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Ready for the count? Back-to-basics review of microbial colony counting
Tim Sandle
Introduction
Microbiological laboratories remain reliant on the accurate determination of the number of
colony forming units (CFUs) on growth media; this is notwithstanding advances with rapid
microbiological methods, at least for some applications. Mainstay methods include pour plates,
spread plates, and membrane filtration. Counting of microbes is important as it enables a
laboratory to estimate the microbial population in a variety of products (bioburden) (1). Yet
there are limitations with plate count methods, including the fact that they only count viable
cells and culturable organisms; moreover, such methods the need a period of time of incubation
to be able to visualize the colonies. There are, of course, other means to assess microbial
numbers (such as flow cytometry, determination of microbial biomass, lab-on-chip
innovations, and indirect mechanism based on fluorescence) or microbial presence (such as
turbidity) (2), yet the low-cost, ease of application, and dominance within the compendia, make
plate counting methods the most commonly used means to derive at a microbial estimate within
pharmaceuticals and healthcare. This paper revisits this subject plate counting in the era of
regulatory focus upon data integrity.
The colony counting process, while economical, is open to human error, both in counting and
transcribing data into a computer. As an example, errors, plus the effect on the person tasked
with the activity of counting can be adversely affected by spending hours hunched over plates
full of bacteria in terms of eyestrain, or with the incorrect recording of the counted result. These
issues are compounded by atypical microbial growth upon agar. One of the disadvantages of
the plate count method is that the growth and distribution of the colonies on the surface of the
agar plate are not always homogeneous: colonies may have different diameters, densities, and
shapes, and/or they may grow to confluence, and colonies may merge or spread (3). This can
be a cause of error, for example the clumping of cells can lead to undercounting of viable cells
as can the clustering of colonies and edge effects towards the edge of the plate (4). To add to
this, incidences of too many or too few colonies on a plate will affect the accuracy of estimating
the viable count (unless serial dilution has been deployed to prevent cases of overcrowding on
a plate from microbial cells).
The activity of colony counting also presents some data integrity concerns, as shown in some
U.S. Food and Drug Administration (FDA) warning letters and as reflected in some data
integrity guidances. To overcome some of the challenges, automated colony counters are
available (as assessed in a companion article to this one, published on the IVT Network:
“Automated, Digital Colony Counting: Qualification and Data Integrity”) (5). However,
automated counters are not without their own challenges in terms of accuracy, validation and
data handling (as briefly looked at towards the end of this paper).
This paper considers controls around manual colony counting. While this is a long-established
practice, the flurry of regulatory issues around this fundamental activity of the microbiology
laboratory suggests, at the very least, a re-examination of the topic is required. The re-review
as presented in this paper could help to improve practices in some laboratories and provide the
basis for a training aid; and perhaps a launching point to assist the microbiology laboratory
with when faced with audit preparation or when addressing audit requests.
FDA warning letters
As indicated in the introduction, regulators have periodically cited deficiencies with plate
courting and in the age of data integrity focus citations are becoming more common. As
examples, issues with plate counting feature regularly in U.S. FDA warning letters, such as:
• Not verifying counts between operators;
• Not using suitable equipment to assess colonies (e.g. appropriate light source);
• Differences in counting between operators;
• Plates not matching official records.
To take an example, from a warning letter:
“The points-of-use for water in the production facility had been checked routinely for germ
count by counting the colony forming units (CFU) on plates. When checking the plates, the
inspector found CFU values that were significantly higher than in the quality control records.
He also found microbial growth on plates with selective medium for which the record had
declared "no growth". The inspector therefore concluded that the lab data was systematically
falsified.”
Two take two further examples: “plate counting, where colony forming units are miscounted”
and “missing samples, such as environmental monitoring samples not being taken or dropped
on transit to an incubator.” While the latter is careless, colony counting errors can occur where
confluent growth occurs (6).
