AOAC Virtual Annual Meeting and Exposition – September, 2020

Probiotic Yeast Enumeration by Flow Cytometry: Method Modification Verification of ISO 19344(B)

Andrzej A. Benkowski, Nathaniel O. Tull and J. David LeganEurofins Microbiology Laboratories2102 Wright Street, Madison, WI 53704Corresponding author: andrzejbenkowski@eurofinsus.com, Phone 608.949.3022

Abstract

Increasingly, probiotic dietary supplement stakeholders are usingflow cytometry to address manufacturers’ and consumers’demands for information about the quantity, quality, and identityof organisms in probiotic products. Previously, we verified ISO19344:2015(E)/IDF 232:2015(E) for enumerating probiotic bacteriain lyophilized powders using acoustic focusing flow cytometry. Thestudy showed that cytometric accuracy, precision, and turnaroundtimes were comparable to or better than those of plate andmicroscopic counting. Here, the utility of flow cytometry isexpanded further by applying modifications to ISO 19344 (ProtocolB) in order to accurately quantify the yeast Saccharomycescerevisiae var. boulardii. Lyophilized yeast materials were evaluatedvia ISO 19344 with modifications to the sample preparation andinstrument settings. The reference method for the study was acultural plate count enumeration. Accuracy, precision(repeatability), and intermediate precision (ruggedness) wereassessed by analysis of data from at least three separate analyticalruns using two concentrations of product, which were counted intriplicate. The data produced by the method modificationverification study met or exceeded AOAC Appendix K acceptancecriteria for accuracy, precision, and intermediate precision.Tailoring the sample preparation for a yeast enumeration followingthe principles of ISO 19344 demonstrated results comparable tocultural enumerations for lyophilized materials.

Introduction

A flow cytometer uses laser light to detect particles in a fluidstream. Emissions scatter at different angles depending on particlesize and internal complexity. The deflected light hits a series ofdetectors and the signals from these detectors are interpreted bycomputer software in real-time.

Figure 1. The Attune NxT flow cytometer.©2016 Life Science Technologies, Thermo Fisher Inc. Used under permission. www.thermofisher.com

A newer innovation, acoustic-assisted hydrodynamic flowcytometry, uses sound vibrations to align the particles and reducesthe time to result.

Materials

Instrument

Invitrogen™ Attune™ NxT acoustic flow cytometer(Thermo Fisher Scientific, Inc.: Figure 1)

Matrix

Lyophilized Saccharomyces cerevisiae var boulardii raw material(S. boulardii)

Methods

Method Modification GuidanceWe followed USP 40-NF 35 <1225>1/ICH Q2 (R1)2 to determineaccuracy, precision and intermediate precision of the assay withthe test matrix. Acceptance criteria for the method verificationwere based on AOAC Appendix K, “Guidelines for DietarySupplements and Botanicals.”3

Flow cytometry results for accuracy and precision testing werecompared with cultural plating derived from the same samplepreparation. Plate counts were generated following Compendiumof Methods for the Microbiological Examination of Foods(Probiotics) 5th Edition, Chapter 20, American Public HealthAssociation: Washington, D.C., (2015). Modified.

AssayISO 19344:2015(E)/IDF 232:2015(E), Protocol B,4 with samplepreparation modifications was evaluated for its ability to providerapid, precise and accurate enumeration of live, injured and deadcells (Figure 3). It should be noted that ISO/IDF considers stainedcells as presumed live, injured or dead and refers to them as active,damaged or non-active, respectively.

Figure 2. Example of a scatter plot showing differentiated live, injured and dead cell populations.

Results

Mean % Recovery (Accceptance range 70 ̶ 125 %)

Day Replicate 1 Replicate 2 Replicate 3 Overall

1 126 105 95 109

2 109 134 92 112

3 102 117 107 109

Average: 110

Table 1. Accuracy Demonstrated by Mean Recovery

Accuracy

The mean recovery by day ranged from 109 to 112 % of the valueobtained by plate count (Table 1).

Precision

Percent relative standard deviation (% RSD) for precision andintermediate precision met the specified acceptance criterion(≤ 15%).

% RSD (Acceptance ≤ 15%)

Test Organism

Precision(n=10)

Intermediate Precision(n=6)

S. boulardii 4.2 3.6

Table 2. Precision and Intermediate Precision Determined by Evaluating S. boulardii Using Modified ISO 19344 Methodology.

Platform Comparison

Flow cytometry results produced a % RSD approximately half thatof cultural plate counts.

Table 3. Total % RSD Observed Between Flow Cytometry and Plate Count Results

% RSD for Mean Counts

Test Organism

Flow Cytometry Live(n=19)

Plating Colony(n=9)

S. boulardii 5.0 9.7

ConclusionsA validated acoustic focusing flow cytometer producesreliable, accurate and reproducible enumeration data forthe probiotic yeast Saccharomyces cerevisiae var. boulardii.With slight modifications, ISO 19344:2015(E)/IDF232:2015(E) can be used to enumerate S. boulardii.Furthermore, flow cytometry generates accurate resultsthat are comparable to cultural plate counting withsuperior precision as shown by relative standard deviationof the data set being approximately half that of culturalplate counting.

Figure 3. Flow diagram of S. boulardii enumeration protocols by flow cytometry and cultural plate count.

1. United States Pharmacopeial Convention (2015). <1225> Validation of compendial

procedures. In: United States Pharmacopeia 40 – National Formulary 35 , General

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2. International Conference on Harmonization of Technical Requirements for Registration

of Pharmaceuticals for Human Use (ICH), step 4 version. 2005. Validation of analytical

procedures: text and methodology Q2(R1). In: ICH harmonized tripartite guideline.

3. AOAC International. 2012. Appendix K: guidelines for dietary supplements and

botanicals. In: AOAC Official Methods of Analysis (19th edition), Gaithersburg, MD.

4. International Organization for Standardization (ISO). 2015. Milk and milk products –

starter cultures, probiotics, and fermented products – quantification of lactic acid

bacteria by flow cytometry. ISO 19344:2015 (E)/IDF 232:2015 (E).