PRESENTATION

Flow Cytometry
Evolution of Microbiological Methods for Probiotics Enumeration
Marco Pane, BS, Serena Allesina, PhD, Angela Amoruso, PhD,
Stefania Nicola, PhD, Francesca Deidda, BS, and Luca Mogna, PhD

for presenting this information. Indeed, another of this

Goals: The purpose of this trial was to verify that the analytical group’s recommendations was “further development of
method ISO 19344:2015 (E)-IDF 232:2015 (E) is valid and methods (in vitro and in vivo) to evaluate the functionality
reliable for quantifying the concentration of the probiotic and safety of probiotics.” Even if the term “probiotic” is
Lactobacillus rhamnosus GG (ATCC 53103) in a finished product
formulation.
usually relative to microorganisms used in food industry and
food supplements, the working group suggestion applies to
Background: Flow cytometry assay is emerging as an alternative Live Biotherapeutic Products as well.
rapid method for microbial detection, enumeration, and Since the literature reports that nonculturable
population profiling. The use of flow cytometry not only permits microorganisms2 and “probiotic derived factors”3 may also
the determination of viable cell counts but also allows for confer a health benefit, it is likely that the definition of
enumeration of damaged and dead cell subpopulations. Results
are expressed as TFU (Total Fluorescent Units) and AFU
probiotics will continue to evolve as more research leads to
(Active Fluorescent Units). In December 2015, the International superior understanding.4
Standard ISO 19344-IDF 232 “Milk and milk products—Starter Over the last 20 years, multiparametric flow cytometry
cultures, probiotics and fermented products—Quantification (FCM) has arisen as a powerful tool in microbiology, par-
of lactic acid bacteria by flow cytometry” was published. This ticularly in biotechnological processing, food preservation,
particular ISO can be applied universally and regardless of the chemical disinfection processes, and pathogen enumeration in
species of interest. clinical and industrial practice. FCM involves generating a
Study: Analytical method validation was conducted on 3 different liquid suspension of bacteria from a food sample that is then
industrial batches of L. rhamnosus GG according to USP39 < 1225 > proceeded within a liquid stream known as sheath fluid. As
/ICH Q2R1 in term of: accuracy, precision (repeatability), inter- each cell passes through a point within the flow path it is
mediate precision (ruggedness), specificity, limit of quantification, interrogated by a laser, which scatters light in 2 major
linearity, range, robustness. directions known as forward angle light scatter (forward
scatter or FSC) and side angle light scatter (side scatter or
Results: The data obtained on the 3 batches of finished product have
significantly demonstrated the validity and robustness of the cyto- SSC). FSC and SSC can, respectively, distinguish cells based
fluorimetric analysis. on their size and granularity and hence are used to pinpoint
cells on a profile known as a cytograph. Forward and side
Conclusions: On the basis of the results obtained, the ISO scattered light and fluorescence from stained cells are split
19344:2015 (E)-IDF 232:2015 (E) “Quantification of lactic acid into defined wavelengths and channeled by a set of filters and
bacteria by flow cytometry” can be used for the enumeration of
mirrors within the flow cytometer. The fluorescent light is
L. rhamnosus GG in a finished product formulation.
filtered so that each sensor will detect fluorescence only at
Key Words: flow cytometry, microbial enumeration, method validation, a specified wavelength. These sensors are called photo-
probiotics multiplying tubes. This is the first step in bacterial enumer-
ation that permits cells to be distinguished from inert matrix
(J Clin Gastroenterol 2018;00:000–000) particles in a sample. The second, and more specific, cell
detection and enumeration method involves gathering of
fluorescence signals radiated at different wavelengths from

