually on-screen by pointing/click-
ing in order to obtain the "gold standard" reference count; the results were 136 (left plate) and
137 (right plate). The respective automatic counts (obtained by processing the image as
described below) were 134 and 139. Manual enumeration of plates like those in Fig 4 required
time in the order of magnitude of one minute per plate even though the diameter ratio of larg-
est to smallest colony was approximately 4, facilitating manual counting. For more details see
below in “Results”.
If the agar plate is segmented counting becomes more challenging, as shown exemplarily in
Fig 2 where approximately 300 colonies mark the practical limit of manual counting. Auto-
mated counting by image processing, however, appeared to be still feasible for such high CFU
numbers. Fig 6 shows an automatically counted segment with almost 200 colonies.

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Fig 5. Manual and automatic enumeration of two non-segmented plates. Top row: original images, middle row: enumeration result of point/click method (counts: 136,
137), bottom row: enumeration by image analysis (counts: 134, 139). Samples inoculated with E. coli DH5α.
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The above-described method resulted in better recognition of confluent colonies than

methods employing binary thresholding and segmentation (using, e.g., watershed separation),

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Fig 6. Example enumerated segment. CFU count of a densely populated 1/6-plate segment, determined by image processing.
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which we tried as alternatives. Advanced shape specific filtering, e.g., Hough transform, might
lead to equivalent or better results [17], but this would be beyond the scope of the simple pro-
cedure we intended to create.
In order to compare the automatic enumeration systematically with the previous counting
method (marking CFUs with a pen on the dish lid) as well as with a reference standard incubated
Petri dishes from multiple series of experiments were then enumerated in three different ways:
• Semi-manual counting by clicking on the colonies as viewed in a digital image of the Petri
dish on a computer screen, in 1:1 representation (screen pixel: image pixel). Numbers were
recorded by software (ImageJ offers specific functions for point click counting of objects).
This represented the "gold standard" and was done by three skilled persons.
• Manual counting by marking colonies on the lid of the Petri dish with a felt marker–the per-
sons performing the task kept the count in memory until finishing one plate (or segment) or
took notes. Three skilled persons enumerated the plates.
• Automatic enumeration using a macro (predefined set of commands) in ImageJ. This macro
comprised the digital image processing steps as described above. All images were processed
using the same parameter set. Automatic enumeration was not repeated; since all parameters
were predefined neither count nor processing time varied (this had previously been estab-
lished by repeated enumeration of several Petri dishes).
For each enumerated plate the CFU count as well as the time required were noted. Prepara-
tion times are not considered here–if large numbers of plates are processed the time required
for daily startup of the camera system is insignificant.

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Results
The above-described method of enumeration by image processing was developed over a
period of several months, using sample Petri dishes as they became available from ongoing
experiments. Once the method had been established and the camera setup had been finalized,
additional experiments were carried out on a number of plates in order to determine count
repeatability and counting time in comparison with traditional methods. About half the num-
ber of plates were inoculated only with one sample on the full area, while the others were split
in six segments (two segments respectively for three dilution levels). The results are discussed
below.
Table 1 shows numerical values of the count ratios, i.e., the CFU numbers determined by
automatic (image analysis) and manual (pen marking) enumeration in relation to the gold
standard count (on screen click marking on a high resolution image). The table also lists the
number of colonies per plate and the counting rates achieved by manual and gold standard
counting. In the case that ROIs were enumerated that represented a 1/6-segment of a plate, the

