Enumeration of Total Yeasts and Molds in Foods by the …€¦ · · 2017-10-17cake mix, cereal, and fresh cheese. Nut meats, frozen fruits, ... DRBC agar was used for all other

FOOD BIOLOGICAL CONTAMINANTS

Enumeration of Total Yeasts and Molds in Foods by theSimPlate® Yeast and Mold–Color Indicator Method andConventional Culture Methods: Collaborative StudyPHILIP T. FELDSINE, ANDREW H. LIENAU, STEPHANIE C. LEUNG, and LINDA A. MUI

BioControl Systems, Inc., 12822 SE 32nd St, Bellevue, WA 98005

Collaborators: S. Al-Hasani; G. Anderson; A. Ashmore; P. Audet; D. Barham; C. Beaulieu; F. Cook; M. Cornec; R. Davis;A. Deans; S. DeLancey; L. DeWinter; M. Divine; S. Drocco; M. Feist; D. Hickson; F. Humbert; L. Humes; J. Jackson;R. Kalinowski; J. Knickerbocker; M. Lisby; C. McDonald; J. Terry; K. Thammasouk; E. Tuncan; A. Vinai; D. Vrana;W. Warren; S. White

The relative effectiveness of the SimPlate Yeastand Mold–Color Indicator method (Y&M–CI) wascompared to the U.S. Food and Drug Administra-tion’s (FDA) Bacteriological Analytical Manual(BAM) method and the proposed International Or-ganization for Standardization (ISO) method,ISO/CD 21527, for enumerating yeasts and moldsin foods. Test portions were prepared and incu-bated according to the conditions stated in boththe BAM and ISO methods. Six food types were an-alyzed: frozen corn dogs, nut meats, frozen fruits,cake mix, cereal, and fresh cheese. Nut meats,frozen fruits, and fresh cheese were naturally con-taminated. All other foods were artificially contami-nated with either a yeast or mold. Seventeen labo-ratories throughout North America and Europeparticipated in the study. Three method compari-sons were conducted. In general, there was<0.3 mean log count difference in recovery be-tween the SimPlate method and the 2 correspond-ing reference methods. Moreover, mean log countsbetween the 2 reference methods were also verysimilar. The repeatability (sr) and reproducibility(sR) standard deviations were comparable betweenthe 3 method comparisons. These results indicatethat the BAM method and the SimPlate method areequivalent for enumerating yeast and mold popula-tions in foods. Similarly, the SimPlate method iscomparable to the proposed ISO method when testportions are prepared and incubated as defined inthe proposed ISO method.

Yeasts and molds can cause various degrees of food de-composition. Their ability to proliferate in a widerange of pH and temperatures, and in foods of various

water contents, make it necessary to detect fungal spoilage atthe early stages. Current methods for enumeration of yeastsand molds include the U.S. Food and Drug Administration’s(FDA) Bacteriological Analytical Manual (BAM) culturemethod (1) and the International Organization for Standard-ization (ISO) method (2). Both methods use agar plates thatare incubated for 5–7 days (120–168 h) before obtaining re-sults. The SimPlate® Yeast and Mold–Color Indicator(Y&M–CI) method uses Binary Detection Technology (BDT)to enumerate yeasts and molds in foods after a minimum of56 h of incubation, thereby offering a significant timesavingadvantage over the current culture methods. The SimPlate de-vice also eliminates the uncertainty in enumeration of platecounts because of factors such as overcrowding, spreadingcolony types, and food particulate interference on conven-tional plate methods. In the SimPlate multiple test format, pre-pared food test portions are placed onto the center of aSimPlate device, and Y&M–CI liquid medium is then added.For the single test format, a premixed test portion/medium ho-mogenate is dispensed into the test device. The test por-tion/medium homogenate is distributed into a fixed number ofindividual incubating wells: 84 for the normal counting rangeSimPlate test and 198 for the high counting range.

With the SimPlate method, foodborne microorganisms aresuspended in a nutritionally defined growth medium. To en-hance performance, medium supplements are provided for usewith certain food matrixes. These supplements balance certaininherent attributes of some matrixes such as increased acidityfound in foods containing high levels of vitamin C. Discretealiquots are separately compartmentalized and isolated fromeach other in the incubating wells where biochemical activi-ties of viable microorganisms are monitored in a liquid envi-ronment. Detection by this biochemical process requiresfewer microorganisms to produce a detectable signal in aSimPlate well than the number required to form a clearly visi-ble colony on an agar plate. Enumeration is measured by a

296 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003

Submitted for publication December 2002.The recommendation was approved by the Methods Committee on

Microbiology and Extraneous Materials as First Action. See “OfficialMethods Program Actions,” (2003) Inside Laboratory Management,March/April issue.

Corresponding author’s e-mail: [email protected].

simple binary reaction; each well is either positive or negative.Any color change from the original background color in eachwell or in the sponge is interpreted as a positive reaction.Yeasts and molds are enumerated by counting the numbers ofwells in each plate that exhibit a color change (positive wells)after incubation. The final count per plate is derived from Ta-ble 1 that is based upon the principle of the Poisson distribu-tion. The large number of wells allows an accurate measure ofthe true microbial population in a test sample.

A recent study compared the SimPlate Y&M–CI methodand BAM method for recovery and quantification of yeasts

and molds in 20 different foods (publication pending). Over-all, the results indicated that the SimPlate Y&M–CI method isequivalent to the BAM reference method. This report de-scribes a separate international multilaboratory collaborativestudy comparing the relative effectiveness of the SimPlateY&M–CI method to the BAM plate method for recovery ofyeasts and molds. The SimPlate method and the proposed ISOculture method were evaluated in this same study.

Collaborative Study

Design of Study

Six food types, representative of a wide range of food cate-gories, were evaluated: frozen corn dogs (frankfurter encasedin cornbread), frozen fruits, cereal, fresh cheese, cake mix,and nut meats. Frozen fruits, fresh cheese, and nut meats werenaturally contaminated. For these foods, 2 test portions from 3different lots were analyzed for each food type (total of 6 testportions per food type). Cereal, cake mix, and frozen corn dogswere artificially contaminated with either a yeast or mold. Inoc-ulation levels in foods artificially contaminated were as fol-lows: high, approximately 10 000; medium, 1000; low100 fungi/g of food. Foods that required Dichloran rose bengalagar (DRBC) spread plates as the reference culture methodwere artificially contaminated at these inoculation levels: high,approximately 100 000; medium, 10 000; low 1000 (minimumdetection level by the BAM method) fungi/g of food. Two testportions from 3 different inoculation levels and uninoculatedcontrols were analyzed for each artificially inoculated food type(total of 8 test portions per food type).

Collaborators were sent 2 sets of randomized test portions;one set was used for analysis by the BAM culture method, us-ing 0.1% peptone diluent, and the corresponding SimPlatemethod (SIM–B). The other set was used for analysis by theISO culture method, using peptone salt solution, and the cor-responding SimPlate method (SIM–I). The cereal, cake mix,and nut meats were stored at room temperature until the day ofanalysis. Fresh cheeses were refrigerated; frozen fruits andcorn dogs were kept frozen (–20�C) until the day of analysis.

For one set of test portions, collaborators were instructed toprepare the initial suspension and further decimal dilutions asrecommended by BAM (1). The appropriate dilutions wereanalyzed by the BAM plate method and the SimPlate method(SIM–B), both incubated at 25�C. Two types of agar plateswere used for the reference method. Dichloran 18 glycerol(DG18) agar was used when the water activity of the food was<0.95; otherwise, DRBC agar was used for all other foodtypes. For the second set of test portions, collaborators pre-pared the initial suspension and further decimal dilutions ac-cording to ISO 6887 (3). The appropriate dilutions were ana-lyzed according to ISO/CD 21527 (2) culture method (DG18agar) and the SimPlate method (SIM–I), both incubated at25�C. The food types with higher water activity (aw > 0.95)were also tested with DRBC agar plates. An overview of theinterlaboratory study design is shown in Figure 1. Results ob-tained were submitted on summary forms with the appropriate

FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 297

Table 1. SimPlate conversion table

No. of positive wells = population per platea

1 = 2 31 = 76 61 = 216

2 = 4 32 = 80 62 = 224

3 = 6 33 = 84 63 = 232

4 = 8 34 = 86 64 = 240

5 = 10 35 = 90 65 = 248

6 = 12 36 = 94 66 = 256

7 = 14 37 = 96 67 = 266

8 = 16 38 = 100 68 = 276

9 = 18 39 = 104 69 = 288

10 = 22 40 = 108 70 = 298

11 = 24 41 = 112 71 = 312

12 = 26 42 = 116 72 = 324

13 = 28 43 = 120 73 = 338

14 = 30 44 = 124 74 = 354

15 = 32 45 = 128 75 = 372

16 = 36 46 = 132 76 = 392

17 = 38 47 = 136 77 = 414

18 = 40 48 = 142 78 = 440

19 = 42 49 = 146 79 = 470

20 = 46 50 = 150 80 = 508

21 = 48 51 = 156 81 = 556

22 = 50 52 = 160 82 = 624

23 = 54 53 = 166 83 = 738

24 = 56 54 = 172 84 = >738

25 = 58 55 = 178 If there are no positivewells and the sponge is

positive, then thepopulation is 1

26 = 62 56 = 184

27 = 64 57 = 190

28 = 68 58 = 196 If there are no positivewells and the sponge is

negative, then thepopulation is <1

29 = 70 59 = 202

30 = 74 60 = 208

a The population reflects the number of microorganisms per plate.To determine the number of microorganisms per gram (mL) foodproduct, see I (Reading and Interpretation of Results).

raw data. A minimum of 8 laboratories submitted valid datafor each food type.

