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The Limitations of CFU: Compliance To CGMP Requires Good Science
The Limitations of CFU: Compliance To CGMP Requires Good Science
The Limitations of CFU: Compliance To CGMP Requires Good Science
Microbiology has a well-deserved reputation for being Microbiology has a well-deserved reputation for
highly variable. Lax attention to precision and accu- being highly variable. Lax attention to precision
racy in measurements helps further this perception. and accuracy in measurements helps further this
The current prevailing confusion between the limit of perception. We have allowed specifications for envi-
detection (1 CFU) and limit of quantification (25 CFU) ronmental monitoring, raw material bioburden, in-
for the plate count method creates a larger degree of process bioburden, and finished product bioburden
variability in microbiology data than is necessary. This to be imposed by regulation without regard for the
discussion looks at the topics of variability, accuracy, ability of the methods to support those specifications
and precision in measurements. specifically in terms of the “countable colonies on a
plate” and the requisite number of replicates needed
for a reasonably accurate estimate.
MICROBIOLOGICAL DATA This discussion looks at the topics of variability,
Most microbiologists would claim that the recorded accuracy, and precision in measurements. Variability
numbers of colony forming units (CFU) were raw is usually discussed in terms of a normally distrib-
data. This is not correct. That recorded number is uted population, where the range of measurements
someone’s (presumably a skilled technician’s) inter- can be represented by a bell-shaped curve with the
pretation of the number of colonies on the plate. Ex- true number at the center of the curve. A population
perience has shown that different technicians (each of measurements that has little variability will give
skilled) can, and frequently do, come up with differ- a narrow bell-shape, and one that is variable will be
ent counts on the same sample. The data recorded in wider. A method providing these measurements will
the lab notebook are an interpretation of the number have two aspects of interest to this discussion: accu-
of colonies on the plate that can be influenced by racy and precision. Accuracy can be thought of as the
the colony morphology, colony density, and the tem- ability of measurements to reflect the true value of
perament of the counter. However, these are the best the population. Precision is the degree of reproduc-
data available to us. ibility among the measurements. Bench technicians
It must also be remembered that the CFU is only an will state that replicate plate counts are not precise.
estimate of the number of cells present. It is a skewed Replicate plate values will vary widely. The accuracy
estimate at best as the only cells able to form colonies of the measured values for a population with a high
are those that can grow under the conditions of the test degree of variability will be influenced greatly by
(i.e., incubation media, temperature, time, and oxygen chance. Even with all plating variables controlled as
conditions). These do not represent a single cell, but is feasible (i.e., plating error, dilution error, sampling
rather those that happened to be well separated on the error, technician counting and transcription error),
plate and so can be distinguished after growth. A col- the level of variability can only be minimized by in-
ony could arise from one cell or several thousand cells. creasing the number of replicate platings.
A second aspect of plate count accuracy is its im- in triplicate) and used them to determine a mean-
portance to the introduction of alternate microbio- squared-error of the estimate for all plates. Their
logical methods into the lab. The basic requirement recommendation at the end of the study was for a
for these alternate methods is that they be at least countable range of 25-250 CFU per plate in tripli-
equivalent to the traditional methods. The establish- cate. Although the authors note that CFU follow
ment of reasonable acceptance criteria for alternate a Poisson distribution, no mention is made of any
methods requires that traditional methods be fully data transformation used to approximate a normal
understood. distribution prior to the use of normal statistical
analytical tools. Tomasiewicz et al. provide excel-
COUNTABLE RANGE ON A PLATE lent cautionary advice: “The data presented herein
The general ranges in common acceptance for count- are not necessarily applicable to other systems. For
able numbers of colonies on a plate are 30–300 and automated equipment, the optimum range may well
25–250. The origin of those ranges is worth exami- vary with the instrument. Furthermore, even if auto-
nation. mation is not used appropriate numbers of colonies
Breed and Dotterrer published a seminal paper that should be on a countable plate can very widely,
on this topic in 1916. They set out to determine the depending on many other variables. With soil fungi
“limit in the number of colonies that may be allowed for example…” (2).