For these reasons, care needs to be paid to the activity of plate counting in the pharmaceutical
microbiology laboratory, and with the process of training operators, in order to reduce the
possibility of falling foul of regulators. The substantive part of this paper provides some good
practice tips. Before this, the nature of colonies and colony counting needs to be considered.
What is a colony?
A viable cell is a cell which is able to divide and form a population (or colony). This provides
the basis for plate counting, where microorganisms, which are culturable under the conditions
of the test (7), are recovered on or within agar (depending on the method used (8). After
incubation, any colonies are counted and, if there has been a dilution performed then from a
knowledge of the dilution used the original number of viable cells can be calculated. The
growth of a single bacterium in a defined location in the agar plate can give rise to a
proliferating colony that is typically visible within one to three days, but incubation times can
easily extend to weeks depending on the species and growth conditions. There are restrictions
on colony sizes since growth is restricted by viscous drag and forces exerted by neighboring
cells leading to reduced growth within the center of the colony and competition for space at the
expanding edge of the colony (9). Because the colony is clonal, with all organisms in it
theoretically descending from a single ancestor, they are genetically identical, except for any
mutations (which occur at low frequencies) (10). However, with many applications of
pharmaceutical microbiology it may not be the case that the colony that forms derives from a
single ancestor.
As it is often unknown whether each colony comes from only one cell, the total number of
viable cells is usually reported as colony-forming units (CFUs) rather than cell numbers (this
is an important point since many text books erroneously portray the colony forming unit as a
single organism) (11). Reasons why a CFU may represent more than one organism include the
fact that many bacteria do not occur naturally as one cell. For example, some bacteria grow in
chains (e.g. Streptococcus) or clumps (e.g. Staphylococcus); the consequence of poor mixing
of a sample before plating out, where cells stick together or become bound to the sample.
Bacillus species, for example, are notorious for clumping; and the method of collection, as with
a settle plate where a CFU develops from where a microbial carrying particle has been
deposited and that particle may have been host to more than one organism.
The generation of a CFU utilizes microorganisms' capability to replicate under the applied
medium, temperature, and time conditions. However, the CFU is an arbitrary estimate at best
since the only cells able to form colonies are those that can grow under the conditions of the
test. These test conditions include: incubation temperature; incubation time; type of culture
media; oxygen conditions and so on.
Nonetheless it remains that the CFU is the primary means of assessing microbial levels on
solid media, whether this is an assessment of the presence or absence of a particular microbial
species; an attempt to enumerate the total count; or with an analysis of non-zero events (as
might be undertaken for aseptic processing).
Colony counting
Since the development of colony-counting assays a century ago, the technique has changed
little (as Figure 1 reevals). Microbial colonies are still grown in conventional Petri dishes or
multi-well plates. Following incubation under conditions appropriate for the microorganism of
choice, the colonies are counted to determine the number of CFUs. This is done manually by
counting colonies on plates illuminated using transmitted light. While this may seem
straightforward, microbiologists need to use their experience, expertise and judgment for test
interpretation; in doing so, even with experienced microbiologists, this can lead to subjective
and variable interpretations and documentation of test results (12).
Figure 1: Plate inspection (Image: Tim Sandle)
Differentiation between colonies and air bubbles inside the agar should be possible without any
difficulties. Beyond this, counting procedures are not simple since colonies must first be
isolated from the background and then, if they overlap, be separated. In addition, such methods
must be capable of rejecting common artefacts such as imperfections in the agar, dust and edges
of Petri dishes (13). Furthermore, colonies can be difficult to count for other reasons (14):
a) The act of colony counting is not only repetitious it can lead to errors and thus problems of
data integrity.
b) In low count assays minor counting errors will have significant effects.
c) A related error is when numbers of CFUs on a plate can lead to false results due to
overcrowding of bacteria.
d) Indistinguishable colony overlap (such as the effect of ‘masking’).
e) Assessing colonies near the plate periphery.
f) Increased density of collected culturable microorganisms.
Some of these factors are discussed below, together with strategies to overcome these issues
and to strengthen manual colony counting.