Preparation of Inocula and Test Portions

Frozen corn dogs, cake mix, and cereal test portions wereinoculated with Candida albicans, Torulaspora delbrueckii,and Aspergillus clavatus, respectively. These fungi were cho-sen because they are commonly isolated from food products.Test cultures were grown on potato dextrose agar (PDA)plates at 25�C for 3–7 days. Growth from these plates was re-suspended into Butterfield’s phosphate buffered dilution wa-ter (BPBD). This suspension was diluted and used to artifi-cially contaminate foods. For cake mix, C. albicans wasgrown on PDA plates at 25�C for a minimum of 3 days. Theviable colonies were collected, washed, and resuspended into10% nonfat dry milk for freeze-drying. Lyophilized cultureswere stored at –20�C until used. Inoculated test portions wereprepared at least 1 week before analysis to allow populationsto stabilize.

The naturally contaminated foods were purchased at the re-tail level in Seattle, WA. The bulk foods were thoroughlymixed and then packaged into 50 g test portions for shipmentto collaborators.

Test Portion Distribution

Each collaborator received 2 sets of 6–8 test portions foreach week the study was conducted. Test products were dis-tributed by an overnight delivery from the United States tolaboratories throughout North America and Europe. Frozenfoods were shipped on dry ice; cereal, cake mix, and nut meattest portions were shipped at room temperature. Fresh cheesewas shipped on ice.

Test Portion Analysis

The BAM and ISO culture methods use different diluentsfor the initial suspension and decimal dilutions of test portions.This required collaborators to set up separate paired sampleswith one test portion prepared with the BAM diluent and the

other with the ISO diluent. Analysts were instructed to followboth BAM and ISO reference procedures without deviation.

Designated food test portions were prepared as specifiedby BAM (8th Ed., Rev. A, Ch. 18; 1). For all food types in-cluding nut meats, 50 g test portions were added to 450 mL0.1% peptone water, and homogenized by stomaching for2 min. Frozen test portions were added directly to the diluent(unthawed) and then stomached to obtain a liquid homoge-nate. After homogenization, suspensions were serially dilutedinto 0.1% peptone water. The number of dilutions necessaryto perform the test depended on the food type being analyzed.Subsequent 10-fold dilutions were prepared by adding 10 mLof the previous dilution to 90 mL sterile diluent and shaken25 times in a 30 cm arc. BioControl provided the suggested di-lution series for each food type the week before analysis wasinitiated.

For low moisture food types (cake mix, cereal, and nutmeats), 1 mL from each appropriate dilution of the test portionwas transferred to a sterile Petri dish in triplicate. A 20 mLamount of DG18 agar, tempered to 42–45�C, was added andswirled to evenly distribute the test dilution and agar. Platecounts of each dilution level were performed in triplicate. Forhigh moisture food types (frozen corn dog, frozen fruits, andcheese), 0.1 mL from each appropriate dilution of the testportion was transferred to a sterile pre-poured DRBC dish intriplicate and spread across the surface of the plate. Platecounts of each dilution level were performed in triplicate.Frozen fruits were spread plated on DRBC agar plates con-taining chlortetracycline. All plates were incubated in the darkat 25�C for 5–7 days. The number of fungi present on eachplate was enumerated after the required incubation. Data wererecorded onto the appropriate worksheets. For the BAMmethod, plates containing 10–150 colonies were counted.

Designated food test portions were also prepared as speci-fied by ISO method 6887. For all food types, 50 g test por-tions were added to 450 mL peptone salt solution and ho-mogenized by macerating for 1 min. After homogenization,aliquots were serially diluted into peptone salt solution. The


1. 50 g Test portion � 450 mL 0.1% peptone water 1. 50 g Test portion � 450 mL peptone salt diluent2. Homogenize test portion 2. Homogenize test portion3. Perform appropriate dilutions 3. Perform appropriate dilutions

SimPlate test FDA’s BAM SimPlate test ISO

� � � �

1.0 mL Diluted sample onto device * 1.0 mL Diluted sample onto triplicate 1.0 mL Diluted sample onto device 1.0 mL Diluted sample onto duplicate+ 9 mL SimPlate Y&M–CI medium Petri plates + 20–25 mL DG18 agar + 9 mL SimPlate Y&M–CI medium Petri plates + 15 mL DG18 agar

OR spread 0.1 mL diluted sample AND, only for foods with aw > 0.95,onto triplicate DRBC agar plates spread 0.1 mL diluted sample onto

duplicate DRBC agar plates

� 22–25�C for 56 h minimum in dark � 25�C for 5–7 days in dark � 22–25�C for 56 h minimum in dark � 23–27�C for 5 days

1. Count number of positive wells 1. Count colonies on agar plates 1. Count number of positive wells 1. Count colonies on agar plates2. Use SimPlate conversion table to 2. Factor in dilution for final CFU/g 2. Use SimPlate conversion table to 2. Factor in dilution for final CFU/g

determine total number of fungi per plate determine total number of fungi per plate3. Factor in dilution for the total 3. Factor in dilution for the total

number of fungi per gram number of fungi per gram

Figure 1. SimPlate Y&M–CI, BAM, and ISO methods for enumeration of yeasts and molds. * = Spread or pour platewas selected based on the water activity of the food analyzed.

number of dilutions necessary to perform the test dependedon the food type being analyzed. Subsequent 10-fold dilu-tions were prepared by adding 10 mL of the previous dilutionto 90 mL sterile diluent and mixed well. BioControl pro-vided the suggested dilution series for each food type theweek before analysis was initiated.

For low moisture food types (cake mix, cereal, and nutmeats), 1 mL from each appropriate dilution of the test samplewas transferred to a sterile Petri dish in duplicate. Approxi-mately 15 mL DG18 agar, tempered to 42–45�C, was addedand swirled to evenly distribute test aliquot and agar. Platecounts of each dilution level were performed in duplicate. Forhigh moisture food types (frozen corn dog, frozen fruits, andcheese), 0.1 mL from each appropriate dilution of test samplewas transferred to a sterile pre-poured DRBC plate in dupli-cate and spread across the surface of the plate. Frozen fruitswere spread plated on DRBC agar plates containingchlortetracycline. Plate counts of each dilution level were per-formed in duplicate. All plates were incubated at 25�C for5–7 days. The number of fungi per plate were enumerated af-ter the required incubation. Data were recorded onto the ap-propriate worksheets.

Diluted test portions prepared for the BAM and ISO refer-ence methods were also used for SimPlate analyses. One set ofSimPlate devices (SIM–B) was set up with dilutions from theBAM method. A separate set of SimPlate devices (SIM–I)was set up with dilutions from the ISO method. A 1 mLamount of diluted test portion was transferred to the center ofthe SimPlate device. The device was overlayed with 9 mLrehydrated Y&M–CI medium, for a final volume of10 ± 0.2 mL. After the plate was covered with the lid, the testsample/medium homogenate was mixed and swirled into thewells. Excess liquid was decanted into the device collectionsponge. All SimPlate devices were incubated in the dark at25�C for a minimum of 56 h. After incubation, the number ofwells with a color change from the original background colorwere counted. Table 1 was used to calculate the yeast andmold population per SimPlate device. The number of positivewells counted was matched to the corresponding populationgiven in Table 1. If a dilution was made to the original foodtest portion before inoculation of the SimPlate device, then thefungi per gram of food was calculated by multiplying the pop-ulation from Table 1 by the dilution factor.

Statistical Analysis

For each lot of food, triplicate (duplicate for ISO) plateswere averaged and reported as colony forming units (CFU)/gfor the BAM or ISO reference methods and the SimPlatemethods. For the BAM method, if none of the plates for a testportion had at least 10 colonies, the exact counts from the leastdilute test sample were averaged.

The base 10 logarithms (log10) of SimPlate counts andCFU/g for each of the reference culture methods were used forstatistical analysis of the test portions. In a few instances, in-definite values were reported for data values for the test por-tions. To perform statistical analysis on these particular datavalues, the indefinite sign was truncated and method perfor-

mance was determined (4). Repeatability (sr) andreproducibility (sR) standard deviations, relative standard de-viations of repeatability (RSDr) and reproducibility (RSDR),and repeatability (r) and reproducibility (R) values were cal-culated according to the methods of Youden and Steiner (5)after the Cochran test was applied to eliminate outliers. TheCochran test was used to remove laboratories showing signifi-cantly greater variability among replicate analyses than otherlaboratories for a particular set of test portions. An F statisticthat computes the ratio of the 2 variances was used to comparerepeatability and reproducibility variances (6, 7). Mean re-sponses between the 2 methods were compared by using a2-sample (paired) t-test (6, 7).