A World Health Organization working group has rec-

ommended that probiotics be defined as: “live micro-
organisms which when administered in adequate amounts
labeled/stained cells as they pass through the laser beam.
Data are collected from individual stained cells and the
uptake degree of a particular stain allows discrimination of
confer a health benefit on the host.”1 What that amount cells into distinct subpopulations. Thus, FCM data may
should be was not indicated. The same working group reflect different features such as functionality of membrane
suggested that product labels should include information on potential, extent of cell membrane integrity, DNA base
“minimum viable numbers of each probiotic strain at the composition, and presence of intracellular enzyme activity.5–7
end of the shelf-life” but notably did not recommend Combinations of dyes are used habitually to generate multi-
adoption of Colony Forming Units (CFUs) as the standard parametric data from individual cells and subpopulations.8
The more significant ones for microbial enumeration relate to
those stains asserting to reflect viability, or “live” and “dead”
From the Biolab Research S.r.l., Novara, Italy.
Supported by Probiotical S.p.A. states. The most common stains used to assess viability are an
M.P., S.A., A.A., S.N., F.D., and L.M. are employees of Biolab association of propidium iodide (PI) and thiazole orange
Research Ltd. (TO). TO or equivalent penetrates all bacteria and stains the
Address correspondence to: Luca Mogna, PhD, Biolab Research Ltd, Via nucleic acid with green fluorescence, whereas PI penetrates
E. Mattei 3, Novara 28100, Italy (e-mail: [Link]@mofi[Link]).
Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. only bacteria with damaged membranes, thus staining with
DOI: 10.1097/MCG.0000000000001057 red fluorescence.9

J Clin Gastroenterol
Volume 00, Number 00, ’’ 2018 [Link] |1

Copyright r 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
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Pane et al J Clin Gastroenterol
Volume 00, Number 00, ’’ 2018

Many attempts have been realized to develop rapid and It seems that FCM is likely to become the rapid
unbiased methods, usually based on the exclusion, uptake, or microbial enumeration method of choice over the next
metabolism of colored, fluorescent, or fluorogenic stains, several years, based on the appearance of standardized
designed to provide information that correlates with viability. methods.11,12
FCM can provide data on different structural and functional In fact, the recent approval of FCM as a tool for cell-
properties of cells, which then enable quantification of viability based processes, products development, and quality control
beyond the traditional reproductive capacity on agar and other by the US Pharmacopeia shows aptitude for more wide-
microbiological media, providing greater insight into the spread acceptance of cytometric profiling as a measure of cell
functional strain-related responses to various applications. number and viability (FCM USP < 1027 > ). Moreover, in
Fluorescent-activated cell sorting (FACS) implicates December 2015, the International Standard ISO 19344-IDF
an enhanced level of FCM-based analytical resolving power 232 “Milk and milk products—Starter cultures, probiotics
for use in food microbiology. Cell sorters are cytometers and fermented products—Quantification of lactic acid bac-
with the ability to physically sample cells originating from a teria by flow cytometry” was published. This particular ISO,
subpopulation of interest.10 as the name suggests, can be applied universally and inde-
FCM has a number of significant advantages over pendently of the species of interest for bacterial enumeration.
other methods: Since neither FCM nor any of the other rapid enu-

It is fast, taking only minutes per sample, instead of days meration methods involves counting microbial colonies, it
as required by most colony-counting methods. follows that, by definition, they would be unable to report in

It can simultaneously gather data about the number of terms of CFUs.
live and dead cells in the counted population. Any guideline should allow for flexibility in the

It can count the cells of all species present in a mixed approach used to determine the “dose” in order to permit
population without the need for a multiple and differently continued innovation in the industry and academic field,
selective growth media. while providing useful information.

TABLE 1. The Most Important Parameters Stated in the USP39 < 1225 > /ICH Q2R1 and Directly Related to Analytical Method Validation
Acceptance Limits USP
Parameters Description No. Samples < 1225 > /ICH Q2R1
Accuracy USP: no method for evaluation of the 3 batches of sample. 3 levels of Recovery not <70%. Estimation of viable
probiotic bacteria count. ISO 19344:2015 concentration. 3 replicates microorganisms calculated with the
(E)-IDF 232:2015 (E) for finished alternative method not <70% of the
product: definition in ICHQ281 “The conventional true value
closeness of agreement between the value
which is accepted either as a
conventional true value and the value
found”
Precision The degree of agreement among individual 3 batches of sample. 1 level of RSD ≤ 10%-15%
(repeatability) test results when the procedure is applied concentration. 6 replicates.
repeatedly to multiple samplings of Same working session
suspensions of laboratory microorganism
across the range of the test. The tests are
performed in the same operating
conditions over a short interval of time
Intermediate Precision expressed within laboratory 3 batches of sample. 1 level of RSD ≤ 10%-15%. The variance of the
precision variations: different reagents, different concentration. 6 replicates. intermediate precision is the sum of the
days, different analysis Different working session variance of the repeatability and the
intergroup variance
Specificity The ability of the method to give reliable 3 batches of sample All microorganisms selected as
results when challenged with extraneous representative are successfully
matter that would generate false positive enumerated
results
Limit of The lowest number of microorganisms that See accuracy/linearity/ —
quantification can be accurately counted precision results
Linearity The ability (within a given range) to obtain 3 batches of sample. 5 levels of Significant estimated slope. R2 ≥ 0.95
test results which are directly concentration. 2 replicates.
proportional to the concentration of 2 working sessions
analyte in the sample
Range The range in the interval between the upper See accuracy/linearity/ —
and lower levels of microorganisms that precision results
have been demonstrated to be
determined with precision, accuracy, and
linearity using the method
Robustness Measure of the capacity of the method to 3 batches of sample. 3 changes Recovery not <70%
remain unaffected by small but deliberate to the standard method
variations in method parameters; it (8 combinations)
provides an indication if its reliability
under a variety of normal test conditions

