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org on September 1, 2010

Evaluation of the ScanRDI® as a Rapid Alternative to the

Pharmacopoeial Sterility Test Method: Comparison of the
Limits of Detection
Ron Smith, Mark Von Tress, Cheyenne Tubb, et al.

PDA J Pharm Sci and Tech 2010, 64 356-363

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Evaluation of the ScanRDI姞 as a Rapid Alternative to the

Pharmacopoeial Sterility Test Method: Comparison of the
Limits of Detection
RON SMITH, MARK VON TRESS, CHEYENNE TUBB, and ERWIN VANHAECKE

Alcon Laboratories Inc., 6201 S. Freeway, Fort Worth, Texas 76134 ©PDA, Inc. 2010

ABSTRACT: Two sterility test methods, the ScanRDI威 rapid sterility test and the United States Pharmacopeia/
European Pharmacopoeia/Japanese Pharmacopoeia (USP/EP/JP) compendial sterility test, were compared with
respect to the limits of detection for the presence of viable microorganisms in aqueous solutions at low inoculation
levels. The ScanRDI威 system employs a combination of direct fluorescent labeling techniques and solid-phase laser
scanning cytometry to rapidly enumerate viable microorganisms from aqueous samples, whereas the compendial
sterility test is a qualitative, growth-based method that uses a visual assessment of turbidity to indicate microbial
contamination. Eight microorganisms were evaluated, seven compendial microorganisms (Clostridium sporogenes,
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Aspergillus niger, Candida
albicans) and the Gram-positive anaerobe Propionibacterium acnes. The number of viable organisms was estimated
using the ScanRDI威 method and the conventional sterility test method using most probable number methodology. The
mean difference between the methods was computed and 95% confidence intervals around the mean difference were
estimated. The ScanRDI威 method was found to be numerically superior and statistically non-inferior to the
compendial (USP/EP/JP) sterility test with respect to the limits of detection for all organisms tested.

KEYWORDS: Rapid sterility test, Chemunex ScanRDI威, Most probable number, Sterility testing

Introduction A sterility test, in its most basic form, is a qualitative

A common experimental approach to demonstrating
functional equivalence between two analytical meth-
ods is to conduct a series of parallel, or side-by-side,
experiments. Such studies are best suited to those
quantitative microbiological methods that characterize
populations. In these methods, the abundance of mi-
crobial cells allows for statistical evaluation of the
experimental outcomes. However, the parallel testing
approach is not well suited to compare methods that
are qualitative in nature, or those that attempt to detect
and quantify rare events (i.e., detecting microbial con-
tamination at extraordinarily low concentrations).
With these methods, the inherent statistical power of
the microbial population is lost, and it must therefore
be provided through increased repetition of the exper-
iment.
Received July 14, 2009.
Please address correspondence to Ron Smith. Phone:
817.551.4975. e-mail: Ron.Smith2@alconlabs.com
assay that is designed to detect the absence of viable
microbial cells in or on a product. The expected out-
come for such a test is zero, or no organisms recov-
ered. In the current pharmaceutical environment, the
likelihood of observing a sterility failure is exceed-
ingly rare, generally on the order of less than 0.1%. As
such, studies designed to demonstrate functional
equivalence for these types of methods, using the
qualitative presence/absence metric, will require inor-
dinately large sample sizes, which become signifi-
cantly larger as the differences between two methods
shrink (1).
An alternate approach to evaluating equivalence be-
tween such qualitative methods is through the appli-
cation of the most probable number (MPN) method.
The MPN method is well-known and widely used for
estimating the density of viable microorganisms based
on the Poisson distribution (2, 3). A sample is diluted
to such an extent that only a small proportion of
sub-samples will contain organisms that will display
positive growth when assayed. It is thus possible to
use a series of qualitative assays, such as a sterility
test, in an MPN series, to provide a quantitative mea-