Table 1. Overview of enumeration results. CFU numbers determined by manual, automatic and gold standard counting of plates. Count ratios of automatic and manual
enumeration in relation to the gold standard method. Count rates of manual and gold standard enumeration. Counts and counting rates in rows 1–25 are based on three
enumerations each.
# Counts/s Count Counts/s Count Count Count ratio Count ratio Segments
Gold Gold Manual Manual Automatic Auto/Gold Manual/Gold /plate
• • • • • • • •
1 1.14 ±0.03 174.0 ±0.0 1.87 ±0.25 168.0 ±6.0 174 1.00 0.97 6
2 1.31 ±0.08 210.0 ±0.0 1.73 ±0.17 208.0 ±3.5 198 0.94 0.99 6
3 1.16 ±0.21 176.0 ±3.5 1.88 ±0.47 188.0 ±13.9 198 1.13 1.07 6
4 1.52 ±0.03 234.0 ±0.0 2.09 ±0.08 196.0 ±12.5 210 0.90 0.84 6
5 0.72 ±0.11 754.7 ±3.8 1.52 ±0.27 714.3 ±22.9 733 0.97 0.95 1
6 1.34 ±0.19 612.0 ±19.0 1.65 ±0.35 578.0 ±15.0 583 0.95 0.94 1
7 1.38 ±0.09 579.0 ±3.5 1.63 ±0.23 547.7 ±26.3 566 0.91 0.88 1
8 1.35 ±0.17 67.3 ±2.3 1.56 ±0.63 63.0 ±5.2 70 1.04 0.94 1
9 1.17 ±0.19 14.0 ±0.0 1.91 ±0.66 14.0 ±0.0 12 0.86 1.00 1
10 0.82 ±0.28 316.0 ±3.5 2.00 ±0.38 284.0 ±9.2 318 1.01 0.90 6
11 1.03 ±0.06 396.0 ±0.0 2.67 ±0.51 304.0 ±12.5 396 1.00 0.77 6
12 0.99 ±0.07 288.0 ±0.0 1.52 ±0.08 276.0 ±6.0 288 1.00 0.96 6
13 0.80 ±0.07 418.0 ±3.3 3.21 ±0.67 404.0 ±12.5 414 0.99 0.97 6
14 0.80 ±0.16 456.0 ±0.0 2.49 ±0.33 364.0 ±9.2 408 0.89 0.80 6
15 0.97 ±0.10 510.0 ±0.0 2.57 ±0.60 442.0 ±15.1 468 0.92 0.87 6
16 1.03 ±0.06 81.3 ±1.1 2.19 ±0.60 76.7 ±2.5 80 0.98 0.94 1
17 1.08 ±0.31 149.7 ±1.5 1.84 ±0.46 141.0 ±8.2 150 1.00 0.94 1
18 0.75 ±0.11 288.0 ±0.9 1.83 ±0.22 272.7 ±3.8 289 1.00 0.95 1
19 0.87 ±0.26 246.7 ±2.0 1.72 ±0.08 210.3 ±23.2 235 0.95 0.85 1
20 1.28 ±0.16 615.3 ±18.5 1.63 ±0.29 564.0 ±7.9 593 0.96 0.92 1
21 1.50 ±0.28 555.0 ±10.5 1.40 ±0.27 459.0 ±149 547 0.99 0.83 1
22 1.01 ±0.11 300.0 ±0.0 1.78 ±0.24 288.0 ±6.0 300 1.00 0.96 6
23 1.14 ±0.30 300.0 ±0.0 1.35 ±0.18 276.0 ±6.0 300 1.00 0.92 6
24 1.20 ±0.0 230.0 ±3.5 1.48 ±0.0 204.0 ±10.4 210 0.91 0.89 6
25 1.21 ±0.12 284.0 ±3.4 2.01 ±0.05 274.0 ±3.5 270 0.95 0.96 6
• • • • • • • •
Average • Average • • Average Average •
1.1/s • 1.9/s • • 0.97 0.92 •
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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

respective CFU count was multiplied by 6 in order to make the results comparable with enu-
meration of complete plates. Counts and count rates are averages of three enumerations each.
Fig 7 shows results of automatic and manual counting, plotted against the gold standard
method, respectively. Horizontal error bars indicate the standard deviation of the three gold
standard repetitions. Vertical error bars in the right diagram indicate the standard deviation of
manual counting. Automatic enumeration (left diagram) always returns the same result for a
given sample, i.e., the y error is zero for all automatic counts. The gold standard method also
returned three identical counts (i.e., zero x error) for 10 out of the 25 samples.
A correlation analysis was carried out, assuming that for small CFU numbers the methods
must match (i.e., forcing the regression lines through the origin as drawn in Fig 7). This
restraint, along with the fact that the standard deviation for some of the individual data points
was zero in either one or both directions, turned out to be a difficulty for linear regression
including error analysis. Instead of using the individual x and y standard deviations of each
data point in the analysis we had to assign average percentaged errors. The gold standard
counts on average had a variation coefficient of 1%, which was assigned as standard x error to
the gold standard values. The manual counts on average had a variation coefficient of 5%,
which was assigned as standard y error to the manual values. A fictitious variation coefficient
of 1‰ was assigned as standard y error to the automatic values. Solutions could then be calcu-
lated using Levenberg-Marquardt’s algorithm.
For manual vs. gold standard a slope of 0.913 with a 95% confidence interval of CI95% =
(0.896, 0.93) was determined. Automatic vs. gold standard correlated with a slope of 0.976 and
CI95% = (0.972, 0.98). For the experiments in this study automatic counting results would thus
be equivalent to the gold standard if multiplied by 1/0.976 = 1.025, and manual results would
be equivalent if corrected by a factor of 1.095.
Fig 8 shows the observed counting rates (markings per second) for manual and gold stan-
dard enumeration. Correlation coefficient values close to zero indicate that there is no relation

Fig 7. Method correlation. CFU counts for 25 samples of plates inoculated with E. coli DH5α. Left diagram: automatic vs. gold standard counts, right diagram: manual vs.
gold standard counts. Error bars in x and y directions represent the standard deviation of three enumerations.
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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Fig 8. Counting rates. Rates (markings per second) for manual and gold standard CFU enumeration achieved for all plates enumerated for this study. All test were carried
out on plates inoculated with E. coli DH5α. Each data point represents the average of three enumerations.
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between the counting rates and the CFU number. One should keep in mind, however, that the
plates enumerated here were produced as part of a small, specific experiment and the persons
who enumerated them did not experience fatigue in the same way as they might in case of
repetitive enumeration of large numbers of plates. Even so, the data for the manual counting
rate show more scatter compared to the gold standard rate.
The counting rate for manual counting was 1.9/s on average, while the gold standard
method was slower at 1.1/s. A counting rate for the automatic method can’t be given, since the
process always requires the same 20 s irrespective of the number of colonies, but as an example
we can compare times for a plate containing 100 colonies: The gold standard method would
require approximately 91 s while manual counting takes 53 s. Fig 9 illustrates the advantage
gained by applying the automatic method for increasing CFU counts.