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Copyright r 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
This paper can be cited using the date of access and the unique DOI number which can be found in the footnotes.
J Clin Gastroenterol
Volume 00, Number 00, ’’ 2018 FCM: Evolution of Microbiological Methods

FIGURE 2. Analytical method validation, precision/repeatability

FIGURE 1. Analytical method validation, accuracy parameter. parameter. Data are expressed as means of 6 replicates for each
Data are expressed as percentage of recovery and are means ± SD batch tested (◊: single result) at the concentration of 10−4.
of 3 replicates for each batch tested. Acceptance limits ≥ 70%. Acceptance limits: relative standard deviation (RDS) ≤ 10% to
15%. AFU indicates Active Fluorescent Unit.
The purpose of our study and activity was to verify that
the analytical method ISO 19344:2015 (E)-IDF 232:2015 (E) of the previous sample to 9 mL of peptone saline. In this
is valid and reliable for quantifying the concentration of the way, sample no. 5 had an expected concentration of 2.5×104
probiotic Lactobacillus rhamnosus GG (ATCC 53103) in a bacterial cells/mL.
finished product formulation.
Sample Staining and FACS Detection
MATERIALS AND METHODS The BD Cell Viability Kit offered an easy-to-use dye
combination to distinguish live and dead cells for analysis
Bacterial Strain Growth Conditions by FCM. The kit contained TO solution to stain all cells and
The Lactobacillus strain was grown overnight in De PI to stain dead cells. BD Liquid Counting Beads is a liquid
Man, Rogosa, and Sharpe (MRS) broth (Difco, BD, MD) suspension of fluorescent beads. The beads were added to a
at 37°C. flow sample to calculate absolute counts.13
Validation of the Analytical Method The kit contained 1 vial of 500 μL 42 μmol/L TO in
dimethyl sulfoxide and 1 vial of 500 μL 4.3 mmol/L PI in
Analytical method validation was conducted on 3 dif-
water. The Liquid Counting Beads were supplied as 1 vial of
ferent industrial batches of L. rhamnosus GG according to
10 mL of fluorescent microspheres in buffer with 0.1%
USP39 < 1225 > /ICH Q2R1 in terms of: accuracy, precision
sodium azide.
(repeatability), intermediate precision (ruggedness), specif-
For staining, 0.5 mL of the fifth decimal dilution of cell
icity, limit of quantification, linearity, range, robustness.
suspension were transferred into a polystyrene round-
The specific criteria applied for each of these parameters are
bottom tube. 2.5 μL of TO and 1.0 μL of PI were added to
detailed in Table 1.
the tube and then vortexed. Before analysis, the bead
Sample Preparation suspension was gently vortexed for 30 seconds and then
The expected count of the industrial freeze-dried 50 μL were added to the tube. Prepared samples were then
batches of L. rhamnosus GG was 2.5×1010 live bacteria/g. acquired on the FACScan cytometer (BD FACSCalibur
The composition of the finished product formulation Software; Becton Dickinson, San Jose, CA) equipped with
encompassed 250 mg/g of a pure culture of freeze-dried 488 nm argon laser excitation and CellQuest software.
L. rhamnosus GG, 50 mg/g of vanilla flavor, 500 mg/g of A SSC threshold was used for microbial cells. Cells
fructooligosaccharides, and 200 mg/g of maltodextrin. To were gated using FSC and SSC. TO fluoresces primarily in
obtain the primary dilution, 2 ± 0.5 g of the test sample were FL1; PI fluoresces primarily in FL3. Therefore, the best
weighed in a sterile plastic bag suitable for stomacher. discrimination of live and dead populations was on an FL1
Sodium borate buffer solution pH 8.4 was added to make a versus FL3 plot.
1:10 dilution, then the bag was mixed well manually and
homogenized in the stomacher circulator for 4 minutes at a
speed of 230 rpm. The theoretical bacterial concentration
was 2.5×109 cells/mL of borate buffer.
Five subsequent decimal dilutions have then been cre-
ated using a peptone saline solution and transferring 1 mL