AOAC Official Method 2002.11Detection and Quantificationof Yeasts and Molds in Foods

SimPlate Yeast and Mold–Color Indicator (Y&M–CI) MethodFirst Action 2002

(Applicable to detection and quantification of yeasts andmolds in chocolate, cake mix, spices, nut meats, dairy foods,red meats, poultry meats, seafoods, fermented meats, frozencorn dogs, cereal, pasta, egg products, flour, prepackagedfresh salad, frankfurters, vegetables, fruits, and fruit juice.)

Caution: Test portion dilutions and incubated SimPlate de-vices from food products could contain patho-genic fungi if the particular test portion was socontaminated. Use standard aseptic microbiologi-cal laboratory technique, including decontamina-tion of any spills with disinfectant.

See Tables 2002.11A–C for the results of theinterlaboratory study supporting acceptance of the method.

A. Principle

SimPlate Yeast and Mold–Color Indicator (Y&M–CI)method is used to detect and quantify yeast and mold popula-tions. The Y&M–CI medium and test sample mixture is dis-pensed into a SimPlate device and incubated for a minimum of56 h. The medium changes color in the presence of yeasts andmolds. The yeast and mold count is determined by countingthe wells with changed color and referring to Table 1.

B. Media and Reagents

(a) Dehydrated Y&M–CI medium.—Individually pack-aged single or multiple test format with supplement(s), avail-able from BioControl Systems, Inc. (12822 SE 32nd St, Belle-vue, WA 98005, www.rapidmethods.com).

(b) SimPlate devices.—In packs of 20 (BioControl).(c) Peptone water diluent, 0.1% (used in BAM

method).—Dissolve 1.0 g peptone in 1 L deionized water. Au-toclave at 121�C for 15 min. Final pH is 7.0 ± 0.2.

(d) Peptone salt solution (used in ISO method).—Use1.0 g enzymatic digest of casein and 8.5 g sodium chloride.Suspend ingredients in 1 L deionized water. Autoclave at121�C for 15 min. Final pH is 7.0 ± 0.2.


C. Apparatus

(a) Incubator.—Maintaining 25�C in dark environment.(b) Micropipetor.—Accurately dispensing 0.1 and

1.0 mL.(c) Pipets.—Glass or plastic sterile pipets, 1 mL with

0.01 mL graduations; and 10 mL with 0.1 mL graduations.(d) Stomacher/masticator.—IUL Instruments (Cincinnati,

OH), or equivalent, for macerating test portions.

D. General Instructions

Do not use expired medium. Store reconstituted mediumbetween 15 and 25�C in the dark and use within 12 h. Disposeof medium in decontamination container, and sterilize beforediscarding.

E. Test Suspension Preparation

(a) Weigh 50 g test portion into 450 mL sterile diluent [e.g.,0.1% peptone water (BAM method) or peptone salt solution(ISO method)]. This is a 1:10 dilution. Macerate mixture.

(b) If alternative test portion size is specified in testing proce-dure, prepare 10% (w/v) suspension.

(c) If necessary, prepare 10-fold serial dilutions appropri-ate for anticipated population of test portion.

F. Y&M–CI Test Procedure, Single Test Medium

For 1.0 mL test suspension.—(a) Suspend powdered medium with 9.0 mL sterile

deionized water containing 1 mL Supplement A per 100 mLwater. Add 1.0 mL prepared test portion and mix well. Do notcount this reconstitution as a dilution.

For 0.1 mL test suspension.—(b) Suspend powdered medium with 9.9 mL sterile

deionized water containing 1 mL Supplement A per 100 mLwater. Add 0.1 mL prepared test portion and mix well. This isan additional 1:10 dilution from E.

The final volume of test portion/medium mixture in thecontainer should be 10 � 0.2 mL.

Note: For raw meats and spices, add 0.1 mL Supplement Mto hydrated medium. For undiluted fruit juice containingvitamin C or dry pet food, add 0.1 mL Supplement V. Forfoods containing spreading mold populations, add 0.1 mLSupplement D. Supplements are available from BioControlSystems, Inc. The final volume of the test portion/mediummixture in the container should be 10 ± 0.2 mL.

(c) Remove lid from the SimPlate device and transfer testportion/medium mixture onto center of plate. Immediately re-place lid. Continue with H.

G. Y&M–CI Test Procedure, Multiple Test Medium

(a) Empty contents of one container, B(a), into 100 mLsterile deionized water containing 1 mL Supplement A per100 mL water. Shake to completely dissolve.

Note: For raw meats and spices, add 1.0 mL Supplement Mto hydrated medium. For undiluted fruit juice containing vita-min C or dry pet food, add 1.0 mL Supplement V. For foodscontaining spreading mold populations, add 1.0 mL Supple-

ment D. Supplements are available from BioControl Systems,Inc.

(b) Remove lid from SimPlate device. Pipet prepared testsuspension onto center of plate. If prepared test suspensionsize is 1.0 mL, overlay test suspension with 9.0 mL medium.Do not count this media addition as a dilution.

(c) For 0.1 mL of prepared test portion, overlay with9.9 mL medium; this is an additional 1:10 dilution of test sus-pension from E.

The final volume of test portion/medium mixture on theplate should be 10 ± 0.2 mL.

Immediately replace lid. Continue with H.

H. Test Procedure for Single and Multiple Tests

(a) Gently swirl to distribute test portion/medium mixtureinto all wells. Hold plate with both hands tilted slightly to helpdistribute medium into wells.

(b) Pour off excess medium by holding lid against plate oneither side of sponge cavity. Tip plate toward you to allow liq-uid to drain into sponge. Observe background color of wells.Background is defined as color of test portion/medium mix-ture inside wells.

(c) Do not invert the SimPlate device. Incubate at roomtemperature (22–25�C) for 56–72 h in the dark. If test portionbeing analyzed is known to contain slow-growing fungal pop-ulations, such as certain mold genera, incubation time may beextended to a total of 72 h. If Supplement V is used, incubateSimPlate devices for 72 h in the dark.

I. Reading and Interpretation of Results

(a) If reading is taken between 56 and 63 h after inocula-tion, place SimPlate device on illuminated background to helpvisualize positive results. After 63 h this is not necessary.

After incubation, observe color change of liquid in wells.Disregard particulate matter if present. Count number of wellsshowing color change from background color. The commoncolor changes produced by microorganisms are orange, peach,red, and white. Note: For mixtures containing Supplement V,count only the number of wells that fluoresce blue by holdinga UV light (365 nm wavelength) approximately 5 cm (2 in.)above the SimPlate device. Do not count nonfluorescent wellsthat only exhibit a color change.

(b) To determine the population, calculate as follows:(1) Count the number of positive wells on the plate; (2) useTable 1 to determine the total population per plate.

(c) To calculate the number of fungi/g, multiply the num-ber in I(b)(2) by the appropriate dilution factor (see E and Ffor single test or E and G for multiple test).

Ref.: J. AOAC Int. 86, 299–300(2003)

Results

The SimPlate Y&M–CI method was compared to theBAM method and the proposed ISO method (ISO/CD 21527)for enumeration of yeasts and molds in foods. Test portionswere prepared and incubated according to the conditionsstated in both the BAM and ISO methods. Seventeen laborato-



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470.

490.

210.

236.

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590.

63

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195.

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800.

560.

540.

240.

216.

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300.

670.

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Hig

h8

4.40

4.48

0.11

0.14

2.50

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3.90

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0.48

Fro

zen

frui

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s12

4.27

4.37

0.35

0.39

8.20

9.00

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11.9

013

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1.42

1.67

Bla

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4.32

4.46

0.29

0.29

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6.60

0.80

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8.30

8.60

1.00

1.08

Str

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3.70

3.74

0.19

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112.

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180.

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29

Hig

h11

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0.82

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7.77

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201.

220.

710.

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366.

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601.

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100.

250.

430.

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273.

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096.

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655.

3010

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1.83

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2.07

1.97

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21.6

026

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1.25

1.43

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1.38

1.59

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122.

502.

430.

310.

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881.

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5320

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Hig

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609.

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610.

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Tab

le20

02.1

1B.

Sta

tistic

alan

alys

iso

fin

terl

abo

rato

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sults

for

tota

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cS

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ISO

cS

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cS

IMd

ISO

cS

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zen

corn

dog

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83.

293.

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5.50

0.37

0.53

0.23

0.22

6.80

6.30

0.63

0.61

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ium

83.

934.

100.

170.

224.

305.

300.

470.

610.

270.

227.

005.

300.

770.

61

Hig

h8

4.36

4.44

0.17

0.13

3.80

2.80

0.47

0.35

0.24

0.21

5.60

4.60

0.68

0.58

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zen

frui

tR

aspb

errie

s12

4.22

4.43

0.29

0.24

6.80

5.40

0.81

0.67

0.51

0.58

12.2

013

.10

1.44

1.62

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ckbe

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s11

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4.49

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3.70

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6.10

7.70

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0.97

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400.

490.

250.

329.

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Hig

h10

3.81

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300.

110.

360.

260.

336.

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400.

730.

91

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ese

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560.

580.

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1.44

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0.57

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561.

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Tab

le20

02.1

1C.

Sta

tistic

alan

alys

iso

fin

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Mc

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dB

AM

cIS

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dB

AM

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Od

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ISO

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AM

cIS

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BA

Mc

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d

Fro

zen

corn

dog

Low

83.

