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FOOD BIOLOGICAL CONTAMINANTS

Two-Day Hydrophobic Grid Membrane Filter Method for Yeast


and Mold Enumeration in Foods Using YM-11 Agar:
Collaborative Study
ENTIS: JOURNAL OF AOAC INTERNATIONAL VOL. 79, NO. 5, 1996
PHYLLIS ENTIS
QA Life Sciences, Inc., 6645 Nancy Ridge Dr, San Diego, CA 92121
Collaborators: A. Athar; M. Ballenger; M.S. Bendeck; W. Birbari; G. Brock; M.S. Curiale; J. Estvander; D.Y.C. Fung; K. Green;
S.C. Ingham; M.M. Jafary; J.A. Jagow; R. Kalinowski; G. Kelley; Y.J. Lee; I. Lerner; C.C.S. Lin; C.K. Mendenhall; J. Tomer;
D. Reyes; G. Rivera; C. Rudolph; J.-H. Ryu; P. Sado; J. Snider; L. Soto-Lopez; R.A.H. Thakur; J. Watson; S.A. White; E.
Wilkin; W.D. Williams

Twenty laboratories participated in a collaborative


study to validate a 2-day hydrophobic grid membrane filter method using YM-11 agar for enumeration of yeast and mold in foods. Six naturally contaminated food products were included in the
study: garlic powder, raw ground beef, walnuts,
flour/meal, orange juice, and yogurt. The test
method produced significantly higher results than
the 5-day pour plate reference method for orange
juice and significantly lower, though numerically
similar, results for walnuts and yogurt. Differences
between the test and reference methods were not
significant for garlic powder, raw ground beef, or
flour/meal. Repeatability and reproducibility were
similar for both the test and reference methods in
all cases. The hydrophobic grid membrane filter
method for enumeration of yeast and mold in foods
has been adopted by AOAC INTERNATIONAL.

While performing additional validation of the PDATR procedure, we found that when dry foods such as spices were filtered, mold colonies did not always develop within 48 h at
25C; rather, reincubation of the filter for an additional 24 h at
25C was often required to obtain results that correlated to the
5-day PDA pour plate method. However, reincubating also resulted in overgrowth of the filter by more rapidly growing components of the flora. Therefore, we designed a new culture medium (YM-11 agar) to improve the development of the slower
growing organisms so that the incubation period could be
maintained at 48 h.
A comprehensive precollaborative study of the 2-day hydrophobic grid membrane filter method incorporating YM-11 agar
was performed with both pure cultures and 26 food product
categories. The new method performed equivalently to the 5day PDA pour plate method (3). After successful completion of
the precollaborative study, we conducted a collaborative study,
the results of which are reported here.
Collaborative Study

n 1982, Brodsky et al. (1) published a 48-h hydrophobic


grid membrane filter method for counting yeasts and molds
in foods. This procedure entailed filtering a portion of food
homogenate and incubating on antibiotic-supplemented potato
dextrose agar (PDA) for 48 h at 25C. At the end of the incubation period, the filter was transferred to a pad soaked with
safranin to stain the yeast and mold colonies for counting.
Though effective, the safranin-staining step was inconvenient.
In 1984, Lin et al. (2) incorporated trypan blue into PDA
(PDATR) to eliminate postincubation staining.

Submitted for publication June 13, 1995.


The recommendation was approved by the Methods Committee on
Microbiology and Extraneous Materials and was adopted by the Official
Methods Board of the Association. See Official Methods Board Actions
(1995) J. AOAC Int. 78, 179A, and Official Methods Board Actions
(1995) The Referee, October issue.

Twenty laboratories participated in this collaborative study.


With the exception of 1 laboratory, all of the participants analyzed all 6 food product categories. Each collaborator received
a complete set of instructions, data reporting sheets, and a set
of 6 naturally contaminated samples (consisting of 3 replicate
pairs) for each food product. The Associate Referee also provided positive control cultures (Saccharomyces cerevisiae and
Aspergillus niger), irradiation-sterilized papain powder, enzymatic soaking detergent, YM-11 agar (including chlortetracyclineHCl supplement), and sufficient number of filters to perform the study. In addition, collaborators not equipped with
filtration units and clamps were provided with a sufficient number of these to perform the study. All other materials were furnished by the participating laboratories.
Six food products, consisting of 3 independent lots per
product, were included in the collaborative study. These were

garlic powder, raw ground beef, walnut pieces, flour/meal


(1 lot each of whole wheat flour, rye flour, and corn meal to
ensure a variety of flora and count levels), orange juice, and
yogurt. Two samples from each independent lot were provided
to each collaborator. All products were naturally contaminated
with yeasts and/or molds.
All products except for the raw ground beef and yogurt were
prescreened by the Associate Referee to verify the presence of
the desired flora and to establish appropriate dilutions to be run
by collaborators. Independent lots of raw ground beef were prescreened and then abused by being refrigerated or held at
room temperature until the flora reached desired levels, and
then 3 lots were chosen, subdivided into individual samples,
and frozen. Because counts decreased during frozen storage,
collaborators were instructed to remove ground beef samples
from the freezer on the day before analysis, hold them at 25C
for 6 h, and then to refrigerate overnight for analysis the following day. This procedure allowed the counts to return to the desired levels.
Yogurt from 3 different manufacturers was purchased well
in advance of the collaborative study and abused by holding
well past the product expiry dates. Yeast and mold counts were
monitored regularly. A few days prior to the scheduled shipping
date for this product, additional yogurt was obtained from the
same 3 manufacturers, and the abused products were diluted
into the freshly obtained material from the corresponding
manufacturers.
The collaborative study was performed over a 6-week period. With the exception of laboratory 6, which was unable to
take part in the orange juice and yogurt analyses, all laboratories participated in the analysis of all 6 products. Collaborators
were instructed to analyze all samples by both the test method
described below and by the 5-day antibiotic-supplemented
PDA pour plate reference method (4). Procedures for these
methods were followed throughout, except that the sample dilutions to be tested by both methods were specified by the Associate Referee. Collaborators calculated and reported the test
method in most probable number (MPN)/g or mL and the reference method in count/g or mL and also reported all raw data.
995.21 Yeast and Mold Counts in Foods,
Hydrophobic Grid Membrane Filter (ISO-GRID)
Method Using YM-11 Agar
First Action 1995
(Applicable to enumeration of total yeasts and molds in all
foods.)
Caution: See Appendix B, safety notes on handling microorganisms. Dispose of waste solvents according to applicable
environmental rules and regulations.
Method Performance:
See Table 995.21A for method performance data.