to grow on a plate without introducing serious errors The compendia have recently harmonized a mi-
in connection with the proposed revisions of stan- crobial enumeration test, and in this test recommend
dard methods of milk analysis.” They note that “the to “select the plates corresponding to a given dilu-
kind of bacteria in the material under examination tion and showing the highest number of colonies less
will have an influence on the size of the colonies, than 250 for Total Aerobic Microbial Count (TAMC)
and consequently, on the number that can develop and 50 for Total Yeast and Mold Count (TYMC)” (3).
on a plate.” They also note that food supply can be In determination of the resistance of biological indi-
an issue, colonies close to each other on the plate cators, the United States Pharmacopeia (USP) recom-
may merge, and that neighbor colonies may inhibit mends a range of “20 to 300 colonies, but not less
growth or conversely stimulate growth. “Because than 6” (4). However, the most complete description
of these and other difficulties, certain plates in any of the countable range is found in the USP informa-
series made from a given sample are more satisfac- tional chapter <1227> (5):
tory for use in computing a total than are others. The “The accepted range for countable colonies on a
matter of selecting plates to be used in computing a standard agar plate is between 25 and 250 for most
count becomes, therefore, a matter requiring consid- bacteria and Candida albicans. This range was estab-
erable judgment” (1). lished in the food industry for counting coliform
Breed and Dotterrer chose their countable plates bacteria in milk. The range is acceptable for com-
from triplicate platings of each dilution, requiring pendial organisms, except for fungi. It is not optimal
acceptable plates to be within 20% of the average. for counting all environmental monitoring isolates.
On this analysis, plates with more than 400 CFU The recommended range for Aspergillus niger is be-
were unsatisfactory as were those of less than 30 tween 8 to 80 CFU per plate. The use of membrane
CFU, with best results in the range of 50-200 CFU filtration to recover challenge organisms, or the use
per plate. of environmental isolates as challenge organisms in
The major paper from Tomasiewicz et al. provides the antimicrobial effectiveness testing, requires vali-
an excellent review of the continued evolution of the dation of the countable range.”
appropriate number of CFU per plate from milk. ASTM provides excellent review of countable
They took data from colony counts of raw milk from ranges on a plate, recommending 20-80 CFU/mem-
three different experiments (each dilution plated brane (when plating membrane filters for determi-
nation of CFU), 20-200 for spread plates, and 30- take is recognized. If the lab wishes to use this “esti-
300 for pour plates (6). The FDA Bacterial Analytical mated count” it should, at a minimum, have it clearly
Manual (BAM) recommends 25-250 CFU per plate described in their “counting CFU” standard operat-
as a countable range (7). ing procedure (SOP) with a rationale as to when the
plate counts are not critical and can be estimated in
Upper Limit this fashion.
The upper limit of plate counts is dependent on a There are methods available to accurately deter-
number of factors, as described previously. The ma- mine the upper limit for a unique plating surface or
jor issues include the colony size and behavior (i.e., a unique colony type. One is presented in the USP
swarming), and the surface area of the plate. The size informational chapter <1227> (5), which is based
particularly comes into play with plating a mem- on a pair-wise comparison of counts from a dilution
brane for determination of CFU as the surface area series. This is based on the assumption that at the
of that membrane is so much smaller than that of a upper limit the observed numbers of CFU will fall
standard plate. off the expected numbers at some point (see Figure
Colony numbers on a plate that exceed the up- 1). This divergence will become significant at some
per limit are referred to as “too numerous to count” point—that point of divergence defines the upper
(TNTC). TNTC can be reported out several ways ac- limit of CFU per plate.
cording to different authorities. ASTM recommends
reporting this out as >”upper limit” (6). For exam- Lower Limit
ple, a 1:10 dilution with more than 200 CFU on a A central concern in this determination is the differ-
spread plate would be reported as “>2,000 CFU/mL ence between the limit of quantification (LOQ) (i.e.,
(or gram). A commonly used practice is to merely the lower limit of plate counts with acceptable accu-
report out TNTC. racy–defined in USP <1227> as 30 CFU with an error
There are a couple of methods that estimate the as 18.3% [5]) against the limit of detection (LOD) (1
number on these crowded plates. FDA’s BAM recom- CFU). This is an important distinction if we are being
mends using the dilution with plates giving counts held to specifications in the lower range.