283.

290.

170.

135.

104.

000.

470.

370.

210.

236.

406.

800.

590.

63

Med

ium

83.

983.

930.

200.

175.

004.

300.

560.

470.

240.

276.

007.

000.

560.

77

Hig

h8

4.40

4.36

0.11

0.17

2.50

3.80

0.31

0.47

0.18

0.24

4.20

5.60

0.52

0.68

Fro

zen

frui

tR

aspb

errie

s12

4.27

4.22

0.35

0.29

8.20

6.80

0.98

0.81

0.51

0.51

11.9

012

.20

1.42

1.44

Bla

ckbe

rrie

s12

4.32

4.37

0.29

0.43

6.60

9.80

0.80

1.21

0.36

0.40

8.30

9.10

1.00

1.11

Str

awbe

rrie

s12

3.70

3.67

0.19

0.16

5.10

4.30

0.53

0.44

0.22

0.26

5.90

7.00

0.61

0.71

Cer

eal

Low

102.

142.

040.

180.

188.

508.

700.

510.

500.

450.

4821

.00

23.4

01.

261.

34

Med

ium

112.

592.

670.

180.

146.

705.

400.

490.

400.

470.

25k

17.9

09.

501.

300.

71

Hig

h11

3.75

3.82

0.14

0.07

l3.

701.

800.

390.

190.

290.

257.

806.

600.

820.

71

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ese

Blu

e12

7.68

7.75

0.15

0.21

2.00

2.70

0.43

0.58

0.23

0.28

3.00

3.60

0.64

0.79

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zare

lla13

7.26

7.25

0.09

0.07

1.20

0.90

0.25

0.19

0.24

0.21

3.30

2.90

0.68

0.58

Che

ddar

146.

096.

150.

120.

111.

901.

800.

330.

320.

320.

265.

304.

200.

900.

72

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2.00

2.05

0.44

0.36

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1.23

1.02

0.53

0.43

26.4

020

.90

1.48

1.20

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132.

462.

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300.

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13.8

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840.

990.

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16.2

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391.

15

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h13

3.20

3.14

0.22

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6.80

9.40

0.61

0.82

0.42

0.46

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mea

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132.

552.

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01.

300.

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640.

7124

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26.4

01.

781.

98

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2.12

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0.12

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215.

609.

900.

330.

570.

270.

3212

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15.5

00.

750.

90

Wal

nut

103.

623.

620.

080.

102.

102.

600.

630.

520.

230.

196.

205.

100.

630.

52

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ries throughout North America and Europe participated in thestudy (Table 2). Eight laboratories analyzed all 6 food types, 5laboratories analyzed 5 food types, one analyzed 3 food types,and 3 analyzed one food type (Table 2). The numbers of fungiper gram of food (reported in log10 values) recovered from in-dividual test portions are presented in Tables 3–8. Repeatabil-ity and reproducibility analyses are presented in Tables2002.11A–C.

Frozen Corn Dogs

Frozen corn dog test portions inoculated at low, medium,and high levels were analyzed (Table 3). Uninoculated con-trols were included on the day of analysis. Thirteen laborato-ries participated in the analysis of frozen corn dogs. Labora-tories 6, 7, 10, 11, and 17 did not follow study instructions.The data from these laboratories were excluded from statisti-cal analysis.

Data generated by the BAM method and the correspondingSimPlate method (SIM–B) were compared statistically. Eightlaboratories submitted valid data for all 3 levels of frozen corndogs. Mean log counts recovered from low, medium, and highlevel test portions were not statistically different between the 2methods. The repeatability (sr) and reproducibility (sR) stan-dard deviations (SDs) of the 2 methods were statistically ana-lyzed (Table 2002.11A). The RSDr and RSDR values were notstatistically different for all 3 levels between the 2 methods(Table 2002.11A).

Data generated by the ISO method and the correspondingSimPlate method (SIM–I) were compared statistically. Eightlaboratories submitted valid data for all 3 levels of frozen corndogs. Mean log counts recovered from medium and high lev-els test portions were not statistically different between the 2methods. The SimPlate method recovered higher mean logcounts than did the ISO method for the low level (p < 0.05).However, the mean counts were very similar, with <0.3 logunits difference between the 2 methods. The RSDr and RSDR

values were not statistically different between the 2 methodsfor all 3 levels (Table 2002.11B).

Data generated by the BAM and ISO methods were com-pared statistically. Eight laboratories submitted valid data forall 3 levels. Mean log counts recovered from all 3 levels werenot statistically different between the 2 methods. The RSDr

and RSDR values were not statistically different for all 3 levelsbetween the 2 methods (Table 2002.11C).

Frozen Fruits

Three types of frozen fruit (strawberries, blackberries, andraspberries) were analyzed (Table 4). These 3 fruits were cho-sen because they are known to contain significant levels offungi contamination. Laboratory 12 did not complete analysisof test portions. The data from this laboratory were excludedfrom statistical analysis.

Data generated by the BAM method and the SimPlatemethod (SIM–B) were compared statistically. Twelve labora-tories submitted valid data for frozen fruit test portions. Themean log counts from the BAM and the SIM–B methods werenot statistically different for the 3 lots analyzed. The RSDr and

RSDR values for the frozen fruits test portions were not statis-tically different between the 2 methods (Table 2002.11A).

Data generated by the ISO method and the SimPlatemethod (SIM–I) were compared statistically. Laboratory 3was determined to be an outlier by the Cochran test for theblackberry test portions. This paired data set was excludedfrom statistical analysis. Twelve laboratories submitted validdata for the strawberry and raspberry test portions, and 11 lab-oratories submitted valid data for blackberry test portions. Themean log counts from the BAM and the SIM–I methods werenot statistically different for the 3 lots analyzed. The RSDr andRSDR values for the frozen fruits test portions were not statis-tically different between the 2 methods (Table 2002.11B).

Data generated by the BAM and ISO methods were com-pared statistically. Twelve laboratories submitted valid datafor all 3 lots of frozen fruits test portions. Mean log counts re-covered from these 3 lots were not statistically different be-tween the 2 methods. The RSDr and RSDR values were not


Table 2. Collaborator participation for SimPlateY&M–Cl interlaboratory study by food typea

LabFrozen

corn dogFrozen

fruit Cereal CheeseCakemix

Nutmeats

1 Y Y Y Y Y Y

2 Y Y Y Y Y Y

3 Y Y Y Y Y Y

4 Y Y Y Y Y Y

5 N Y Y Y Y Y

6 Yb Y Y Y Y Y

7 Yb Y Y Y Y Y

8 N Y Y Y Y Y

9 N Y Yb Y Yc Y

10 Yb Y Y Y Y N

11 Yb Y Y Y Y Y

12 Y Yd Yd Y N Y

13 Y Y Y Y Y Y

14 N N N Y Y Y

15 Y N N N N N

16 Y N N N N N

17 Yb N N N N N

Totale 13 13 13 14 13 13

a Y = Collaborator analyzed this food type; N = collaborator did notanalyze this food type.

b Laboratory did not follow study instructions. Results were notincluded in the statistical analysis for the designated food types.

c Laboratory did not follow the SimPlate directions for use. Resultswere not included in the statistical analysis for the designated foodtypes.

d Laboratory did not complete/start analysis. Results were notincluded in the statistical analysis for the designated food types.

e Total number of laboratories participating in the analysis of thisfood type.

statistically different for all 3 lots between the 2 methods (Ta-ble 2002.11C).

Cereal

Cereal test portions inoculated at a low, medium, and highlevel were analyzed (Table 5). Uninoculated controls were in-cluded on the day of analysis. Laboratory 9 did not follow theSimPlate directions for use. Laboratory 12 did not completeanalysis of the test portions. The data from these laboratorieswere excluded from statistical analysis. Throughout the analy-sis of cereal test portions, some laboratories reported platecounts for certain test portions that were below the suitablecounting range for the BAM method. These data were esti-mated and used for statistical analysis.

Data generated by the BAM and SimPlate methods werecompared statistically. Eleven laboratories submitted validdata for all 3 levels. The mean log counts from the BAM andSIM–B methods were not statistically different for all 3 levelsanalyzed. The RSDr values from the BAM and SIM–B meth-ods were not significantly different for the medium and hightest portions. The BAM method’s data reported lower RSDr

for the low test portions (p < 0.05). The RSDR values from the

data for the BAM and SIM–B methods were not statisticallydifferent for the all 3 levels.

Data generated by the ISO and SimPlate methods werecompared statistically. Laboratories 11 and 4 were determinedto be outliers by the Cochran test for the low and high test por-tions, respectively. These paired data were excluded from sta-tistical analysis. Eleven laboratories submitted valid data forthe medium level and 10 laboratories submitted valid data forthe low and high levels. The mean log counts from the ISOand SIM–I methods were not statistically different for all 3levels analyzed. The RSDr values from the ISO and SIM–Imethods were not significantly different for the medium leveltest portions. The ISO method’s data showed lower RSDr forthe low and high test portions. The RSDR values from the ISOand SIM–I methods were not statistically different for the all 3levels (Table 2002.11B).