A. Principle
Method uses membrane filter imprinted with hydrophobic
material in grid pattern. Hydrophobic lines act as barriers to

spread of colonies, thereby dividing membrane filter surface


into separate compartments of equal and known size. Squares
occupied by colonies are counted and converted to most probable number (MPN) value of organisms.

B. Apparatus, Media, and Reagents


(a) Hydrophobic grid membrane filter (filter).Pore size,
0.45 m; imprinted with nontoxic hydrophobic material in grid
pattern.
(b) Filtration units for hydrophobic grid membrane filter.Equipped with 5 m mesh prefilter to remove food particles during filtration. Use one unit/test sample.
(c) Pipets.1.0, 5.0, and 10.0 mL, serological with
0.1 mL graduations. Instead of 1.0 mL serological, 1.1 or
2.2 mL milk pipets may be used.
(d) Blender.Multispeed with low-speed operation at
10 00012 000 rpm equipped with 250 mL glass or metal
blender jars with covers. Use one jar/test sample.
(e) Vacuum pump.H2O aspirator vacuum source is satisfactory.
(f) Manifold or vacuum flask.
(g) Peptone diluent.Dissolve 1.0 g peptone (gelatin hydrolysate peptone) in 1 L H2O. Dispense enough volume of
peptone diluent into dilution bottles to obtain 90 1 mL or 99
1 mL after autoclaving 15 min at 121C.
(h) PeptoneTween 80 (PT) diluent.Dissolve 1.0 g peptone (gelatin hydrolysate peptone) and 10.0 g Tween 80 in 1 L
H2O. Dispense enough volume of PT diluent into dilution bottles to obtain 90 1 mL or 99 1 mL after autoclaving 15 min
at 121C.
(i) YM-11 agar.Dilute 20.0 g soy peptone, 20.0 g tryptone, 5.0 g D-glucose, 5.0 g NaCl, 2.4 g K2HPO4, 0.03 g trypan
blue, 0.1 g chloramphenicol, and 15.0 g agar to 1 L with H2O.
Heat to boiling while stirring. Autoclave 15 min at 121C and
then temper to 4550C in H2O bath. Temper chlortetracyclineHCl supplement, (j), to 4550C. Add 20 mL supplement to 1 L YM-11 medium and mix well. Aseptically pour
sufficient volume of complete YM-11 agar into small weighing
boat or Petri dish to obtain 3 mm layer of agar. Allow agar to
solidify.
Check pH with flat-surface combination electrode. Adjust
pH, if necessary, to 7.0 0.2 by adding sterile 1N NaOH or 1N
HCl, as required. Aseptically pour another small portion of medium and check pH as before. Continue adjustments until pH
is within specified range.
Dispense ca 1820 mL agar portions into 15 100 mm Petri
dishes. Allow agar to solidify. Surface-dry medium before use,
by inverting partly open Petri dishes and placing them in incubator 1520 min at 35C.
(j) ChlortetracyclineHCl supplement.Dissolve 0.5 g
chlortetracyclineHCl in 100 mL H2O. Stir without heating until dissolved. Filter-sterilize solution. Unused portion of supplement can be stored up to 2 months at 46C, if protected from
evaporation.
(k) Papain stock solution.16 000 FCC (Food Chemical
Codex) papain units/mg. Reconstitute 10.0 g presterilized pa-

pain powder with 100 mL sterile H2O. Unused portion of solution can be stored frozen up to 3 months.
(l) Hemicellulase stock solution.1500 HCU (viscometric
hemicellulase unit)/g. Reconstitute 20.0 g presterilized
hemicellulase powder with 100 mL sterile H2O. Unused portion of solution can be stored frozen up to 3 months.
Items (a), (b), (i), (j), (k), and (l) are available from QA Life
Sciences, Inc., 6645 Nancy Ridge Dr., San Diego, CA 92121.