TABLE 2. Specificity Results (AFU/mL) Using the 3 Samples

Reported
Sample Results (AFU/mL)
Peptone saline, pH 6.8-7.2 Not detectable
PBS solution Not detectable FIGURE 3. Analytical method validation, intermediate precision
Placebo (maltodextrin) Not detectable parameter. Data are 6 replicates for each batch tested and for
single analyst at the concentration value of 10−4. Orange dotted
AFU indicates Active Fluorescent Unit; PBS, phosphate-buffered saline line represents the mean of all results. Acceptance limits: relative
containing 0.01% TWEEN 20 and 1 mmol/L EDTA. standard deviation (RDS) ≤ 10% to 15%. AFU indicates Active
Fluorescent Unit.

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Pane et al J Clin Gastroenterol
Volume 00, Number 00, ’’ 2018

range as 105 to 107 cells/mL derived from a dilution of 10−3

to 10−5 of the initial sample. In the case of finished products
with a concentration above 109 cells/g, it is necessary to
further dilute in order to stay in the optimal instrumental
range.
Regarding the range, the data obtained on the 3
batches allowed to define the lowest level (limit of quanti-
fication) as equal to 105 cells/mL. According to the linearity
trial performed, the detection limit could be set at a value of
104 cells/mL.
Taken as a whole, these data have significantly dem-
onstrated the validity and robustness of the cytofluorimetric
FIGURE 4. Analytical method validation, linearity parameter. The analysis (Figs. 2–5).
graph was built using 5 different concentrations ranging from
2.5×104 to 2.5×108 AFU/mL. Acceptance limits: R2 ≥ 0.95. AFU
indicates Active Fluorescent Unit. DISCUSSION
Bacterial viability at present refers to capability of a cell
Calculation and Expression of Results to grow and replicate itself under a set of definite environ-
To determine the concentration of the cell populations, mental conditions.14 The features of viable cells include the
the following equation was used: presence and running of a range of structural, metabolic,
n cell region n beads=tube physiological, and genetic properties. However, this tradi-
 dilution factor ¼ cells number: tional definition requires that a single “viable” cell grows and
n beads region test volume
successively generates a colony of cells that can be measured
For freeze-dried samples, results were expressed in optically either in liquid or solid media, thus establishing the
number of cells/g. basis of Koch’s principles.15 Hence, viability is generally
The measurement of uncertainty was based on the equated with the ability to reproduce and cultivability.
parameters reported in Table 1. Another classification is centered on the term “vitality”
or the degree to which a cell can achieve various aspects of
RESULTS physiological, metabolic, and genetic functionality and the
The data obtained on the 3 batches of L. rhamnosus GG magnitude of structural and morphologic integrity.16 In this
are reported in Figure 1 (accuracy), 2 (precision/repeatability), case, cell vitality is not exclusively related to reproducibility
3 (intermediate precision), 4 (linearity), 5 (robustness). or cultivability and in certain cases a cell can be described
Specificity is reported in Table 2. with a viable but nonculturable state.17
For what concerns the limit of quantification, the data On the basis of these different states, the enumeration
obtained on the 3 batches allowed to define the optimal of bacterial cells has evolved into 2 general methodologies:

FIGURE 5. Analytical method validation, robustness parameter. Deliberate variations in method parameters were applied, as described.
Acceptance limits: ≥ 70%.

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J Clin Gastroenterol
Volume 00, Number 00, ’’ 2018 FCM: Evolution of Microbiological Methods

(1) culture dependent and (2) culture independent. consumers may have little ability to compare the probiotic
Approximately only 0.1% to 1.0% of the total bacteria can content of 1 product with that of another in any
be enumerated by the plating procedure. meaningful way.
Culture-independent methodologies for bacterial enu-
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Copyright r 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
This paper can be cited using the date of access and the unique DOI number which can be found in the footnotes.