Data generated by the BAM and the ISO methods werecompared statistically. Laboratory 11 was determined to be anoutlier by the Cochran test for the low level test portions.These paired data were excluded from statistical analysis. Tenlaboratories submitted valid data for the low level and 11 labo-ratories submitted valid data for the medium and high levels.


Table 3. Yeast and mold counts for frozen corn dog test portions (log10 CFU/g) by SimPlate Y&M–CI (SIM–B and –I),BAM culture method, and ISO culture methoda

Lab

Uninoculated Low

Test portion 1 Test portion 2 Test portion 3 Test portion 4

SIM–B BAM SIM–I ISO SIM–B BAM SIM–I ISO SIM–B BAM SIM–I ISO SIM–B BAM SIM–I ISO

1 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 3.08 3.08 3.04 3.04 3.18 3.11 3.49 3.04

2 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 3.46 3.30 3.56 3.20 3.34 3.23 3.41 3.34

3 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 3.08 2.78 3.11 3.00 3.53 3.26 3.34 3.18

4 <2.00 <2.00 1.60 <2.00 <2.00 <2.00 2.34 <2.00 3.67 3.34 3.60 3.57 3.62 3.60 3.59 3.62

12 <2.00 2.11 <2.00 2.43 <2.00 2.23 <2.00 2.43 3.18 3.46 3.72 3.11 3.51 3.28 3.48 3.30

13 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 3.49 3.32 3.32 3.23 3.84 3.64 3.76 3.61

15 <2.00 <2.00 <2.00 <2.00 1.60 <2.00 1.78 <2.00 3.43 3.20 3.76 3.62 3.42 3.18 3.66 3.45

16 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 3.53 3.32 3.26 3.23 3.68 3.45 3.46 3.11

Medium High



1 3.92 3.64 3.95 3.73 4.18 3.96 3.81 3.70 4.32 4.20 4.38 4.15 4.53 4.45 4.28 4.23

2 4.15 4.00 4.15 4.04 4.00 3.65 3.83 3.73 4.26 4.34 4.57 4.51 4.61 4.38 4.20 4.11

3 3.59 4.20 4.23 3.81 4.00 4.53 3.67 3.34 4.23 4.08 4.08 4.11 4.32 4.15 4.15 3.86

4 3.89 3.91 3.99 4.04 4.00 4.18 4.15 4.15 4.36 4.49 4.49 4.26 4.68 4.82 4.51 4.62

12 4.00 4.00 4.36 3.66 4.04 4.04 3.94 3.72 4.34 4.30 4.38 4.32 4.32 4.34 4.28 4.30

13 4.04 4.00 4.23 4.04 3.57 3.70 4.08 4.30 4.63 4.49 4.59 4.48 4.66 4.53 4.66 4.72

15 4.23 4.04 4.34 4.23 4.08 3.70 4.08 4.00 4.48 4.41 4.70 4.63 4.60 4.30 4.75 4.65

16 4.15 4.04 4.43 4.26 4.41 4.18 4.28 4.15 4.67 4.52 4.58 4.52 4.70 4.57 4.43 4.34

a SIM–B = Test portions prepared according to the FDA’s BAM method; SIM–I = test portions prepared according to the proposed ISO method.


Table 4. Yeast and mold counts for frozen fruits test portions (log10 CFU/g) by SimPlate Y&M–CI (SIM–B and –I),BAM culture method, and ISO culture methoda

Lab

Raspberries

Test portion 1 Test portion 2

SIM–B BAM SIM–I ISO SIM–B BAM SIM–I ISO

1 3.51 3.59 3.96 3.79 3.51 3.54 4.49 3.99

2 4.66 4.68 4.65 4.65 4.66 4.68 4.58 4.56

3 3.20 3.11 3.15 3.15 3.20 3.90 3.00 3.04

4 3.97 4.08 4.08 4.00 3.97 5.04 4.08 3.89

5 4.46 4.11 4.98 4.38 4.46 3.96 4.85 4.04

6 5.08 4.83 4.54 3.93 5.08 4.99 5.18 4.93

7 4.75 4.52 4.97 4.91 4.75 4.08 4.65 4.57

8 4.60 4.18 3.91 3.94 4.60 4.28 4.11 3.86

9 4.76 4.77 5.08 4.97 4.76 4.80 4.98 4.63

10 3.83 3.88 4.04 3.93 3.83 4.08 4.32 4.36

11 5.18 4.69 4.51 4.11 5.18 3.92 5.15 4.72

13 4.00 4.00 4.56 4.51 4.00 4.72 4.43 4.36

Blackberries



1 4.04 4.11 4.15 4.04 4.30 4.18 4.04 4.04

2 4.40 4.23 4.52 4.51 4.48 4.51 4.54 4.49

3 4.18 3.92 5.64b 5.72b 3.34 3.20 3.58b 3.72b

4 4.81 4.64 4.72 4.70 4.63 4.48 4.51 4.52

5 4.51 4.64 4.48 4.23 4.30 4.04 4.61 4.41

6 5.46 4.63 3.91 4.11 4.59 4.34 4.43 4.54

7 4.57 4.82 4.20 4.08 3.99 3.96 4.28 4.08

8 4.38 4.04 4.26 4.04 4.32 4.15 4.26 3.99

9 4.71 4.51 4.80 4.63 4.92 4.68 4.52 4.45

10 4.40 4.65 4.46 4.52 4.41 4.49 4.51 4.45

11 4.65 4.28 4.48 4.11 4.43 4.04 4.58 4.18

13 4.66 4.59 5.15 4.90 4.49 4.43 5.46 4.52

Strawberries



1 2.94 3.28 3.53 3.48 3.26 3.53 3.43 3.41

2 3.83 3.80 3.67 3.83 3.79 3.86 3.83 3.83

3 3.32 3.08 3.18 3.04 4.23 3.91 3.08 3.20

4 3.84 3.76 3.45 3.70 3.93 3.96 3.59 3.67

5 3.64 3.72 3.93 3.73 3.89 3.72 3.90 3.58

6 3.76 3.89 3.89 3.94 4.00 3.79 3.71 3.46

7 3.49 3.56 3.48 3.63 3.40 3.54 3.34 3.79

8 3.77 3.79 3.83 3.77 3.63 3.70 3.88 3.70

9 4.15 3.95 4.18 3.95 4.11 3.98 4.08 4.00

10 3.49 3.59 3.64 3.97 3.45 3.56 3.52 3.60

11 3.53 3.54 3.34 3.28 3.62 3.67 3.79 3.63

13 4.61 3.75 3.81 3.89 4.00 3.89 3.89 3.88

a SIM–B = Test portions prepared according to the FDA’s BAM method; SIM–I = test portions prepared according to the proposed ISO method.b Outlier; data not used in analysis for method comparison of SIM–I and ISO.

The mean log counts from the BAM and ISO methods werenot statistically different for all 3 levels analyzed. The RSDr

values from the ISO and BAM methods were not significantlydifferent for the low and medium level test portions. The ISOmethod’s data reported lower RSDr for the high test portions(p < 0.05). The RSDR values from the ISO and BAM methodswere not statistically different for the low and high test por-tions. The ISO method’s data showed lower RSDR for the me-dium test portions (p < 0.05; Table 2002.11C).

Fresh Cheese

Three types of fresh cheese (blue, mozzarella, and cheddar)were analyzed (Table 6). Data generated by the BAM and

SimPlate methods were compared statistically. Laboratory 6was determined to be an outlier by the Cochran test for blueand mozzarella cheese test portions; therefore, the paired datawere excluded from statistical analysis. Thirteen laboratoriessubmitted valid data for blue cheese and mozzarella cheesetest portions and 14 laboratories submitted valid data for ched-dar cheese test portions. The mean log counts from the BAMand SIM–B methods were not statistically different for testportions of all 3 cheeses analyzed. The BAM method’s datashowed lower RSDr mozzarella and cheddar cheese test por-tions (p < 0.05). The SimPlate method’s data, however, re-ported lower RSDr (p < 0.05) for the blue cheese test portions.The RSDR values from the BAM and SIM–B methods were


Table 5. Yeast and mold counts for cereal test portions (log10 CFU/g) by SimPlate Y&M–CI (SIM–B and –I), BAMculture method, and ISO culture methoda

Lab

Uninoculated Low



1 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 2.83 2.62 2.23 2.40 2.68 2.54 2.45 2.15

2 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 2.46 2.36 2.26 2.15 2.40 2.34 2.15 2.11

3 <1.00 0.52 1.30 1.30 2.51 <1.00 <1.00 <1.00 1.78 1.00 <1.00 1.00 1.30 1.23 1.30 0.70

4 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 2.67 2.70 2.26 2.32 2.69 2.51 2.94 2.32

5 1.74 <1.00 1.85 <1.00 1.74 <1.00 1.78 <1.00 2.77 2.46 2.11 2.30 2.15 2.04 2.26 1.81

6 2.11 1.00 2.73 1.54 1.30 1.30 2.08 1.43 2.23 2.11 2.38 2.20 2.08 2.04 2.08 1.88

7 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 1.78 <1.00 2.40 2.41 2.26 2.26 2.00 2.18 2.53 2.30

8 1.60 <1.00 <1.00 <1.00 1.60 <1.00 <1.00 <1.00 2.00 2.11 2.08 2.15 2.00 1.63 2.34 2.23