closest to 250 and counting only a portion of the ASTM provides guidance in this aspect of plate
plate, estimating the total number and then using counting, recommending that the technician focus
that number as the estimated aerobic count. The US be on the LOD, and urging that the reported value
Department of Agriculture (USDA) discusses this be “< the dilution value if no colonies are recovered
in their lab manual and recommends using a grid (i.e., <10 CFU/mL reported for a 1:10 dilution with
to segment the counting area, then determining the zero colonies; <100 CFU/mL if no colonies are seen
average CFU per grid and multiplying this average on the 10-2 dilution and this is the most concentrated
CFU per grid by the number of grids on the plate plating available). According to the ASTM procedure,
(8). It is not clear to the author how either of these if colonies are present but below the countable range,
methods is greatly superior to guessing. they should be counted and reported as an estimated
The reason there is an upper limit to CFU per count (6).
plate is that the colonies begin to compete for space USP <1227> does not have a specific recommenda-
and nutrients, thereby skewing the count. This is an tion on how to report out these low numbers, but does
invalid plating and may serve as the basis to invali- note “Lower counting thresholds for the greatest dilu-
date the entire test. This may be a hardship to the tion plating in series must be justified” (5).
lab personnel, who were trying to reduce the plating FDA BAM recommends a different reporting for-
load initially by not plating out excessive dilutions. mat. In the FDA BAM method, all counts are recorded
However, making a mistake initially is not a reason- in the raw data, but the information is reported out as
able excuse to avoid doing it correctly after the mis- <LOQ (7). For example, a 1:100 dilution that yields
Figure 1: Figure 2:
Estimated vs. observed plate counts. Error estimates of low plate counts.
10000 1
0.9
1000 0.8
0.7
Error as % of Mean
CFU/Plate
Expected
100
Observed 0.6
0.5
10
0.4
1 0.3
Dilution 0.2
0.1
other words, all plates were counted and each plate’s n=2 n=3 n=5 n=10
CFU count was used to estimate the original CFU/
mL. Each estimate was evaluated, and if the estimate
for each plate was within 30% of the mean, it was example, plating 10 x 1 mL samples on 10 different
deemed acceptable. plates, and then reporting it as if a 10 mL sample
Establishment of QC limits for plate counts works was plated. This approach is flawed in that it ig-
best if there are at least three replicate plates for each nores several sources of variability in plating includ-
dilution and the relationship between accuracy and ing sampling, growth, and counting errors (9, 10).
the number of CFU/plate is understood. Weenk The correct interpretation for this situation is that
developed these discussions in an understandable you have just plated 1 mL 10 times, not 10 mL once.
discussion in relation to media growth promotion The numbers might be averaged—they cannot be
studies (9). His analysis assumed a 5.5% dilution er- added.
ror from pipetting, and estimated the ability of par-
allel plating experiments (new media vs. standard) ROUNDING AND AVERAGING
to distinguish between two populations. The results Discussion of rounding and averaging requires de-
are shown in Figure 3 (recalculated from Weenk’s termination of what significant figures might be in
equations). the measure. For raw colony counts, common prac-
This treatment clearly shows the effects of increas- tice determines that the CFU observed determine
ing the number of plates in replicate—increasing our the significant figure, and that the average is one
ability to distinguish between similar populations. decimal place to the right of that number (sticklers
In a similar manner, we are able to distinguish small- for accuracy will report the geometric mean rather
er differences (the counting becomes more accurate) than the arithmetic mean given the Poisson distri-
as the number of CFU per plate increases. Therefore, bution followed by CFU). That number is then mul-
trying to establish a QC guideline for CFU/replicate tiplied by the dilution factor. In other words, if the
(e.g., each replicate must be within 30% of the mean) 10 -2 plates have 125 and 114 colonies, the average is
may be problematic at lower CFU per plate counts. 119.5 times 102, or 11950. In reporting, it is common
The method used to QC individual plate counts, if practice to report out as scientific notation using two
used, should be documented and justified in SOP, significant figures (in our example, 1.2 x 104). This
along with the response to finding variant counts. requires rounding.