C. Preparation of Test Sample


Note: See Table 995.21B for diluents and enzyme treatments of various foods.
(a) Liquid egg.Thoroughly mix sample with sterile
spoon or spatula. Dilute sample 1:10 by aseptically weighing
11 g egg material into sterile wide-mouth, screw-top bottle.
Add 99 mL PT diluent, B(h), and 1 tablespoonful sterile glass
shot. Thoroughly agitate sample suspension (1:10 dilution) to
ensure complete solution or distribution of egg material in diluent as follows: Shake each bottle rapidly 25 in 7 s, by using
up-and-down movement of ca 30 cm for each shake. Let bubbles escape. If enzyme treatment is needed (see Table 995.21B), combine 5 mL sample homogenate (1:10 dilution) with 1 mL appropriate enzyme stock solution, mix well,
and incubate 2030 min in H2O bath at 3537C. Correct for
additional dilution by filtering 1.2 mL enzyme-treated sample
homogenate.
(b) Other liquid samples.Thoroughly mix contents of
sample container. Prepare 1:10 dilution by aseptically transferring 10 mL sample into 90 mL peptone diluent, B(g), or 90 mL
PT diluent, B(h) (see Table 995.21B), in sterile wide-mouth,
screw-top bottle. Mix contents by shaking bottle 25 through
30 cm arc in 7 s. If enzyme treatment is needed, combine
5 mL sample homogenate (1:10 dilution) with 1 mL appropriate enzyme stock solution, mix well, and incubate 2030 min
in H2O bath at 3537C. Correct for additional dilution by
filtering 1.2 mL enzyme-treated sample homogenate.
(c) Whole-egg powder.Thoroughly mix sample with
sterile spoon or spatula. Dilute sample 1:10 by aseptically
weighing 11 g egg material into sterile wide-mouth, screw-top
bottle. Add 99 mL PT diluent, B(h), and 1 tablespoonful sterile
glass shot. Shake bottle rapidly as described in (a) to ensure
complete solution or distribution of egg material in diluent.
Prepare 1:100 dilution by aseptically transferring 10 mL
sample homogenate (1:10 dilution) into 90 mL PT diluent.
Combine 10 mL of 1:100 dilution with 1 mL papain stock solution, B(k), mix well, and incubate 2030 min in H2O bath at
3537C. Filter entire 11 mL volume of enzyme-treated
1:100 dilution.
(d) Other powders.Thoroughly mix sample with sterile
spoon or spatula. Prepare 1:10 dilution by aseptically weighing
10 g sample into sterile wide-mouth, screw-top bottle. Add
90 mL peptone, B(g), or PT diluent, B(h) (see Table 995.21B).
Shake each bottle rapidly 25 in 7 s, by using up-and-down
movement of ca 30 cm for each shake. Let bubbles escape. If
enzyme treatment is needed, combine 5 mL sample homogenate (1:10 dilution) with 1 mL appropriate enzyme stock solution, mix well, and incubate 2030 min in H2O bath at 35

37C. Correct for additional dilution by filtering 1.2 mL enzyme-treated sample homogenate.
(e) Other foods.Prepare 1:10 dilution by aseptically
weighing 10 g sample into sterile blender jar. Add 90 mL peptone, B(g), or PT diluent, B(h) (see Table 995.21B), and blend
2 min at low speed (10 00012 000 rpm). If enzyme treatment
is needed, combine 5 mL sample homogenate (1:10 dilution)
with 1 mL appropriate enzyme stock solution, mix well, and
incubate 2030 min in H2O bath at 3537C. Correct for additional dilution by filtering 1.2 mL enzyme-treated sample homogenate.

D. Analysis
See Figures 986.32A and 986.32B for filtration unit and filtration unit clamp.
Turn on vacuum source. Place sterile filtration unit on manifold or vacuum flask. Open clamp A. Rotate back funnel portion C. Aseptically place sterile filter, B(a), on surface of
base D. Rotate funnel forward. Clamp shut by sliding jaws L of
stainless steel clamp over entire length of flanges B that extend
from both sides of funnel C and base D and rotating moving
arm K into horizontal (locked) position.
Aseptically add ca 1520 mL sterile H2O to funnel. Pipet
1.0 mL sample homogenate (1:10 dilution) or appropriate volume of enzyme-treated sample homogenate into funnel. Apply
free end of vacuum tubing E to suction hole F to draw liquid
through prefilter mesh G. Aseptically add additional 1015 mL
sterile H2O to funnel and draw through mesh as before. Close
clamp A to direct vacuum to base of filtration unit and draw
liquid through filter.
Rotate moving arm K of stainless steel clamp into unlocked
(ca 45 angle) position and slide jaws L off flanges B. Rotate
back funnel C. Open clamp A.
Place filter on surface of predried YM-11 plate, B(i). Avoid
trapping air bubbles between filter and agar. Incubate plates
50 2 h at 25 1C.
Count all squares containing one or more colonies (positive
squares). Colonies are usually some shade of blue. Examine
filter with illuminated magnification, as some colonies may be
only pinpoint in size. To confirm that filter does not contain any
positive squares, hold up Petri dish and examine horizon of
filter for raised bumps or areas that reflect light differently.
These may be either very small or very pale colonies. Although
hydrophobic lines act as barriers to spread of colonies, some
fast-growing molds produce too much mycelium to remain
completely confined within a single square. If sample contains
fast-growing molds, some colonies may cover more than one
square (spreaders). In case of spreader, count only the square of
origin, which usually has the densest or tallest growth. Do not
count each individual square into which colony has spread.
Count squares containing one or more colonies as described
above. Convert total number of positive squares to MPN index
as follows:
MPN = 1600 loge

1600
1600 x

where x = number of positive squares.

Multiply MPN by reciprocal of dilution factor, round to


2 significant figures, and report as yeast and mold count/g or
mL.
Ref.: J. AOAC Int. 79, 1069(1996)
Results and Discussion
All reported yeast and mold count data were checked for
correct determination of MPN indices and reference method
average counts and for correct choice of dilution factors and
were rounded to 2 significant figures. These data are reported
in Tables 16. All data were converted to log10 for statistical
analysis. Statistical analyses performed were a rank score test
(5) to identify statistical outliers, a 3-way analysis of variance
(ANOVA) to test for significance of differences between methods, and determination of repeatability and reproducibility statistics for both the test and reference methods for each food/lot
combination (5).
Determination of the appropriate dilution(s) to use for calculation of the reference method counts/g or mL were made
according to the reference method and based on the following
priority series: (1) if one and only one dilution had 3 replicate
counts in the range of 10150 colonies, then this dilution was
used to determine the count/g or mL; (2) if none of the dilutions had 3 replicate counts in the range of 10150 colonies and
all of the dilutions had one or more replicate counts exceeding
150 colonies, then the highest dilution was used to determine
the count/g or mL, and the result is reported as an estimated
count; (3) if none of the dilutions had 3 replicate counts in the
range of 10150 colonies and all of the dilutions had 1 or more
replicate counts below 10 colonies, then the lowest dilution
was used to determine the count/g or mL, and the result is reported as an estimated count; (4) if none of the dilutions had
3 replicate counts in the range of 10150 colonies, 1 dilution
had 1 or more replicate counts exceeding 150 colonies, and the
next higher dilution had 1 or more replicate counts below
10 colonies, then both of these dilutions were used to determine
the count/g or mL, and the result is reported as an estimated
count; and (5) if none of the dilutions had any colonies, then
the result was reported as less than {reciprocal of the lowest
dilution}/g or mL.
Data exclusions due to significant method deviation and statistical outliers identified by the rank score test are described
below for each food product. Data from samples for which
counts were indeterminate for both methods (i.e., either both
the test method and the reference method were indeterminately
high, or else both methods were indeterminately low) were also
omitted from statistical analysis, as were data from samples for
which the results from one method were indeterminate and the
second method produced a determinate result beyond the range
of the first method (e.g., one method produced a log10 count of
>7.000/g and the second method produced a log10 count of
7.150/g, or one method produced a log10 count of <1.000/g and
the other method produced a log10 count of 0.634/g). In all cases
of data exclusion, data from both the reference and the test
methods were excluded from statistical analysis.