10 <1.00 <1.00 <1.00 <1.00 1.60 <1.00 <1.00 <1.00 2.46 2.11 1.90 1.63 1.60 1.83 2.28 1.96

11 1.60 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 2.91 2.54b <1.00 1.30b 2.46 2.43b 2.93 2.73b

13 1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 1.78 2.32 2.48 2.57 2.56 2.23 2.56 2.45

Medium High



1 3.04 2.97 2.72 2.77 2.81 2.76 2.73 2.79 3.92 4.04 4.08 3.88 4.00 3.95 4.11 3.96

2 2.96 2.80 2.71 2.82 2.99 2.85 2.71 2.79 4.15 3.99 3.76 3.99 4.11 4.00 3.71 3.93

3 1.78 1.63 2.00 2.15 1.30 1.00 2.00 1.96 3.28 3.04 3.34 3.15 3.40 3.04 3.40 3.08

4 2.52 2.61 2.90 2.78 2.94 2.74 2.89 2.91 4.11 4.14 4.30c 4.04c 3.98 3.89 3.94c 3.78c

5 2.83 2.46 2.92 2.85 2.64 2.68 2.41 2.60 3.99 3.85 3.93 3.77 3.82 3.65 3.67 3.73

6 2.60 2.45 2.66 2.54 2.86 2.54 2.64 2.36 3.15 3.30 3.34 3.78 3.66 3.72 3.60 3.77

7 3.11 2.87 3.08 2.91 2.79 2.63 2.63 2.61 3.93 3.80 4.08 3.90 3.90 3.74 3.86 3.85

8 2.71 2.54 2.56 2.58 2.66 2.48 2.49 2.64 3.84 3.66 3.81 3.80 3.83 3.65 3.93 3.77

10 2.94 2.83 3.04 2.98 3.11 2.90 2.63 2.58 4.32 4.04 4.23 3.99 3.83 3.64 4.00 3.90

11 2.92 2.79 3.30 2.87 3.40 3.08 3.08 2.64 3.98 3.79 4.48 4.00 3.87 3.90 4.34 3.98

13 3.04 2.68 2.72 2.81 3.00 2.73 2.81 2.75 4.26 3.91 3.93 3.96 3.74 3.85 4.18 3.99

a SIM–B = Test portions prepared according to the FDA’s BAM method; SIM–I = test portions prepared according to the proposed ISO method.b Outlier; data not used in analysis for method comparison of BAM and ISO.c Outlier; data not used in analysis for method comparison of SIM–I and ISO.


Table 6. Yeast and mold counts for cheese test portions (log10 CFU/g) by SimPlate Y&M–CI (SIM–B and –I), BAMculture method, and ISO culture methoda

Lab

Blue



1 7.94 7.86 7.78 7.62 7.85 7.77 7.56 7.32

2 7.96 7.78 8.76 8.71 7.81 7.67 7.96 7.85

3 7.34 7.28 7.70 7.51 7.48 7.48 7.58 7.61

4 7.87 7.87 7.89 7.80 8.00 7.85 7.85 7.81

5 7.77 7.80 8.41 7.79 7.63 7.92 8.08 7.94

6 8.51b <6.00b,c 8.61 7.79c 8.48b 7.32b,c 9.04 7.76c

7 7.95 7.75 8.04 7.78 7.70 7.70 7.81 7.67

8 7.38 7.63 7.28 7.66 7.36 7.60 7.51 7.61

9 8.96 7.36 9.23 7.60 8.23 7.40 9.11 7.92

10 7.83 7.53 7.86 7.41 7.65 7.51 7.76 7.26

11 7.98 7.83 7.93 7.81 8.00 7.15 7.95 7.79

12 8.68 9.88c 7.83d 8.73c,d 7.75 7.79c <6.00c 7.76c,d

13 8.15 8.04 8.04 7.95 7.97 7.95 8.08 7.96

14 8.00 7.84 7.81 7.85 7.79 7.74 7.77 7.73

Mozzarella



1 7.53 7.32 7.23 7.11 7.32 7.41 7.34 7.15

2 7.60 7.43 7.75 7.34 7.48 7.32 7.26 7.32

3 7.00 6.91 7.08 6.91 6.86 6.92 7.15 7.00

4 7.57 7.52 7.58 7.46 7.52 7.49 7.60 7.63

5 7.34 7.32 7.73 7.46 7.51 7.58 7.26 7.40

6 9.38b 7.45b,c 8.20 7.23c 7.84b 6.87b,c 8.89 7.11c

7 7.36 6.96 7.43 7.15 7.20 7.15 7.30 7.04

8 7.08 7.11 7.23 7.28 7.32 7.11 7.23 7.32

9 7.62 6.76 7.28 6.86 7.56 6.87 7.59 7.04

10 7.18 7.08 7.30 7.08 7.34 7.18 7.23 7.08

11 7.58 7.52 7.59 7.41 7.65 7.53 7.61 7.41

12 7.04 7.32 7.00 7.08 6.93 7.11 7.08 7.15

13 7.48 7.42 7.62 7.49 8.08 7.40 7.65 7.45

14 7.45 7.53 7.38 7.32 7.45 7.40 7.62 7.46

Cheddar



1 5.83 6.32 5.97 6.04 5.90 6.11 5.66 5.93

2 6.51 6.34 6.20 6.48 6.40 6.38 6.30 6.38

3 5.08 5.40 5.46 5.57 5.08 5.04 5.54 5.64

4 6.18 6.41 6.53 6.28 6.40 6.30 6.30 6.36

5 5.76 5.92 6.23 6.08 5.89 5.97 5.96 5.87

6 6.11 5.81 7.00 6.11 6.98 5.97 6.93 5.92

7 5.97 6.18 6.00 6.51 5.93 6.04 5.98 6.48

8 5.38 6.11 5.89 5.91 5.71 5.84 5.91 6.04

9 7.46 5.95 7.43 5.90 7.23 5.94 7.36 6.32

10 6.45 6.15 6.28 6.15 6.36 6.15 6.34 6.11

11 6.41 6.38 6.23 6.32 6.36 6.15 6.38 6.43

12 4.70 6.00 4.92 6.11 5.11 6.11 4.72 5.97

13 6.52 6.48 6.61 6.48 6.49 6.48 6.60 6.43

14 6.52 6.42 6.18 6.20 6.18 6.28 6.04 6.11

a SIM–B = Test portions prepared according to the BAM method; SIM–I = test portions prepared according to the proposed ISO method.b Outlier; data not used in analysis for method comparison of SIM–B and BAM.c Outlier; data not used in analysis for method comparison of ISO and BAM.d Outlier; data not used in analysis for method comparison of SIM–I and ISO.

not statistically different for the blue and mozzarella cheesetest portions. The BAM method’s data reported lower RSDR

for the cheddar cheese test portions (Table 2002.11A).

Data generated by the ISO and the SimPlate methods werecompared statistically. Laboratory 12 was determined to be anoutlier by the Cochran test for blue cheese test portions. Thesepaired data were excluded from statistical analysis. Overall,13 laboratories submitted valid data for blue cheese test por-tions and 14 submitted valid data for mozzarella and cheddar

cheese test portions. The mean log counts from the ISO andSIM–I methods were not statistically different for the cheddarcheese test portions. The SimPlate method recovered highermean log counts for the blue and mozzarella cheese test por-tions than did the ISO method (p < 0.01). However, the meancounts were very similar, with <0.3 log units difference be-tween the 2 methods. The ISO method’s data showed lowerRSDr values for mozzarella cheese test portions (p < 0.01).The RSDr values were not statistically different for the blue


Table 7. Yeast and mold counts for cake mix test portions (log10 CFU/g) by SimPlate Y&M–CI (SIM–B and –I), BAMculture method, and ISO culture methoda

Lab

Uninoculated Low



1 <1.00 <1.00 <1.00 1.15 <1.00 <1.00 <1.00 <1.00 2.23 2.23 2.34 2.15 3.38 3.52 3.18 2.18

2 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 2.36 2.18 <1.00 2.08 2.41 1.80 2.49 2.38

3 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 1.60 1.30 1.30 2.38 1.30 1.43 1.78 1.88

4 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 2.68 2.86 1.90 2.41 2.40 2.23 2.15 2.52

5 <1.00 1.74 1.74 <1.00 <1.00 1.74 2.04 <1.00 2.11 2.30 1.85 2.00 1.78 1.76 1.85 1.63

6 2.58 1.11 <1.00 1.18 1.30 0.83 <1.00 <1.00 2.11 2.15 1.60 1.96 2.11 1.67 1.30 1.93

7 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 1.90 2.04 2.92 1.76 1.90 2.20 2.59 2.60

8 <1.00 <1.00 <1.00 <1.00 <1.00 1.30 <1.00 <1.00 1.60 2.11 <1.00 1.90 1.90 2.15 <1.00 2.15

9 —b 0.85 —b <1.00 —b <1.00 —b 1.00 —b 1.00 —b 1.30 —b 1.48 —b 1.30

10 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 2.08 2.11 1.30 1.60 1.30 1.67 2.81 2.69

11 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 2.45 2.48 <1.00 1.30 <1.00 1.30 1.60 1.65

13 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 1.30 2.00 2.15 2.26 2.66 2.49 2.26 2.51