356 PDA Journal of Pharmaceutical Science and Technology

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surement of the number of viable organisms in a
solution. More importantly, the MPN method provides
a means to utilize a quantitative non-inferiority crite-
rion for a qualitative test method. Non-inferiority stud-
ies are designed to show that a particular test method
is no worse than a standard method by a given margin.
Non-inferiority testing further permits the conclusion
of superiority.
In theory, the presence of a single viable cell should be
sufficient to generate a positive result in a sterility test.
Consequently, the limit of detection is the single most
important attribute of a sterility test method. In this
study we employed an MPN scheme to compare the
ability of two sterility test methods to detect microor-
ganisms when they were exceedingly rare. We com-
pared a rapid detection approach using the ScanRDI威
microbial analysis system and the reference sterility
test method described in the current US/EP/JP phar-
macopeias (United States Pharmacopeia/European
Pharmacopoeia/Japanese Pharmacopoeia). We also
evaluated the robustness of the methods by replicating
the experiments using three discrete teams of analysts
in three different laboratories. The data generated
from these studies support the conclusion that the
ScanRDI威 sterility test method is non-inferior to the
compendial sterility test with respect to the limits of
detection.
Materials and Methods
Overview of the ScanRDI威 System
The ScanRDI威 microbial analysis system employs a
combination of direct fluorescent labeling and solid-
phase laser scanning cytometry to rapidly enumerate
viable microorganisms from aqueous samples. The
system has sufficient sensitivity to detect a single
viable microorganism in 2–3 h, without the need for
growth and multiplication. Cells are collected by fil-
tration onto a single, 0.4-␮m-porosity polyester mem-
brane and treated with a proprietary combination of
background and viability stains. The viability stain
consists of a membrane-permeant, non-fluorescent
substrate, similar to fluorescein diacetate, which freely
crosses the cell membrane. The substrate is cleaved by
nonspecific esterases into a membrane-impermeant
chromophore. As a result, cells with intact membranes
accumulate the chromophore in the cytoplasm while
those with compromised membranes are unable to
retain the fluorescent probe. A processed filter is sub-
sequently transferred into the cytometer where the
Vol. 64, No. 4, July–August 2010
filter is scanned by a high-speed, 488-nm argon laser.
The entire filter surface is scanned in a pattern that
ensures that each section of the membrane is illumi-
nated twice. Fluorescent light is detected by multiple
photomultiplier tubes and processed through a number
of discrimination parameters that enable the instru-
ment to differentiate between valid signals (labeled
microbes) and background noise (electronic, optical,
and/or auto-fluorescent particles). The result of the
scan is displayed in the form of a membrane scan map
that identifies the position of each fluorescent event.
Each event is verified by visual examination using an
epifluorescent microscope with an automated motor-
ized stage. Those events that possess morphology con-
sistent with microbial cells are subsequently consid-
ered viable microorganisms. Amorphous particles and
auto-fluorescing crystals are invalidated. The com-
bined speed and sensitivity of the ScanRDI威 system
make it ideally suited for industrial applications, par-
ticularly for ascertaining the sterility of filterable so-
lutions (4 – 6).
Sterility Test Methods
Organisms and Culture Conditions: Organisms,
ATCC identification, and growth conditions used in
each MPN series are given in Table I. Organisms, with
the exception of Propionbacterium acnes, were ob-
tained as lyophilized Bioballs™ from BTF Precise
Microbiology (Pittsburgh, PA). Bacillus subtilis, Clos-
tridium sporogenes, and Aspergillus niger were ob-
tained as preparations of spores, while the remaining
organisms were vegetative cells. Bioballs were dis-
solved in sterile saline to yield an initial concentration
of 30 cfu/mL⫺1. Subsequent dilutions were performed
in saline to yield three dilutions: 10⫺1, 10⫺2, and
10⫺3. P. acnes was serially diluted in sterile saline to
yield cell concentrations ranging between 0.03 and 3
cfu/mL⫺1. Culture conditions (media, temperature)
were based on the compendial requirements for steril-
ity testing and are indicated in Table I.
ScanRDI威 Test Method: At each dilution, the con-