Garlic Powder
The collaborative study results for garlic powder are presented in Table 1. Collaborator 11 was a statistical outlier, having reported results for all 6 garlic powder samples 23 orders
of magnitude higher than any other collaborator for both the
test and reference methods. Data from this collaborator were
excluded from statistical analysis.
Contamination levels were very low in all 3 independent
lots of this food and consisted virtually entirely of molds.
Counts obtained by the test and reference methods did not differ significantly from each other (Pr > F of 0.4610), and the
repeatability and reproducibility results were similar by both
methods (Table 995.21A).

Raw Ground Beef


The collaborative study results for raw, ground beef are presented in Table 2. The following data were excluded from statistical analysis: Collaborator 12 was a statistical outlier for the
reference method, on the basis of the rank score test (5), and
collaborator 14 reported a technique error for the reference
method. This collaborator used a single pipet to transfer all dilutions from a sample into Petri dishes, beginning with the lowest dilution. As a result, significant inoculum was carried over
from the lower to the higher dilutions, invalidating the data
from those samples for which the lowest dilution could not be
counted. Collaborator 19 was a statistical outlier for the test
method and was excluded from statistical analysis.
Contamination levels were both higher and more varied in
the ground beef than in the garlic powder and consisted mainly
of yeasts. Although all 3 lots produced higher mean counts by
the test method than by the reference method, the 2 methods did
not differ significantly from each other (Pr > F of 0.0601), and
the repeatability and reproducibility statistics were similar for
both methods (Table 995.21A).

Walnut Pieces
The collaborative study results for walnut pieces are presented in Table 3. The following data were excluded from statistical analysis: Collaborators 1 and 5 were found to be statistical outliers for the reference method by the rank score test (5),
and collaborators 12 and 14 were statistical outliers for the test
method.
The flora in these samples consisted almost entirely of
molds. The test and reference methods produced results which,
although numerically not very different (differences between
the log10 means were 0.15 or less for each of the 3 lots), were
significantly different based on ANOVA (Pr > F of 0.0001).
Repeatability and reproducibility values were similar for both
methods (Table 995.21A)

Flour/Meal
The collaborative study results for flour/meal are presented
in Table 4. Flora in these samples consisted principally of
molds. There were no significant method deviations or statistical outliers associated with this food product. Differences
between the test and reference methods were not statistically

significant (Pr > F of 0.2905), and repeatability and reproducibility results were similar for both methods (Table 995.21A).

Orange Juice
The collaborative study results for orange juice are presented in Table 5. The following data were excluded from statistical analysis: Collaborator 1 set up the orange juice samples
approximately 1 week late. Data from this collaborator were
found to be statistical outliers for both methods by the rank
score test (5). Collaborator 2 was a statistical outlier for the orange juice for both the test and reference methods, and collaborator 4 was a statistical outlier for the orange juice for the test
method, on the basis of the rank score test (5). Collaborator 14
reported a significant technique error for the reference method
(see description of the error in Raw Ground Beef).
The majority of the flora in these samples consisted of
yeasts, although some molds were also present. The test
method produced results that were significantly higher than the
reference method, based on ANOVA (Pr > F of 0.0001). Results from 1 lot were similar by both methods, but the test
method produced counts more than one-half of a log10 cycle
higher than the reference method for the other 2 lots. Repeatability and reproducibility values were in a similar range for
both methods (Table 995.21A).

Yogurt
The collaborative study results for yogurt are presented in
Table 6. The natural flora in all 3 lots of yogurt multiplied far
more rapidly in the product than expected between the time of
prescreening in the coordinating laboratory and the day specified for initiation of analyses. As a result, many collaborators
reported indeterminately high counts for the test method, which
had an absolute upper counting limit of 107/g, based on the
dilution specified for analysis by the Associate Referee,
whereas the reference method was often able to yield estimated
counts beyond 107/g. Collaborator 1 set up the yogurt samples
approximately 1 week late, and data from this collaborator
were excluded from statistical analysis. Collaborator 14 reported a significant technique error with the reference method
(see description of the error in Raw Ground Beef), and data
from this collaborator were also excluded from statistical
analysis.
Results from 2 of the 3 independent lots of yogurt were numerically higher by the reference method, and results from the
third lot were numerically higher by the test method. The largest difference between the method log10 means for any lot was
0.20. Although the numerical differences were not large, the
methods differed significantly, on the basis of ANOVA (Pr > F
of 0.0465). Repeatability and reproducibility values were in a
similar range for both methods (Table 995.21A).
Yogurt was one of 26 products included in the precollaborative study (3), and differences between the test and reference
methods were not significant for the precollaborative study
samples. Other dairy products included in the precollaborative
study were sour cream, cream cheese, and grated Parmesan
cheese. In all of these cases, differences between the methods
were not statistically significant.