14 <1.00 0.83 <1.00 <1.00 <1.00 0.83 <1.00 1.30 1.30 1.60 1.30 1.90 1.30 2.04 2.38 2.80

Medium High



1 2.00 2.34 3.32 3.45 3.00 2.72 1.90 2.36 3.66 3.65 2.99 3.04 3.53 3.66 2.76 3.08

2 2.32 2.32 2.56 2.71 2.40 2.60 2.23 2.54 2.71 3.11 2.38 3.00 3.00 3.23 3.08 3.30

3 1.78 1.80 1.00 2.38 2.08 2.04 2.23 2.18 3.04 3.00 2.51 2.54 3.15 3.08 2.34 2.79

4 3.85 4.00 3.11 3.23 2.56 2.74 2.15 2.51 3.57 3.76 3.08 3.48 3.04 3.43 3.62 3.97

5 2.28 2.18 2.18 2.34 1.95 2.08 2.73 2.97 2.75 2.91 2.59 2.66 2.59 2.90 2.99 3.18

6 2.28 2.40 2.41 2.92 2.11 2.20 2.38 2.48 3.04 3.08 3.15 3.48 3.87 3.76 2.94 3.11

7 2.79 2.76 2.45 2.45 3.28 2.95 2.20 2.38 3.30 3.59 2.94 3.08 3.00 3.18 3.32 3.15

8 2.15 2.38 1.78 2.45 2.32 2.61 2.32 2.60 2.57 3.18 2.99 3.23 3.32 3.60 3.61 3.76

9 —b 1.98 —b 2.00 —b 2.08 —b 2.40 —b 2.30 —b 2.42 —b 2.00 —b 1.70

10 2.11 2.23 2.46 2.49 2.04 2.28 2.08 2.36 3.18 3.38 3.11 3.18 3.32 3.49 3.18 3.15

11 1.60 2.08 1.30 2.04 2.26 1.65 1.30 1.65 3.08 2.90 3.57 3.65 2.46 2.95 2.53 2.94

13 2.23 2.68 2.15 2.26 2.85 3.00 2.34 2.75 3.32 3.20 3.15 3.51 3.00 3.32 2.81 3.15

14 2.90 3.11 2.26 2.84 3.23 2.86 3.20 3.34 2.83 3.04 2.88 3.28 2.83 3.43 2.88 3.15

a SIM–B = Test portions prepared according to the FDA’s BAM method; SIM–I = test portions prepared according to the proposed ISO method.b Invalid data for SIM–B or –I methods; laboratory did not follow SimPlate directions for use.


Table 8. Yeast and mold counts for nut meat test portions (log10 CFU/g) by SimPlate Y&M–CI (SIM–B and –I), BAMculture method, and ISO culture methoda

Lab

Hazelnut


SIM-B BAM SIM-I ISO SIM-B BAM SIM-I ISO

1 2.85 2.78 2.18 2.41 2.52 2.58 2.54 2.36

2 2.62 2.08 3.45 3.15 2.72 2.43 3.18 2.96

3 2.15 2.08 2.76 2.61 2.23 2.00 1.60 1.60

4 3.08 3.00 3.32 3.76 3.18 2.95 3.43 3.49

5 2.15 2.08 2.00 1.79 2.51 2.20 2.40 1.83

6 2.38 2.08 3.20 2.64 3.30 2.74 2.51 2.28

7 1.60 2.04 2.32 2.04 1.30 2.08 1.78 2.52

8 2.71 2.28 2.86 2.92 3.56 3.90 3.00 2.89

9 2.20 1.60 4.15 2.38 4.26 2.60 3.45 2.20

11 2.26 2.30 2.15 2.15 2.08 2.04 2.26 2.18

12 2.90 3.79 2.86 2.80 3.61 4.04 3.41 3.88

13 2.88 2.64 4.20 4.00 3.32 3.08 4.15 4.08

14 3.36 2.99 2.20 2.46 2.34 2.00 1.78 2.08

Pecan



1 2.65 2.36 2.28 2.28 2.45 2.26 2.53 2.18

2 2.51 2.38 2.46 2.11 2.45 2.30 2.32 2.32

3 2.11 1.94 2.34 1.93 2.20 2.08 2.28 2.00

4 2.46 2.36 3.04 2.45 2.83 2.26 2.91 2.45

5 2.11 2.00 2.34 2.00 2.28 1.72 2.49 2.00

6 2.15 2.04 1.30 1.70 2.00 2.11 1.30 1.78

7 1.30b 1.52b 1.30 1.72 <1.00b 1.30b <1.00 1.48

8 2.66 2.26 2.00 2.00 2.34 2.04 2.00 1.18

9 3.88 2.28 4.30 1.89 3.60 2.26 3.75 2.26

11 2.79 1.86 2.54 2.15 2.87 2.23 2.38 1.86

12 3.15 2.30 3.20 2.32 3.23 2.28 3.15 2.20

13 2.62 2.28 2.32 2.30 2.73 2.26 2.73 2.54

14 1.78 2.18 2.26 2.30 2.00 2.15 2.41 2.23

Walnut



1 4.26 3.77 4.34 3.74 4.04 3.64 3.98 3.72

2 3.87 3.78 4.04 3.69 4.04 4.04 4.04 3.76

3 3.94 3.56c 3.23 3.26c 2.30 3.11c 2.78 2.3c

4 4.51 3.79 4.79 3.76 4.52 3.79 4.15 3.76

5 4.57 3.48 4.00 3.28 3.90 3.53 3.90 3.54

6 5.34 4.64c 3.52 3.26c 4.36 3.46c 3.28 3.08c

7 4.23 3.20 2.78 3.15 3.15 3.15 4.08 3.36

8 4.36 3.54 4.08 3.73 4.45 3.52 4.75 3.60

9 —d 3.18c —d 4.61c —d 4.51c —d 4.54c

11 4.65 3.56 4.62 3.66 4.51 3.58 4.04 3.77

12 4.73 3.38 —e 3.53 4.64 3.48 —e 3.52

13 3.90 3.75 4.56 3.81 4.28 3.73 4.45 3.76

14 3.56 3.76 3.69 3.72 3.53 3.88 3.59 3.56

a SIM–B = test portions prepared according to the FDA’s BAM method; SIM–I = test portions prepared according to the proposed ISO method.b Outlier; data not used in analysis for method comparison of SIM–B and BAM.c Outlier; data not used in analysis for method comparison of BAM and ISO.d No end point determined for these test portions; data from these paired test portions were not used for comparison of SimPlates and BAM or ISO methods.e Did not set up test for these test portions, not enough SimPlates.

and cheddar cheese test portions. The ISO method’s data re-ported lower RSDR values for all 3 lots of cheese analyzed(Table 2002.11B).

Data generated by the BAM and ISO methods were com-pared statistically. Laboratory 6 was determined to be an out-lier by the Cochran test for blue and mozzarella cheese testportions. Laboratory 12 was determined to be an outlier for theblue cheese test portions. These paired data were excludedfrom statistical analysis. Overall, 12, 13, and 14 laboratoriessubmitted valid data for blue, mozzarella, and cheddar cheesetest portions, respectively. The mean log counts from theBAM and ISO methods were not statistically different for all 3types of cheese analyzed. The RSDr and RSDR values werenot statistically different for all 3 types of cheese between the2 methods (Table 2002.11C).

Cake Mix

Cake mix test portions inoculated at a low, medium, andhigh level were analyzed (Table 7). Uninoculated controlswere included on the day of analysis.

Data generated by the BAM and SimPlate methods werecompared statistically. Laboratory 9 did not correctly followthe SimPlate directions for use. These data were not includedfor statistical analysis. Twelve laboratories submitted validdata for all cake mix test portions. The difference in mean logcounts between the BAM and SIM–B methods were not sta-tistically significant for the low and medium test portions. TheBAM method recovered higher mean log counts (p < 0.05)than did the SIM–B method. However, the mean log countswere very similar, with <0.3 log units difference between the 2methods. The RSDr and RSDR values for all 3 levels were notstatistically different between the 2 methods (Ta-ble 2002.11A).

Data generated by the ISO and SimPlate methods werecompared statistically. Laboratory 9 did not correctly followthe SimPlate directions for use. These data were not includedfor statistical analysis. Twelve laboratories submitted validdata for all cake mix test portions. The mean log counts fromthe ISO and SIM–I methods were statistically not different forthe low test portions. The ISO method recovered higher meanlog counts (p < 0.05) than did the SIM–I method for the me-dium and high test portions. The ISO method’s data showedlower RSDR values for the low level test portions. The RSDr

and RSDR values for all medium and high levels were not sta-tistically different between the 2 methods (Table 2002.11B).

Data generated by the BAM and ISO methods were com-pared statistically. Overall, 13 laboratories submitted validdata for all cake mix test portions. The mean log counts fromthe BAM and ISO methods were not statistically different forall levels. The repeatability and reproducibility standard devi-ations for all levels were not statistically different between the2 methods (Table 2002.11C).