In addition to numbers, colonies can also be described and examined for typical / atypical
growth patterns, such as (15):
• Form - What is the basic shape of the colony? For example, circular, filamentous, etc.
• Size – The diameter of the colony. Tiny colonies are referred to as punctiform.
• Elevation - This describes the side view of a colony. Turn the Petri dish on end.
• Margin/border – The edge of a colony. What is the magnified shape of the edge of the
colony?
• Surface - How does the surface of the colony appear? For example, smooth, glistening,
rough, wrinkled or dull.
• Opacity - For example, transparent (clear), opaque, translucent (like looking through
frosted glass), etc.
• Color - (pigmentation) - For example, white, buff, red, purple, etc.
Countable size of colonies
A study undertaken by LeBlanc and bioMerieux looked at the accuracy of the human eye to
count colonies (16). The study considered differences between people and it looked at
differences across three different laboratories. For this inquiry into the challenges associated
with colony counting, beads were used some that an accurate size determination could be made
(with beads placed at different locations onto an agar plate). The beads ranged in size between
50 and 500 µm, with some beads colored black and some beads colored white. The number of
beads per agar plate ranged from zero to 12 (designed to represent low levels of contamination,
as might be recovered from a Grade A / ISO class 5 or Grade B / ISO class 7 cleanroom
environment).
The study was designed to examine the false positive rate (overcounting colonies) and the false
negative rate (under-counting colonies). To assess this, around 1,000 plates were read per
operators across the three laboratories and some 13,058 plates were read in total. The plates
had been designed so that 79% were ‘negative’ (in that no beads were placed onto the agar
plates) and 21% were ‘positive’ (in that a plate contained one or more beads).
The results showed:
1. A variation between operators (with some operators misreading positive pates by up to
50%).
2. Negative plates were generally read satisfactorily.
3. The results from the three laboratories were generally very similar, with equivalent
terror rates.
When errors occurred:
1. Errors were more common with smaller colonies than with larger colonies.
2. The biggest influence over correct counting was the position of the colony on the plate.
3. Counting errors were greater for lighter colonies (as simulated by the white beads) than
with darker colonies (as represented by the black beads).
With reference to colony size, the inference was:
1. Colonies between 53 and 63 µm cannot be counted as they cannot be seen by operators.
2. Colonies between 90 and 166 µm can be seen with a strong contrast; however, errors
in counting are common.
3. The counting of colonies sized between 212 and 250 µm was better; however, the
position on the agar was a variable that affected counting accuracy.
4. Greatest counting accuracy was obtained for colonies in the size range 425 and 500 µm.
Here the position of the colony on the agar plate made little difference.
The final review of the results attributed the errors to the limitations of the human eye. The
errors can be addressed with control of the light source; the use of magnification; and through
operator training, in terms of manual methods. In the presentation of the results it was noted
that automated colony counters were the answer to overcoming the errors in a consistent
manner.
Light source
A key component of our colony counting system is the choice of illumination. The optimal
orientation of light is from behind the Petri dish, although there are some points to consider
with background illumination as discussed below. Hence lighting can additionally be provided
some the side, or even from the top, to assist with viewing and discerning colonies. With light
direction, inhomogeneity of the agar thickness can cause issues in relation to colony
discrimination; therefore, optimizing the light direction is important.
The light source should allow for the correct identification of mucus secreting colonies and to
assess different pigmentation. In addition, the light should enable any decrease of turbidity
caused by calcium carbonate inside the agar when working with acid producing
microorganisms to be visually detected. It is important that the light pattern is uniform and that
and shadowing effects are avoided.
The ability to dim the light can sometimes be useful, for adjusting the light intensity to the
needs of the user and accounts for surrounding light inside the laboratory. However, care needs
to be taken that the level of light reduction does not lead to colonies being missed. LEDs can
overcome the issue of stray light or blinding, which can arise when using fluorescent lamps.