The overall performance of the 2-day hydrophobic grid


membrane filter (ISO-GRID) method with YM-11 agar was
very comparable with that of the 5-day pour plate reference
method using antibiotic-supplemented PDA. Statistical analysis of the data revealed no significant differences between the
2 methods for 3 of the 6 foods: garlic powder, raw ground beef,
and flour/meal. The 2-day test method produced significantly
higher results for orange juice than did the reference method,
with 2 of the 3 lots differing by greater than 0.5 on a log10 cycle
basis. The reference method produced significantly higher results for walnuts than did the test method, with the 3 lots differing by less than 0.15 on a log10 cycle basis, and for yogurt, with
the 3 lots differing by less than 0.20 on a log10 cycle basis. For
one yogurt lot, the test method produced a higher mean recovery than the reference method. In the precollaborative study, the
test method and reference method did not differ significantly
for orange juice, walnuts, or yogurt (3).
Repeatability and reproducibility of results were also similar for both methods (Table 995.21A). The test method, which
specified a 10 g sample and a single filtration per dilution, produced a lower repeatability coefficient (RSDr) in 7 of the
18 food/lot combinations than the reference method, which required a 50 g sample and triplicate platings per dilution. The
test method produced a lower reproducibility coefficient
(RSDR) than the reference method in 8 of the 18 food/lot combinations. RSDr measures the consistency of results between
replicate samples analyzed by the same analyst at the same
time. RSDR measures other sources of variability including, for
example, variability among laboratories (5).
The test method has very few critical control points. Principal among these are the incubation time (50 2 h) and temperature (25 1C). Room temperature incubation is not
adequate, as it differs uncontrollably among laboratories and
even from day to day in the same laboratory. Incubation outside
the specified range will result in slower growth of at least a
portion of the yeast and/or mold population. Although YM-11
agar contains potassium phosphate buffer, it is still necessary to
verify that the medium, when prepared, is within the specified
pH range of 7.0 0.2.
Collaborators Comments
Comments received from collaborators were not extensive.
Some collaborators found counting colonies more difficult on
the filter than in the pour plate, especially when very large numbers of colonies were present. Conversely, one collaborator also
found the pour plate counts difficult, notably with samples containing spreading mold colonies or large numbers of particles.
Several collaborators mentioned that, with the garlic powder
samples, the entire filter became blue because of large numbers
of fine particles deposited on the filter. This deposit was due to
the small size of the particles, which were not entirely screened
out by the 5 m prefilter. Although the blue color did not affect
adversely the results (see Table 995.21A), it reduced the contrast somewhat between the colonies and the filter.
The consistency of performance of the test method is noteworthy in that several collaborators had never worked with any

hydrophobic grid membrane filter method prior to this study,


the method does not use replicate plating, and the initial sample
size is smaller than that used for the reference method. Several
factors contribute to the performance of test method. Fundamental to the performance is the hydrophobic grid membrane
filter and the filtration system. The filtration unit, which incorporates a 5 m prefilter screen, eliminates most particulate
food debris, greatly facilitating interpretation by removing the
need to differentiate between colonies and food particles. Any
water-soluble components of a food that might interfere with
outgrowth of the natural flora are eliminated during filtration
instead of being introduced into the culture medium, as is the
case with the reference method. Also, use of the filter enables
incorporation of a nontoxic dye in the culture medium, greatly
enhancing visibility of colonies by increasing contrast between
colonies, which are colored blue by the dye, and their surroundings. Finally, the YM-11 agar was formulated specifically to
encourage rapid growth of most yeasts and molds. All of these
factors cooperate to produce a 2-day hydrophobic grid membrane filter enumeration method which is equivalent both in
sensitivity and in reproducibility to the conventional 5-day
method.
Recommendation
On the basis of the results of this study, it is recommended
that the proposed 2-day hydrophobic grid membrane filter
(ISO-GRID) method using YM-11 agar for total yeast and
mold enumeration be adopted first action for all foods.
Acknowledgments
I thank the following microbiologists who took part in this
study:
A. Athar and M.S. Curiale, Silliker Laboratories Group, Research Services, Chicago Heights, IL
M. Ballenger, Amway Corporation, Ada, MI
M.S. Bendeck and M.M. Jafary, U.S. Food and Drug Administration, Los Angeles, CA
W. Birbari and G. Rivera, ABC Research Corporation,
Gainesville, FL

G. Brock and J. Snider, Dairy and Food Labs, Inc., Modesto, CA


J. Estvander, Savannah Cocoa, Inc., Savannah, GA
D.Y.C. Fung and R.A.H. Thakur, Kansas State University,
Dept. of Animal Science & Industry, Manhattan, KS
K. Green and W.D. Williams, The Coca-Cola Co., Atlanta, GA
S.C. Ingham and J.-H. Ryu, University of Wisconsin-Madison, Madison, WI
J.A. Jagow and P. Sado, U.S. Food and Drug Administration, Bothell, WA
R. Kalinowski and J. Watson, U.S. Food and Drug Administration, Alameda, CA
G. Kelley, Midwest Laboratories, Milwaukee, WI
Y.J. Lee, Amway Corporation, R&D Division, Ada, MI
I. Lerner, QA Life Sciences, Inc., San Diego, CA
C.C.S. Lin and J. Tomer, S & J Laboratories, Inc., Kalamazoo, MI
C.K. Mendenhall, U.S. Food and Drug Administration,
Minneapolis, MN
D. Reyes, Strasburger & Siegel, Inc., Hanover, MD
C. Rudolph and E. Wilkin, Leprino Foods, Denver, CO
L. Soto-Lopez, U.S. Food and Drug Administration, San
Juan, PR
S.A. White, Oregon Freeze Dry, Inc., Albany, OR
I thank Z. Toy and M. Gorin for their help in organizing and
preparing for this study and F.D. McClure of the U.S. Food and
Drug Administration, Washington, DC, for performing the statistical analysis. I also acknowledge the assistance of M.P. Entis, J. Zehner, and R. Armock.
References
(1) Brodsky, M.H., Entis, P., Entis, M.P., Sharpe, A.N., & Jarvis,
G.A. (1982) J. Food Prot. 45, 301304
(2) Lin, C.C.S., Fung, D.Y.C., & Entis, P. (1984) Can. J. Microbiol. 30, 14051407
(3) Entis, P., & Lerner, I. (1996) J. Food Prot. 59, 416419
(4) Mislivec, P.B., Stack, M.E., Koch, H.A., & Bandler, R.
(1992) in FDA Bacteriological Analytical Manual, 7th Ed.,
AOAC INTERNATIONAL, Arlington, VA, pp. 227234
(5) Youden, W.J., & Steiner, E.H. (1975) Statistical Manual of
the AOAC, AOAC, Arlington, VA