Discussion
The automatic method returned counting results that were on average only slightly below the
gold standard count, performing better in this respect than the manual method. The linear
regression against the gold standard also returned a smaller 95% confidence interval for auto-
matic counting compared to manual counting. Automatic counting time is independent of the
number of colonies, while gold standard and manual counting times grow proportionately
with the CFU count. Automatic counts are independent of operator’s skill and fatigue, which

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Fig 9. Required counting time as a function of colony count. Lines based on constant enumeration time of 20 s for
automatic method, and count rates for manual and gold standard counting as shown in Fig 8 and Table 1.
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makes the method particularly attractive for laboratories that need to enumerate large num-
bers of the same type of samples recurringly. However, the output strongly depends on the
chosen values of various parameters that control the image processing operations. When set-
ting up an image processing system for the first time, or when a new type of samples is intro-
duced, an initial series of tests involving automatic and gold standard enumeration is thus
required. One should determine a parameter set for the automatic method that leads to a good
match, and the correction factor to achieve equivalence between gold standard and automati-
cally determined results. From our point of view it would also be good practice to repeat such
comparisons at intervals.
In this study we examined E. coli DH5α, which forms circular colonies and has a fairly uni-
form growth rate–at the end of the incubation period the colonies were between 0.5 and 4 mm
in diameter. The results (and the values of the command parameters used) should be transfer-
able to the enumeration of other microorganisms, which behave similarly.
It is also recommendable to carry out a final visual check of the detection performance by
comparing the original image and the result of ImageJ’s Find Maxima operation for densely
populated plates (e.g., more than 200 colonies for a complete dish). The Single Points_Output
option may be applied to deliver single point marks of the CFU centers, which can be made
well visible by one or two steps of dilatation. Visual checking is then greatly facilitated by either
creating an overlay of original image and detected points map, or by combining both to an
image "stack" and cycling between the two quickly. Fig 10 shows an example of a plate segment
with dots marking the centers of detected CFUs.
Another very useful feature of ImageJ is its ability to type results as text directly into an
image, as shown in Fig 10. It is thus possible to create an automated command sequence or
macro [18], which does not just count the CFUs but also creates a result image containing
detected CFU centers as well as the numeric count for each region of interest. The saved data
will then contain the original image, which may be re-evaluated again, as well as detailed
results. It is also possible to append a documentation of all processing steps carried out, and
the respective parameter values, to each image.
Our expectations regarding the potential benefits to be earned from a comparatively small
investment in image processing hardware and software were fulfilled. The equipment
described here is of industrial quality and can thus be expected to have an extended life span.
The automatic enumeration

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Fig 10. Example for visualization of results. Plate segment processed and detection results copied as white spots into the original image. Numerical counting result typed
to a text field.
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• returns results which are closer to the gold standard than the previously used manual
method,
• saves a considerable amount of time in the laboratory,
• improves our quality management because plate images are stored, so that counting results
can be proofed and reproduced,
• was implemented with purchase cost for equipment even below our initial budget.

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

Conclusion
We describe the development of an experimental setup and data processing steps for auto-
mated imaging and counting of E. coli DH5α colonies grown on 110 mm Petri dishes. The
hardware used is easily available and, at less than €2,000, purchase cost is only a small fraction
of that of a comparable commercial system. Image processing was done using the public
domain software package ImageJ. Processing steps for fast and reliable detection of E.coli
DH5α colonies are described in detail, allowing readers to adapt the method to their needs by
reproducing–and, if necessary, adjusting–these processing steps. Fast evaluation of large num-
bers of samples can be achieved with the equipment used here by creating an automated com-
mand sequence. The resulting system can greatly reduce the time required for CFU
enumeration compared to manual counting. It allows CFU counting independent of subjective
influence and creation of a database of samples and enumeration results.

Supporting information
S1 Data.
(XLSX)
S1 File.
(PDF)
S2 File.
(PDF)
S3 File.
(PDF)
S4 File.
(PDF)

Author Contributions
Conceptualization: Stefan H. Hogekamp.
Data curation: Lola Hogekamp.
Formal analysis: Stefan H. Hogekamp.
Funding acquisition: Mario R. Stahl.
Investigation: Lola Hogekamp.
Methodology: Lola Hogekamp, Stefan H. Hogekamp.
Project administration: Mario R. Stahl.
Resources: Mario R. Stahl.
Software: Lola Hogekamp, Stefan H. Hogekamp.
Supervision: Mario R. Stahl.
Validation: Lola Hogekamp.
Visualization: Lola Hogekamp, Stefan H. Hogekamp.
Writing – original draft: Stefan H. Hogekamp.
Writing – review & editing: Stefan H. Hogekamp, Mario R. Stahl.

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PLOS ONE Method for automatic enumeration of bacterial colonies on agar plates

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