For some colored agar, blue dark field illumination may provide the best discrimination of
colonies.
Light in relation to different colony types
Transmission illumination uses a light source below the plate and is most useful when the plate
is nearly transparent with opaque, relatively high-contrast colonies. High transparency of
colonies can be best visualized using an LED-light source. It is important with the light source
to avoid any heat being transferred to the sample. Positioning LED lights to the side can further
assist with the detection of transparent colonies, given the three-dimensional structure of each
colony. Furthermore, light that only comes from the back can lead to some colonies (especially
lighter-colored colonies) from being missed.
With other types of illumination, reflective illumination uses a light source above the colonies
and is effective with opaque media such as blood agar. Dark field illumination uses light
sources below the plate at an angle that would, if the plate and colonies were entirely
transparent, miss the system's optics entirely, giving a dark field-of-view. This type of
illumination is especially useful for imaging small colonies, because their light scattering is
more visible against the dark background than it would be with transmitted or reflected light.
A black contrast disk can assist with working in dark fields.
Magnification
To aid the viewing of colonies, magnification is recommended (and this needs to be considered
in the context of the limitations of the human eye, as described above). The typical magnifying
glass that comes with a colony counter is configured to x 1.5 or x 3.0 (sometimes greater),
which should be sufficient to visualize smaller sized colonies (with limitations of colony
pigmentation, translucence and contrast noted).
Countable range
The countable range varies according to the test method (for a pour plate, for example, this is
generally placed within 25 to 250 CFU or 30 to 300 range; with membrane filtration this
generally accepted as being between 20 to 80). The countable range is important since it avoids
an overcrowding error occurs from individual colonies inhibiting the formation of other
colonies nearby (17). Sutton has provided some excellent overviews of the countable range
issue in relation to plate counting (18). Here he notes that factors affecting the upper countable
range include colony size and behavior (possible swarming, as the case with organisms like
Proteus mirablis and some Bacillus species, such as Bacillus cereus), as well as the surface
area of the plate. P. mirabilis provides a classic example; it is well-known in laboratories as
the species that swarms across agar surfaces (in a striking bulls’-eye pattern), overtaking any
other species present in the process (19).
At the lower end can be added the difference between limit of quantification (which is what
arguably really matters, and with a standard 9 centimeter plate is 25 CFU) and limit of detection
(which many setters of 'specifications' assume can be reliably recovered, often going down as
a low as 1 CFU). Error arises with the lower limit because the CFU’s follow Poisson
distribution where the error of the estimate is the square root of the mean (20).
Hence, the interpretation of microbial CFU is affected by the number of colonies present, where
there is too high a number leads to counting errors as a result of confluence or overcrowding
across the surface of the plate; and where the number is too low error arises because the CFU’s
follow Poisson distribution. A distinction also needs to be drawn between the Limit of
Detection for microbiological agar plates (assumed to be 1 CFU, but in reality untestable with
conventional methods) and the Limit of Quantification (which is based on the countable range,
which is partly a function of the size of the test plate).
The reasons for these targets is because dilution factors may exaggerate low counts (less than
25 CFU), and crowded plates (greater than 250 CFU) may be difficult to count or may inhibit
the growth of some bacteria, resulting in a low count. It is typical to report counts less than 25
or more than 250 colonies as estimated plate counts (21). To address high numbers, some
organizations divide up plates for counting. Hence, plates with over 200 colonies are usually
counted by dividing the plates into equal sectors (from 1/2 up to 1/8). After counting one sector,
the count was multiplied with the total number of sectors to estimate whole plate CFU count.
The limitation here is with the distribution across the plate, which is not often normal and hence
the selected area may or may not be representative of other areas on the plate.
Atypical growth patterns
Atypical growth patterns on media can cause counting difficulties and it cannot always be
assumed that colonial growth is evenly distributed across a given plate. Atypical growth
patterns are considered below.