Table 995.21A. Method performance for enumeration of yeast and mold in foods by ISO-GRID and reference
methods

Food
Garlic powder

Lot

Method

Log10 mean,
count/g or
mL

ISO-GRID
Reference

1.23
1.08

0.95
0.90

1.12
1.23

0.34
0.32

0.40
0.44

27.49
29.12

32.09
40.19

0.76

1.01

0.27

0.36

22.35

30.03

1.20
1.01

0.35
0.19

0.43
0.36

28.95
16.11

36.10
30.42

Reference

1.20

1.34

1.48

0.48

0.53

40.00

44.18

ISO-GRID
Reference

1.20
1.18

ISO-GRID

3.61

0.76

1.06

0.27

0.38

7.50

10.60

Reference
ISO-GRID

3.59
3.91

0.81
0.95

1.06
1.32

0.29
0.34

0.38
0.47

8.23
8.70

10.61
11.99

Reference

3.84

0.64

1.01

0.23

0.36

5.94

9.36

ISO-GRID
Reference

4.43
4.28

1.09
0.81

1.68
1.46

0.39
0.29

0.60
0.52

8.82
6.88

13.60
12.03

Overall

ISO-GRID

3.99

Reference
ISO-GRID

3.90
3.57

0.67

0.73

0.24

0.26

6.77

7.15

Reference

3.68

0.53

0.53

0.19

0.19

4.93

5.07

ISO-GRID
Reference

4.08
4.23

0.28
0.22

0.64
0.45

0.10
0.08

0.23
0.16

2.47
1.90

5.59
3.81

ISO-GRID

3.66

0.62

0.70

0.22

0.25

5.97

6.74

3.79
3.76

0.59

0.59

0.21

0.21

5.43

5.43

Overallb

Reference
ISO-GRID
Reference

3.90

ISO-GRID
Reference

3.36
3.52

1.20
1.01

1.20
1.01

0.43
0.36

0.43
0.36

12.80
10.23

12.80
10.23

ISO-GRID

4.63

0.59

1.18

0.21

0.42

4.45

9.05

Reference

4.76

0.76

0.92

0.27

0.33

5.66

6.97

ISO-GRID
Reference

3.38
3.20

1.01
0.73

1.18
1.09

0.36
0.26

0.42
0.39

10.57
8.19

12.46
12.23

Overall

ISO-GRID

3.80

Reference
ISO-GRID

3.83
3.11

0.53

0.64

0.19

0.23

5.91

7.19

Reference

3.32

0.42

0.64

0.15

0.23

4.39

6.85

ISO-GRID
Reference

7.20
6.66

1.04
1.12

1.18
2.10

0.37
0.40

0.42
0.75

5.16
6.06

5.87
11.31

ISO-GRID

6.00

0.73

1.74

0.26

0.62

4.39

10.26

5.45
5.45

1.29

1.54

0.46

0.55

8.35

10.08

Overallb

Reference
ISO-GRID
Reference

5.15

ISO-GRID
Reference

6.54
6.75

0.48
0.31

1.37
1.82

0.17
0.11

0.49
0.65

2.51
1.59

7.44
9.58

A
B

ISO-GRID

6.46

0.45

1.85

0.16

0.66

2.46

10.25

Reference
ISO-GRID

6.61
6.72

0.34
0.59

2.38
1.48

0.12
0.21

0.85
0.53

1.81
3.07

12.85
7.83

Reference

6.66

0.62

1.93

0.22

0.69

3.25

10.36

ISO-GRID
Reference

6.60
6.67

Overallb

RSDR %

0.98
0.53

RSDr %

1.20

Yogurt

sR

1.20
1.18

Orange juice

sr

ISO-GRID

Flour/meal

Reference
ISO-GRID

Walnuts

Overall
Raw ground beef

Precision estimates

Precision estimates were calculated after conversion of the data to log10; r = 2.8 sr; R = 2.8 sR; sr = repeatability standard deviation; sR =
reproducibility standard deviation; RSDr = repeatability relative standard deviation; RSDR = reproducibility relative standard deviation.
Difference between the methods is statistically significant at the 5% level.

Table 995.21B. Diluents and enzyme treatments for


a
various foods
Food
Flour/meal
Black pepper
Onion powder
Garlic powder
Soy beans
Pinto beans
Rice
Coffee beans
Cocoa beans
Cream cheese
Other cheeses
Sour cream
Vegetable juices
Fruit juices
Raw tomato
Frozen strawberries
Fresh blackberries
Fruit preserves
Walnuts
Peanuts
Raw ground beef
Summer sausage
Roast beef
a

Diluent

Enzyme

Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
PTb
Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
Peptone
PT
PT
PT

None
None
Papain
Papain
None
None
None
None
None
Hemicellulase
Papain
Papain
None
None
None
None
None
None
Papain
Papain
None
None
None

Based on analysis of 1 mL 1:10 dilution. Foods tested at dilutions of


1:100 or higher do not usually need enzyme treatment. See Table
986.32 for recommended enzyme treatments of foods not listed in
this table .
PeptoneTween 80 diluent.