tents of a tube were briefly vortexed and a 1-mL
aliquot was filtered through a fluorassure integrated
filtration unit (FIFU) (Chemunex 220-C2104-02). The
filter was incubated aerobically on resuscitation media
for 3.0 h ⫾ 10 min at 30 –35 °C prior to labeling with
the viability reagent. This resuscitation step is re-
quired to activate the metabolic activity of dormant
spores and to induce germination such that the spores
can be labeled with the viability reagent (6, 7, 9).
357
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TABLE I
Microorganisms and Culture Conditions
Strain
Organism Numbera
Clostridium sporogenes 11437
Propionibacterium acnes 6919
Escherichia coli 8739
Pseudomonas aeruginosa 9027
Staphylococcus aureus 6538
Bacillus subtilis 6633
Aspergillus niger 16404
Candida albicans 10231
a
American Type Culture Collection.
b
FTM ⫽ fluid thioglycollate medium; TSB ⫽ tryptic soy broth.
Under these conditions both aerobic and anaerobic
bacteria spores as well as mold spores can be quanti-
tatively detected by the ScanRDI威 system with no
effect on the vegetative forms. While longer incuba-
tion periods may improve the recovery and detection
of spores, extended periods of incubation adversely
affect the ability to efficiently label and detect other
microorganisms, particularly those that have been
physically damaged or subjected to metabolic and
environmental stressors. As such, the parameters (time
and temperature) of the pre-labeling step were opti-
mized to achieve accurate recovery of all the test
organisms, and by inference, the broadest spectrum of
organisms possible. Following the pre-labeling step,
the filters were aseptically transferred onto an absor-
bent pad saturated with the proprietary fluorogenic
stain (Chemunex 201-R2007-03) and incubated
(45– 60 min at 30 –35 °C, aerobic conditions). The
processed filters were transferred into the cytometer
and scanned using discrimination parameters deter-
mined by the manufacturer. Each fluorescent event
was verified by visual examination using an epifluo-
rescent microscope with an automated motorized stage
at a magnification of 500⫻. Those events morpholog-
ically consistent with microbial cells were considered
viable microorganisms and enumerated by the analyst.
For the purpose of this study, the presence of one or
more validated fluorescent events was scored as a
positive sterility result.
Direct Sterility Test Method: Test solutions were
assessed for sterility using a direct inoculation test
method (9 –11). This increased assurance that the ap-
propriate levels of organisms were delivered into the
358
Incubation
Growth Temperature
Mediumb (°C)
FTM 30–35
FTM 30–35
FTM 30–35
FTM 30–35
FTM 30–35
TSB 20–25
TSB 20–25
TSB 20–25
assay. For enumeration, a 1-mL aliquot from each
dilution tube in the MPN series was directly inocu-
lated into 100 mL of the appropriate culture medium
and incubated for 14 days at the recommended tem-
perature (Table I). Positive growth was determined by
a visual assessment of turbidity.
Quantitation by MPN
To create the MPN series, a suspension of each mi-
croorganism was serially diluted, as described previ-
ously, to yield suspensions containing approximately
3.0, 0.3, and 0.03 cfu/mL. From each dilution tube, a
series of 1-mL aliquots was aseptically transferred into
each of five FIFU assemblies, and similarly into each
of five culture vessels, thus creating two 5-tube MPN
series (see http://www.cfsan.fda.gov/⬃ebam/bam-
a2.html) (12). The concentration of microorganisms in
one MPN series (FIFU) was determined using the
ScanRDI威 method, while estimates of the microorgan-
ism concentrations in the other series were determined
using the reference sterility test method.
Statistical Methods
Test Method and Early Stopping Rule: A paired t-test
was used to test the hypothesis that the concentration
of microorganisms detected by ScanRDI威 is less than
or equal to 70% of the concentration of microor-
ganisms detected and enumerated by the conventional
sterility test method (i.e., the ([ScanRDI威 mean
log10(MPN)] ⫺ [Conventional mean log10(MPN)]) ⬎
(log10(0.7))) is not inferior to that of the compendial
sterility test method) (13). The experimental designed
PDA Journal of Pharmaceutical Science and Technology
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allowed up to 25 pairs of MPN determinations to be
collected for each organism. A confidence limit of
95.25% is required to control for an overall type I
error using this number of samples. Consequently, a
test of the hypothesis was performed by comparing a
lower, one-sided 95.25% confidence limit on the