Merged colonies
A merged or oblong colony if formed from the merger of two colonies that were near each
other when they started to grow (sometimes this is a ‘full merger’ at other times a degree of
overlapping takes place); as with figures 2 and 3. Merged colonies are often the consequence
of the numbers of colonies on the plate being too high relative to the size of the agar plate, for
the higher the density of colonies on a plate, the more likely it is that two bacteria will fall so
close to each other that their two colonies merge and appear to be a single colony, resulting in
an undercount. This can also emerge as the result of poor of a sample mixing or the way a plate
is inoculated (where a poor plate method is performed). The problem with merging colonies is
that they result in an underestimation of the number of colonies counted (22). As well as
miscounting, merged colonies also affect the number of colonies that form through limitations
of the food supply, in that colonies close to each other on a plate merging together cause
neighbor colonies to either inhibit growth or alternatively to stimulate growth (23).
Figure 2: Growth across an agar plate, with merging of colonies (Image: Tim Sandle)
Figure 3: Another example of the merged colony effect (Image: Tim Sandle)
Edge effects
Colonies that grow around the edge of Petri-dishes are difficult to count because the edge has
similar density as that of colonies, as illustrated in figures 4 and 5. This can be common because
many cell colonies tend to have some affinity for the edges (18).
Figure 4: Colonies growing on the edge of an agar plate and hence easily missed (Image: Tim
Sandle)
Figure 5: A plate with colonies growing close to the edge of the plate (Image: Tim Sandle)
Spreaders
Spreaders are colonies of bacteria which grow in such a way that they appear to be “spread”
across the plate. The sliding ability of some species of bacteria is provided by the expansive
forces of a growing culture in combination with special surface properties of the cells resulting
in reduced friction between the cell and its substrate (24).
Spreading colonies are usually of three distinct types:
• A chain of colonies, not too distinctly separated, that appears to be caused by
disintegration of a bacterial clump.
• One that develops in film of water between agar and bottom of dish.
• One that forms in film of water at edge or on surface of agar.
One example of spreading is provided with figure 6, below.
Figure 6: The spreading effect (due to a Bacillus species) (Image: Tim Sandle)
If plates prepared from sample have excessive spreader growth so that either:
• The area covered by spreaders, including total area of repressed growth, exceeds 50%
of plate area, or
• The area of repressed growth exceeds 25% of plate area
Then it is typical to report plates as spreaders (and if the colonies form a continuous sheet (a
"lawn”), then it is not possible to obtain a count. When it is necessary to count plates containing
spreaders not eliminated by the above, the preferred practice in many laboratories is to count
each of the three distinct spreader types as one source. For the first type, if only one chain
exists, then count it as a single colony. If one or more chains appear to originate from separate
sources, then count each source as one colony. It is not good practice to count each individual
growth in such chains as a separate colony. In other cases, distinct colonies may be able to be
discriminated and need to be counted as such. For the final result combine the spreader count
and the colony count to compute the total count.
There are occasions outside of plate enumeration where spread or ‘lawn’ plates are required,
as the heavy, often confluent growth of culture spread evenly over the surface of the growth
medium is useful to test the sensitivity of bacteria to many antimicrobial substances, for
example, mouthwashes, garlic, disinfectants and antibiotics.
Desiccation and cracked plates
The major issue facing operators when exposing settle plates in high airflow environments,
such as laminar airflow cabinets; class II microbiology safety cabinets and isolators, is the
desiccation of the medium during the four-hour exposure time, which is the maximum
recommended exposure time. It is important during exposure that the agar does not shrink
from the edge of the petri dish or crack down the middle (or any other location, as figure 7
reveals). This is to ensure the maximum surface area is constantly exposed to the environment.
Possibly more importantly, is that the agar is still able to show good recovery of regular test
organisms; both post exposure and post incubation (25).