Table 1. Collaborative study results for yeast and mold counts in garlic powder by test and reference methods
Results of reference method, count/g
1 (C)a

Laboratory
1
2
3
4
5
6
7
8
9
10
d

11

12
13
14
15
16
17
18
19
20
a
b
c
d

2 (A)

1.0 101b

3.3 10

1b

2.3 101b

6.7 100b

1.7 10

3.3 100b

1.0 10

1b

6.7 101b

3.3 10

0b

3.3 101b

3.3 10

0b

5.5 103
b

1.7 10

<1.0 101
b

2.7 10

1.2 104

2b

1.0 101b

1.0 10

<1.0 10

3.3 100b

<1.0 10

1.3 101b

3.3 10

1b

<1.0 10

1.3 102b

1.7 10

<1.0 10

2.3 101b
4.7 10

1b

3.3 100b

6.7 10

6.7 100b
0b

<1.0 10

<1.0 10
b

1.7 101b
6.1 10

6.7 100b

3.0 10

1.0 10

2b

3.3 100b

6.7 10

0b

0b

9.4 103
b

2.7 101b
1b

6.7 10

1.7 101b

3.3 10

1b

1.0 102b

6.7 10

0b

0b

3.3 10

8.9 103
b

2.3 101b

3.3 101b

3.3 10

0b

3.3 100b

1.0 10

3.3 10

1b

2b

1b

3.3 10

1b

6.0 103

1.7 101b

1.7 101b

<1.0 10

4.3 10

6.7 101b

1.0 10

1b

1.0 101b

3.3 100b

1b

3.3 100b

1.7 10

6.7 10

0b

3.3 100b

0b

1b

1b

3.3 10

3.3 100b

1.7 10

4.0 101b

1.0 10

1.0 101b

4.7 10

1.0 102b

1b

1b

1b

3.3 10

1.0 101b

1.0 104b

3.3 101b

b
b

1.0 10

1.0 10

1b

1.7 10

6.7 10

1.3 101b

6.7 101b
1b,c

6.7 100b

1.0 10

1 (C)

5.7 10

1b

1b

b,c

1.3 101b

1b

3.0 10

<1.0 101

3.3 100b

6 (A)

1b

2b

1b

8.3 10

1b

6.7 100b

1b

2.7 10

3.3 100b
1b

<1.0 10

2.0 101b

2.7 101b

3.3 10

5 (C)

0b

0b

6.7 10

1b

3.3 100b

b
b

1.0 10

1b

<1.0 101
3.7 10

2.3 101b

1.0 10

4 (B)

1b

<1.0 101
1b

3 (B)

Results of test method, count/g

3.3 10

1b

4.3 101b

<1.0 10

3.3 100b

4.7 10

1b

3.3 100b

<1.0 10

2.0 101b

3.3 10

0b

2 (A)

3 (B)

4 (B)

5 (C)

6 (A)

1.0 101

2.0 101

<1.0 101

4.0 101

<1.0 101

<1.0 101

2.0 102

6.0 10

1.0 10

7.0 10

<1.0 10

1.0 10

<1.0 101

<1.0 101

<1.0 101

<1.0 101

1.0 101

<1.0 101

2.0 10

1.0 10

<1.0 10

1.0 10

1.0 10

2.0 101

6.0 101

1.0 101

7.0 101
<1.0 10

2.0 101
<1.0 10

2.0 101
<1.0 10

1.0 101
<1.0 10

<1.0 10

1.0 101

1.0 101

<1.0 101

2.0 101

3.0 101

<1.0 101

<1.0 101

<1.0 101

<1.0 10

1.0 10

<1.0 10

<1.0 10

<1.0 10

2.0 101

2.0 101

1.0 101

2.0 101

1.0 101

2.0 101

1.0 101

8.9 103

9.4 103

3.0 10

5.8 103
<1.0 10

1.3 10

1.2 104
<1.0 10

4.0 10

1.9 104
5.0 10

1.0 10

6.0 103
<1.0 10

<1.0 10
<1.0 10

<1.0 101

<1.0 101

<1.0 101

1.0 101

1.0 101

3.0 101

5.0 101

2.6 102

1.0 101

2.0 101

<1.0 101

<1.0 101

<1.0 101

<1.0 10

<1.0 101

1.2 10

1.1 10

3.0 10

1.0 101

4.0 101

4.0 101

<1.0 10

1.0 101
<1.0 10

1.0 10

3.0 101
<1.0 10

<1.0 10

1.0 101
<1.0 10

1.1 10
c

1.0 101c
1.0 10

1.0 101
<1.0 10

2.5 10
<1.0 10

<1.0 101

<1.0 101

<1.0 101

<1.0 101

<1.0 101

1.0 101

1.0 10

<1.0 10

1.0 10

1.0 10

<1.0 10

3.0 101

Letter in parentheses refers to the lot from which the sample was drawn.
Estimated count. Counts on one or more pour plates fall outside the specified counting range of 10150 colonies/plate.
Estimated count due to spreader.
Statistical outlier for both methods. Data excluded from statistical analysis.

Table 2. Collaborative study results for yeast and mold counts in raw ground beef by test and reference methods
Results of reference method, count/g
1 (A)a

Laboratory

2 (A)

3 (B)

4 (C)

Results of test method, count/g


5 (C)

3 (B)

4 (C)

5 (C)

6 (B)