paired difference (log-transformed [log10] bacterial
concentration determined from the MPN series using
ScanRDI威 minus the log-transformed concentration of
bacteria determined using the reference sterility test
method) to the index of non-inferiority (⫺0.1549 ⫽
log10(0.7)).
Fewer than 25 pairs of samples for each organism
were collected if there was at least a 95% probability
of rejecting the hypothesis using fewer samples; how-
ever, the minimum sample size was 10. An early
stopping rule was established using the test statistic z n
and critical values determined according to Jennison
and Turnbull (14 [see p 207, eq 10.3]). The test
statistic was calculated according to
共d៮ ⫹ 0.1549兲 冑n
zn ⫽
sd
where d៮ is the difference in the means between the
log-transformed (log10) bacterial concentration deter-
mined from the MPN series using ScanRDI威 minus
the log-transformed concentration of bacteria deter-
mined using the reference sterility test method, n is the
number of samples, and s d is the standard deviation of
the paired differences.
Determination of Limits of Detection: The limit of
detection was the dilution level at which there is
97.5% confidence that there was less than 10% chance
of detecting organisms in a sample. The 97.5 % con-
fidence level stems from the upper limit of a two-sided
95% confidence interval for the probability of detect-
ing an organism. The limit of detection was deter-
mined from repeated measures logistic regression us-
ing three dilutions (as above), five tubes per dilution,
and the number of tubes indicating the presence of
microorganisms. The predictor variable was the log10
of the dilution. Four thousand two-sided 95% confi-
dence limits on the predicted probability of response
were computed, and the log value producing the larg-
est upper confidence limit less than or equal to 0.1
(10%) was selected as the limit of detection. The data
analysis was generated using SAS/STAT software,
Version 9.1 of the SAS System for Windows (15).
Vol. 64, No. 4, July–August 2010
Robustness Evaluation: Experiments were replicated
in three locations (Alcon Research and Development
Lab in Fort Worth, Texas; Alcon Quality Assurance
Lab in Fort Worth, Texas; and Alcon Quality Assur-
ance Lab in Kaysersberg, France; 120 tests per site)
with three teams of analysts. An analysis of variance
was used to estimate site-to-site variability. The model
is given by
Y l共ijk兲 ⫽ ␮ ⫹ T i ⫹ ␥ j ⫹ ␦ k共 j兲 ⫹ ε l共ijk兲
where Y l(ijk) is a measurement of log MPN, ␮ is an
intercept, T i is the fixed treatment effect, ␥ j is a
random effect for sites, ␦ k( j) is a random effect for
treatments within sites, and ⑀ l(ijk) is a random effect
for the individual measurements of log10 (MPN). The
random effect for sites, ␥ j , is assumed to be normally
distributed with a mean of zero and a variance for sites
of ␴␥2 , which is tested to be zero. If the site-to-site
variance is not significantly greater than zero, then the
statistical inference may be considered robust to vari-
ation among sites.
Results
The results presented in this report indicate that the
ScanRDI威 method is numerically superior and statis-
tically non-inferior to the reference sterility test
method with respect to the limits of detection for all of
organisms evaluated (Table II). The differences be-
tween mean MPN values were always positive, indi-
cating that the ScanRDI威 method was consistently
better at detecting viable organisms at exceedingly
low concentrations (Figure 1). However, the confi-
dence interval around the mean difference for Clos-
tridium sporogenes includes zero, which indicates that
both methods are at least equivalent in their ability to
detect this organism. Since the lower one-sided
95.25% confidence limit on the paired difference for
each organism was greater than ⫺0.1549, the accep-
tance criteria were satisfied and the non-inferiority
null hypothesis of inferiority was rejected. Addition-
ally, the robustness of the ScanRDI威 method was
assessed using analysis of variance to estimate the
site-to-site variability and determine if it was signifi-
cantly different from zero. The site-to-site variance
component in this analysis measures the method’s
consistency among laboratories. The results of this
analysis are presented in Table III and indicate that the
ScanRDI威 sterility test method remains superior or
equivalent to the reference sterility method in the
determination of mean MPN after adjusting for vari-
359
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TABLE II
Paired T-Tests of Non-Inferiority of the ScanRDI威 and Compendial Sterility Test