ASTM and USP both round up at 5 if 5 is the num-
Plating 10 x 1 mL Samples to ber to the right of the last significant figure (6, 11).
Plate a Total of One 10 mL Sample FDA BAM has a more elaborate scheme, rounding up
There have been suggestions that a larger volume if the number is 6 or higher, down if 4 or lower (7).
of material may be plated across several plates, and If the number is 5, BAM looks to the next number to
the results reported out for the larger volume. For the right and rounds up if it is odd, down if it is even.
All laboratory personnel should perform calcula- IMPACT OF THE CONCEPT “COUNTABLE RANGE OF
tions in the same way. Be sure to include direction CFU/PLATE” ON METHOD SUITABILITY STUDIES
and its justification in the “Counting CFU” SOP if it AND QUALIFICATION OF ALTERNATE MICROBIO-
does not already exist in a separate SOP. LOGICAL METHODS
We are trained to conduct replicate platings in dupli-
IMPACT ON SPECIFICATIONS AND cate and instructed by the compendia that method
ENVIRONMENTAL MONITORING CONTROL LEVELS suitability studies should be done with <100 CFU/
If you are faced with a finished product bioburden plate. However, are these really the best parameters
of Not More Than (NMT) 100 CFU/gram, and your for these types of studies?
method suitability study requires a 1:100 dilution of In both suitability and qualification of alternate
the product to overcome any antimicrobial effects, microbiological methods, we are trying to compare
then how are you to test it? Common practice is to two treatments. These treatments might be the in-
perform the 1:100 dilution, perform a pour plate oculum vs. the recovered count (method suitability
of 1 mL in duplicate. If two colonies grow on each study) or the traditional method’s results vs. the al-
plate, the product fails specification. This common ternate method’s results. At the core of the study is a
practice is scientifically unsupportable as it relies on comparison between two measurements.
data generated below the LOQ (25-30 CFU/plate) to Weenk’s comparison of growth promotion meth-
determine compliance with a product specification. ods included an extraordinarily good description of
However, this is common industry practice. this problem (9). In this analysis, he assumed that
the number of colonies on a plate followed the Pois-
Environmental Monitoring Alert and son distribution and that the dilution error from pi-
Action Levels for Aseptically Produced Products petting was about 5.5% (see Figure 3 and previous
Hussong and Madsen published a thoughtful review of description in the section on QC limits for replicate
environmental monitoring (EM) alert and action levels plate counts).
forasepticallyproducedproductswheretheyarguethat These calculations show the effect of reducing the
the levels of acceptable CFU for many room classifica- number of replicate platings (10 > 5 > 3 >2) and the
tions are below the noise level plate count technology number of CFU/plate (300 → 30) combine to greatly
(e.g., in the range of 1-2 CFU/m3) (12). In addition, en- decrease the ability of the lab to “see” a difference
vironmental data is extremely variable—much more between two populations. In other words, we might
than in controlled lab studies as the numbers of micro- not be able to tell if one treatment (or media batch) is
organisms, the physiological state of the isolates, and better or worse. Perhaps more troubling, as the num-
even the species are completely out of the control of the ber of replicate plates decreases with the number of
investigator. In addition, the numbers do not conform CFU/plate, chance plays a bigger and bigger role in
to a normal distribution, as there are sporadic counts whether the validation study is successful. Ideally,
with a count of “zero” CFU predominating (especially we should be using at least five replicate plates and
in the Grade A/B areas). Hussong and Madsen conclude have challenge inocula of approximately 200 CFU/
that because the numbers are unreliable, the trend in plate for the best results, balancing the theoretical
the data is the only important consideration, and that accuracy of the counts and replicates with practical
EM counts cannot be used for product release criteria. considerations of time required for counting and our
A separate treatment of this subject was presented by ability to accurately estimate inocula concentration.
Farrington who argues that the relationship between It should be noted that all compendia recommend a
EM data and finished product quality is a widely held range of <100 CFU/plate for these studies (presum-
but unproven belief, compounded by the problems in ably a hold-over from the established limits for the
accuracy with the low counts generated by plate count sterility test study).
methodology (13).