Nut Meats

Three types of nut meats (hazelnut, pecan, and walnut)were analyzed (Table 8). These nutmeats were chosen be-cause they contained significant levels of naturally contami-

nating fungi compared to other nutmeats screened. Through-out the analysis of nut meat test portions, some laboratories re-ported plate counts for certain test portions that were belowthe counting range for the BAM method. Data from thesepaired test portions were estimated and used for statisticalanalysis.

Data generated by the BAM and SimPlate methods werecompared statistically. Laboratory 9 had no end point for theSimPlate analysis for walnut test portions. Laboratory 7 wasdetermined to be an outlier by the Cochran test for pecan testportions; therefore, the paired data were excluded from statis-tical analysis. These data were not included for statistical anal-ysis. Thirteen laboratories submitted valid data for hazelnuttest portions and 12 laboratories submitted valid data for pe-can and walnut test portions. The mean log counts from theBAM and SIM–B methods were not statistically different forhazelnut test portions. The SimPlate method recovered highermean log counts for pecan and walnut test portions. The BAMmethod’s data showed lower RSDr and RSDR values for thewalnut test portions. The BAM method’s data showed lowerRSDR for the pecan test portions (p < 0.01). The RSDr andRSDR values were not statistically different for hazelnut testportions.

Data generated by the ISO and SimPlate methods werecompared statistically. Laboratory 9 had no end point for theSimPlate analysis for walnut test portions. Laboratory 12 didnot have enough SimPlate devices to analyze the walnut testportions. No SimPlate data were submitted for these test por-tions. These data were not included in the statistical analysis.Overall, 13, 13, and 11 laboratories submitted valid data forhazelnut, pecan, and walnut test portions, respectively. Themean log counts from the BAM and SIM–I methods were notstatistically different for the hazelnut test portions analyzed.The SimPlate method recovered higher mean log counts forthe walnut and pecan test portions. The RSDr were not statisti-cally different for the 3 lots of nut meats analyzed. The ISOmethod’s data showed lower RSDR for the pecan test portions.The RSDR values were not statistically different for the hazel-nut and walnut test portions.

Data generated by the BAM and ISO methods were com-pared statistically. Laboratories 3, 6, and 9 were determined tobe outliers by the Cochran test for the walnut test portions.These paired data were excluded from statistical analysis.Thirteen laboratories submitted valid data for the hazelnut andpecan test portions, and 10 laboratories submitted valid datafor the walnut test portions. The mean log counts from theBAM and ISO methods were not statistically different for anyof the 3 types of nut meats analyzed. The BAM method’s datareported lower RSDr values for the pecan test portions(p < 0.05). The RSDr values were not statistically different forthe hazelnut and walnut test portions between the 2 methods.The RSDR values were not statistically different for all 3 lotsof nut meat test portions.


Discussion

In this international interlaboratory study, the SimPlateYeast and Mold–Color Indicator method was compared to theFDA’s BAM method for enumeration of total yeasts andmolds. Additionally, the proposed ISO method (ISO/CD21527) for enumerating total yeasts and molds was analyzed.The BAM and ISO methods use the same growth media(DRBC and DG18 agars); however, they differ in the diluentsused. The BAM method uses 0.1% peptone water, whereasthe ISO method uses peptone salt solution. Because of this dif-ference between the 2 reference methods, an additional set ofSimPlate devices was set up according to ISO requirements,for a total of 4 methods being tested.

Six food types were evaluated by the 4 methods in thisstudy. Three lots of naturally contaminated test portions wereanalyzed for frozen fruits, cheese, and nut meats. Three foodtypes (frozen corn dog, cereal, and cake mix) did not containsignificant levels of natural fungi and were artificially con-taminated with either a yeast or a mold. For these 3 food types,3 contamination levels and uninoculated controls were ana-lyzed. In total, 21 food lots, comprising naturally contami-nated, artificially inoculated, and uninoculated controls, wereanalyzed per method. Comparing the SimPlate method(SIM–B) and the BAM method, recovery (mean log counts)of total yeasts and molds was statistically different for 3 of the18 contaminated food lots analyzed (Table 2002.11A). Themean log counts were actually similar for one of these 3 lots,with <0.30 mean log difference in recovery. The SimPlatemethod recovered an average mean log count of 0.41 and 0.52higher than the reference method for pecans and walnuts, re-spectively. A possible explanation for this may be that liquidmedium is more suitable than agar plates for incubating thefungi present in the nutmeats.

The recovery of yeasts and molds from the SimPlate testsprepared with the ISO diluent (SIM–I) had good correlationwith the ISO method. Mean log counts for 7 of the 18 contami-nated food lots analyzed were statistically different betweenthe 2 methods (Table 2002.11B). Four of these 7 lots had simi-lar results, with a difference of 0.30 or less mean log counts.

A comparison of the BAM and ISO method’s data for re-covery of yeasts and molds indicates very good correlation be-tween the 2 methods. There were no statistical differences inrecovery between the 2 methods.

For uninoculated frozen corn dogs, cake mix, and cerealtest portions, both SimPlate methods (SIM–B and –I), theBAM or ISO methods sometimes produced measurable butlow counts of naturally occurring fungi. These fungi were be-low the appropriate levels needed for the study.

Conclusions

In general, there was <0.3 mean log count difference in re-covery between the BAM method and the correspondingSimPlate method (SIM–B), the ISO method and the corre-sponding SimPlate method (SIM–I), and the BAM methodand the ISO method. Higher mean log counts (>0.3 mean log

count difference) were recovered by the SIM–B/SIM–Imethod for walnut and pecan test portions than by the BAMand ISO methods. Higher mean log counts were recovered bythe SIM–I method for one level of cake mix than by the ISOmethod. The RSDr and RSDR values were similar for the 3methods.

Recommendations

The data generated in this interlaboratory study indicatethat the SimPlate Yeast and Mold–Color Indicator method iscomparable to FDA’s BAM method and to the ISO methodfor the recovery of yeasts and molds from foods. It is recom-mended that the SimPlate Yeast and Mold–Color Indicatormethod be adopted Official First Action for enumeration offungi in foods.

Acknowledgments

The participation of the following collaborators is ac-knowledged with appreciation:

Sami Al-Hasani, Erdal Tuncan, and David Vrana,ConAgra Foods Inc., Columbia, MO

Gloria Anderson, Minnesota Valley Testing Laboratories,Inc., New Ulm, MN

Alan Ashmore and Dellah Hickson, Direct Laboratory Ser-vices Ltd., Wolverhampton, UK

Pascal Audet and Chantal Beaulieu, Centre de Recherchésur les Aliments, Pavillon Jacqueline-Bouchard, Universotede Moncton, Moncton, Quebec, Canada

Frederick K. Cook and Jake D. Knickerbocker, ConAgraFoods Inc., Omaha, NE

Robert Davis and Diane Barham, Davis Research, Avon,MS

Matt Feist and Khamphet Thammasouk, U.S. Food andDrug Administration, Pacific Regional Laboratory North-west, Bothell, WA

Florence Humbert and Marie Pierre Cornec, Laboratoirede Developpement et d’Analyses 22, Ploufragan, France

Jab Jackson, Piknik Products, Montgomery, AL

Ralph Kalinowski and Lorraine Humes, U.S. Food andDrug Administration, Alameda, CA

Morten Lisby, The Danish Veterinary and Food Adminis-tration, Regional Veterinary and Food Control Authority,Nordostsjaelland, Denmark

Julia Terry, Amy Deans, and Sarah DeLancey, BioControlSystems, Inc., Bellevue, WA

Anna Maria Vinai and Sandra Drocco, MicrobiologicalLaboratory, Soremartec s.r.l., Alba, Italy

Wendy Warren, Michael Divine, and Colbert McDonald,Food Safety Net Services, Ltd., San Antonio, TX

Sandy White and Leanne DeWinter, Canadian Food In-spection Agency, Calgary, Canada


References

(1) U.S. Food and Drug Administration (2001) BacteriologicalAnalytical Manual, 8th Ed., Rev. A, AOAC INTERNA-TIONAL, Gaithersburg, MD

(2) International Organization for Standardization (2001) Hori-zontal Method for the Enumeration of Yeasts andMolds—Colony Count Technique, ISO/CD 21527, December,Geneva, Switzerland

(3) International Organization for Standardization (1999) Micro-biology of Food and Animal Feeding Stuffs—Preparation ofTest Samples, Initial Suspension and Decimal Dilutions for

Microbiological Examination, ISO 6887-1, Geneva, Switzer-land

(4) McClure, F.D., Cole, M.E., Philips, J.G., Lee, J.K., Thomas,L.C., Newell, R., Wiest, S.C., Chen, J., Champaneri, A.M.,Wehling, P., Lindberg, K. (2001) J. AOAC Int. 84, 301–302

(5) Youden, W.J., & Steiner, E.H. (1975) Statistical Manual ofthe AOAC, AOAC, Arlington, VA

(6) Christian, G.D. (1980) Analytical Chemistry, 3rd Ed., JohnWiley and Sons, Canada, Ltd., Etobicoke, Ontario, Canada

(7) Glantz, S.A. (1992) Primer of Bio-Statistics, 3rd Ed.,McGraw-Hill, Inc., San Francisco, CA


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Enumeration of Total Yeasts and Molds in Foods by the …€¦ · · 2017-10-17cake mix, cereal, and fresh cheese. Nut meats, frozen fruits, ... DRBC agar was used for all other