Difference Lower
between Standard Confidence
Organism Mean MPNa Deviation n zn P Limitb
Clostridium sporogenes 0.05 0.29 24 3.518 0.0009 ⫺0.050
Propionibacterium acnes 0.58 0.40 15 7.128 ⬍0.0001 0.398
Escherichia coli 0.20 0.30 25 5.812 ⬍0.0001 0.092
Pseudomonas aeruginosa 0.95 0.37 13 10.972 ⬍0.0001 0.762
Staphylococcus aureus 0.39 0.29 14 7.067 ⬍0.0001 0.251
Bacillus subtilis 0.43 0.23 12 8.904 ⬍0.0001 0.308
Aspergillus niger 0.67 0.41 16 8.115 ⬍0.0001 0.490
Candida albicans 0.64 0.46 13 6.278 ⬍0.0001 0.412
a
Mean log10 MPN for ScanRDI威 minus mean log10 MPN for compendial sterility method.
b
The acceptance criteria were satisfied if the lower one-sided 95.25% confidence limit on the paired difference was
less than ⫺0.1549, which is log10(0.70).

ation among sites. Moreover, the site-to-site variabil-
ity in MPN measurement is not significantly different
from zero for any organism tested in the limit of
detection study.
Figure 1
Difference between mean concentrations of bacte-
ria detected by ScanRDI威 and the compendial
methods for each of eight bacteria. Data points
falling within the shaded region would indicate that
the ScanRDI威 method was inferior to the compen-
dial sterility test method for detecting contami-
nants. Error bars represent the two 1-sided 95%
confidence intervals.
Because the study was designed to estimate MPN, the
data could not support a comparison of accuracy,
precision, and specificity between methods, nor could
a false-positive rate be established. However, the data
indicate that the overall variability (as range in percent
positives) of the reference sterility test method ap-
peared to be greater than that of the ScanRDI威 method
when microbes were inoculated into an MPN series at
a cell concentration of 3 cells/mL⫺1. The overall vari-
ability of the reference method appeared to decrease
when cells were present at lower concentrations (0.03
cells/mL⫺1). However, this may be misleading be-
cause the reference method rarely recovered bacteria
at low cell abundance (high frequency of zero values)
but cells were more often recovered by the ScanRDI威
(Figure 2, percent positives greater than zero). For
example, when all eight organisms were considered,
the percentage of positive MPN tubes containing 0.03
bacteria/mL⫺1 was 2.12% for the reference method
but 17.88% for the ScanRDI威. The difference in the
ability of the two methods to recover bacteria is
clearly revealed in Figure 2. Not only was the
ScanRDI威 better able to reveal the presence of bacte-
ria when they were very rare, analysis of the data
indicate that the limit of detection of the ScanRDI威 is
about an order of magnitude lower than that of the
reference sterility test method (Table IV).
Discussion
One of the challenges for any sterility test is the ability
to detect or recover contamination, even when con-
taminants are exceedingly rare. In this study we es-

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TABLE III
Analysis of Variance for Robustness Evaluation

One-Sided
Lower 95.5%
Confidence
Limit Site
Organism Log10(MPN)a Variance P > 円Z円b
Clostridium sporogenes ⫺0.050 0.0093 0.2522
Propionibacterium acnes 0.415 0.0857 0.1780
Escherichia coli 0.092 0.0000 1.0000
Pseudomonas aeruginosa 0.771 0.0218 0.2366
Staphylococcus aureus 0.264 0.0138 0.2227
Bacillus subtilis 0.308 0.0054 0.3167
Aspergillus niger 0.490 0.0014 0.4544
Candida albicans 0.438 0.0270 0.2392
a
The confidence coefficient selection reflects a 0.5% adjustment for interim analysis. The lower confidence limits are
slightly different than those presented in Table II because the standard errors were estimated differently in the
robustness model.
b
P-value for the test whether the site-to-site variance component is significantly different from zero. All are greater
than 0.05, indicating that none can be declared significantly different from zero.