Figure 7: Image of a settle plate with a crack, due to desiccation (Image: Tim Sandle)
Where desiccation occurs, there could be suppressed growth or loss of colonies that were
forming until the cracking in the agar occurred. To avoid this from occurring, users need to
work with culture media manufacturers to improve the media formulation, such as having
plates intended to be used as settle plates with additional media added during the plate filling
stage. It may also be the presence of neutralizers, required where plates are used for taking
finger dabs, may be inappropriate additives where plates are intended to be used as settle plates.
Data integrity
An important area for data integrity in microbiology is with culture media (26). Plate reading
and counting is an area of data integrity concern. Plates can be incorrectly read by analyst;
misinterpreted; or incorrectly counted. The manual process can be partly addressed by training
staff appropriately in plate reading, including training in typical colonial morphology and how
to interpret colonies that have merged together (confluent growth) or where spreading, by a
motile organism has occurred. Understanding such limitations is the basis of good
microbiology laboratory practices.
Consideration can also be given to second checks or random checks performed by a supervisor.
In addition, any sample at alert or action level should be subject to a confirmatory check as part
of local out-of-limits / specification procedures (27). With sterile manufacturing, it can be
equally as important to reassess some plates that do not appear to have any colonies (zero
counts), especially when assessing EU GMP Grade A / ISO 14644 class 5 environments. One
limitation with the concept of second checks is whether this only ends up with collusion
between two operators? Even where errors are flagged, companies need to develop procedures
to describe what should happen when differences between operators are flagged. Should this,
for example, require some type of quality record to be raised?
Automated colony counters
Although not part of the scope of this article, automated colony counters are worthy of
consideration. The technology is based on the use of digital cameras or scanners to image the
cell colonies in agar media in Petri dishes, where the colonies are enumerated using an image
processing algorithm (28). The automatic methods can eliminate noises outside agar plate
discounting the plate rim and wall, identify and separate clustered or overlapped colonies, and
count colonies by using techniques like connected region labelling, distance transform (a
derived representation of a digital image), and watershed algorithms. Such devices can provide
a faster and more reproducible means to capture microbial test results (29, 30). A key practical
advantage is with automatically transferring results to a computerized system. Central to this
the validation, and microbiologists need to be confident that the automated colony counting
technology is as accurate (or better) than a manual counting method and that the data is secure
and cannot be manipulated (31).
With such instruments in place, greater accuracy of counts can probably be obtained together
with data security. However, automated plate counters will be subject to some validation issues.
Such issues include microorganisms growing at different rates and limitations with counters
being able to assess different colony shapes, sizes, and colors; plus, the presence of spreading
colonies or where colonies are embedded within other colonies, which can cause issues for
accurate recording of colony numbers.
Summary
With the counting of colonies, the operator has a difficult task to perform counts which are then
susceptible to miscalculation and erroneous id entifications. This relates to the way the colonies
grow; the color of colonies (and degree of contrast), the limitations of the human eye, and as a
consequence of the mental and visual fatigue caused by the process itself.
The complexities and associated data integrity matters are apparent in observations made by
regulatory authorities. Most notably there have been regulatory comments where colony
forming units have been miscounted. Often errors are attributed to the use of inappropriate light
or absence of magnification; a failure to use a suitable counting device; the incorrect
multiplication of a dilution and so on.
Although advances in imaging and automatic software can perform an objective analysis so
that the variabilities with colony counting are reduced, it remains that a sizeable proportion of
plate counting in microbiology laboratories is performed manually (32). In order to improve
counting reliability, good practice measures need to be adopted, such as the use of white light;
using colony counters with the ability to use a dark background; and applying magnification.
It is additional important to consider data integrity, both through the training of operators in
counting techniques and with understanding atypical colony growth, and with introducing
second checks as required, especially with plates relating to samples where one or very few
colonies can make a significant difference to the associated risk (such as with samples from
Grade A / ISO class 5 environments) (33).
While microbiologists can endeavor to improve and to address data integrity concerns, it is
important methods for enumeration of microorganisms can only have the objective of
providing the best indicator possible of the microbial bioburden but not the absolute bioburden.
What remains of greatest importance is investing quality by design and contamination control
measures.
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