tablished initial conditions in which the concentration

of bacteria ranged more than 100-fold, from common
(3 cells/mL⫺1) to rare (0.03 cells/mL⫺1). The
ScanRDI威 and the reference sterility test methods
were used to indicate presence/absence of growth in a
five-tube MPN procedure. Microbes were enumerated
using an MPN procedure because the MPN method
produces a quantitative measure of the concentration
of microorganisms in a sample even when microor-
ganisms are present at very low concentrations. The
microorganisms chosen for comparison were those
recommended by USP/EP/JP pharmacopeias and sup-
plemented with two additional organisms, E. coli and
P. acnes. E. coli was included because it is recom-
mended as a test organism for total viable aerobic
count (16), and P. acnes was included because it is a
slow-growing, anaerobic to aerotolerant bacterium Figure 2
that is frequently associated with contamination from
human flora. As such, the aerobic nature of the Percent of culture tubes showing the presence of
ScanRDI威 assay method imposes a severe metabolic microorganisms at three dilution levels. Open sym-
stress on this organism. It is clear that the limit of bols depict data collected using the ScanRDI威
detection for P. acnes was not adversely affected by method, while closed symbols depict data collected
the additional metabolic stress. We thus employed a using the reference compendial sterility test
range in the types of organisms including anaerobes method. Values alongside the names of organisms
and aerobes, Gram-negatives and Gram-positives, in the legend indicate the total number of tubes
those with fermentative metabolism and those with tested for a given organism. Horizontal lines
respiratory metabolism, and prokaryotes and eu- (dashed or dotted) depict the overall mean for all
karyotes. organisms at a given dilution.

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TABLE IV
Comparison of the Limits of Detection
(cells/mLⴚ1)
ScanRDI威 Compendial
Organism Method Method
Clostridium sporogenes 0.000070 0.002992
Propionibacterium acnes 0.000153 0.002805
Escherichia coli 0.000805 0.003846
Pseudomonas aeruginosa 0.001172 0.017179
Staphylococcus aureus 0.000489 0.002871
Bacillus subtilis 0.000192 0.001832
Aspergillus niger 0.000690 0.003199
Candida albicans 0.000178 0.005370
In a recent commentary, Moldenhauer and Sutton (1,
17) outlined some of the flaws associated with the
standard sterility test. They point out that it is well
within the limits of current technology to conduct
sterility testing without the necessity of culturing or-
ganisms and the attendant problems of culturing tech-
niques (medium type, incubation conditions, detection
limits, etc.). The ScanRDI威 effectively avoids many of
the problems associated with detection of microbes by
culture approaches. It links two metrics of cell viabil-
ity—the action of nonspecific esterases and the pres-
ence of an intact-functioning cell-membrane—with
the production of a fluorochrome. Cells containing the
fluorochrome are easily detected by laser and subse-
quently detected (and enumerated) by computer. The
approach is rapid and sensitive and allows for the
detection of a single cell on the surface of a 25-mm
membrane (5).
When routinely used, the ScanRDI威 requires neither
lengthy incubation times nor specific culture media.
However, by combining requirements of the reference
sterility test (specific organisms, growth in specific
media, incubation conditions, and duration) with an
MPN enumeration protocol we were able to directly
compare the two approaches to sterility testing. The
ScanRDI威 method for detection of microbes was dem-
onstrated to be statistically non-inferior to the refer-
ence sterility test and numerically superior in that it
had a likelihood of detecting microbes that was sig-
nificantly greater at all dilution levels. As such, the
ScanRDI威 method is appropriate for use as a rapid
alternative to the growth-based sterility test method.
362
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