4.7 103

3.4 104

5.8 10

3.5 103

4.5 103

1.7 104

5.6 10

1.3 10

1.1 10

1.8 103

3.1 103

2.5 103

1.0 104

3.2 103

6.4 103

3.5 103

7.2 103

2.3 103

4.5 104

2.5 10

1.8 10

5.2 10

1.0 10

4.5 10

2.9 10

1.0 10

4.4 10

2.7 10

1.1 10

3.9 103

2.9 103

1.3 104

3.2 104

1.3 105

9.6 103

1.0 103

1.6 103

8.4 103

5.9 104

1.1 105

3.9 103

8.6 10

5.6 103

3.7 103

10

3.4 10

1.4 104b

11
12

1.7 10

13

5.7 103

5.4 10

3.5 103
b

1.5 10

3b

1.4 10

7.3 10

6.1 10

5.6 10

4.3 103

1.4 105

8.4 104

1.7 104

7.2 103

2.9 10

9.8 10

2.8 10

4.2 10

2.5 10

3.6 103

1.4 104

9.0 104

9.1 104

7.3 103

3.1 10

7.1 10

4.3 10

5.0 10

1.0 10

1.1 10

3.1 10

7.5 10

4.6 103

3.7 104

5.8 103

5.6 10

2.6 10
b

1.6 104b
7.6 10

1.8 104

1.0 10

4.1 104
b

2.7 10

4b

3.9 10

2.5 10

5.6 10

1.7 10

1.6 10

5.0 10
1.2 10

5b

1.7 104

1.3 104

2.7 103

1.4 104

7.9 103

8.0 103

3.2 103

7.2 103

2.1 103

4.1 10

7.2 10

2.7 10

8.7 10

2.9 10

1.1 10

1.8 104b

9.1 103

2.9 104

2.5 104

9.7 103

3.1 104

3.1 104

9.1 103

6.2 10

1.8 10

3.5 10

5.7 10

8.1 10

1.7 10

1.7 10

2.1 103

4.1 103

4.3 103

1.4 104

6.1 103

2.0 104

1.5 103

2.3 103

3.3 104

2.0 10

4.1 10

6.9 10

4.3 10

4.1 10

7.0 104

2.1 103

2.5 104

4.4 103

5.4 103

3.0 103

4.1 103

6.1 103

3.8 104

4.5 104

4.1 103

16

6.2 103

17
18

2.0 10

3.9 104

2.2 103

2.1 103

3.8 105

2.1 104

4.4 104

1.4 104

5.4 10

2.9 10

6.5 10

1.9 10

7.3 10

5.1 10

3.1 10

1.8 10

1.9 10

7.3 10

7.0 10

1.4 103

20

2.4 10

2.1 10

2.0 10

9.6 10

3.0 10

3.9 104

2.0 104

4.8 10

1.9 104

1.2 104

2.3 10

1.4 104

3.0 103

1.2 10

1.3 103

5.2 103

5.7 10

3.3 102b

19

9.7 10

N/A

2.6 103

8.0 10

N/A

4.2 103

15

5.6 10

N/A

N/A

2.4 10

N/A

6.1 104

14

3.9 10

N/A

1.1 104

3.0 103

3.8 103

2.2 103

6.7 103

2.5 104

9.6 103

8.7 10

2b

1.4 104

7.8 10

2.5 10

4.1 104b

4.1 103

1.2 104b
4

9.7 10

1.7 104

2 (A)

3.4 103

2b

4.4 104

1 (A)

5.3 10

1.5 105

6 (B)

1.4 104b

3.5 103

1.4 103

<1.0 102

<1.0 102

<1.0 102

1.4 104

2.0 103

5.9 10

9.8 10

2.6 10

3.7 10

2.4 10

1.9 10

6.2 10

1.8 104

Letter in parentheses following sample number refers to the lot from which the sample was drawn.
Estimated count. Counts on one or more pour plates fall outside the specified counting range of 10150 colonies or number of positive squares on filter exceeded 1520 (>95% of squares positive).
Statistical outlier for the reference method. Data excluded from statistical analysis.
N/A, valid counts not available for reference method. Collaborator used a single pipette to transfer all dilutions into Petri dishes and proceeded from lowest dilution to highest dilution, resulting in
carryover of significant inoculum from each dilution to the next higher. Counts on lowest dilution were too numerous to count on all 6 samples, and results of higher dilutions were compromised by
this technique error. Data excluded from statistical analysis.
Statistical outlier for test method. Data excluded from statistical analysis.

Table 3. Collaborative study results for yeast and mold counts in walnut pieces by test and reference methods
Results of reference method, count/g
1 (A)a

Laboratory

2 (A)

3 (B)

4 (B)

Results of test method, count/g


5 (C)

6 (C)

1 (A)

2 (A)

3 (B)

4 (B)

5 (C)

6 (C)

9.4 103

3.1 104

2.5 104

3.1 104

2.2 104

1.8 104

1.4 104

4.1 103

1.1 104

8.7 103

4.8 103

4.1 103

3.8 10

3.9 103

2.3 103

7.9 103

2.5 104

2.4 104

2.7 103

9.4 103

1.6 103

1.0 104

2.2 104

2.3 104

2.8 103

5.3 103

3.1 10

3.0 10

4.6 10

8.5 10

6.6 10

3.3 10

2.5 10

9.0 10

4.9 10

3.5 10

3.8 10

1.0 103

4.5 10

1.6 10

1.6 10

4.7 10

4.9 10

3.7 10

1.3 10

1.2 10

3.9 10

2.4 103

2.7 103

5.3 103

4.7 103

2.5 103

5.5 10

3.8 10

1.9 10

1.8 10

1.3 10

5.3 103

2.9 103

1.5 104

5.8 10

4.5 103

3.4 103

2.5 104

2.5 104

5.1 103

7.4 103

2.0 103

3.2 103

9.4 103

10

1.1 10

11

1.9 104

4.3 103

1.7 104

1.7 104

2.0 104

5.6 103

1.2 104

4.4 103

1.3 104

12

3.9 10

2.0 10

2.2 10

2.8 10

3.6 10

5.3 10

9.0 10

7.0 10

4.8 10

13

2.8 103

4.0 103

1.9 104

2.0 104

4.3 103

5.4 103

2.4 103

3.4 103

7.7 103

6.9 103

<1.0 10

5.3 10
5.6 10

1.5 10
2.1 10

1.8 104c
1.8 10
2.3 10

1.2 10

4c

3.4 103

3.1 103

8.7 103

8.5 103

4.6 103

6.1 103

1.4 10

3.3 10

9.7 10

7.2 10

4.1 10

4.5 103

3.2 103

5.3 103

3.4 103

3.5 103

1.3 104

2.9 104

2.8 103

6.0 103

3.0 103

1.1 104

4.3 103

7.5 103

5.7 103

1.0 104

2.2 104

5.7 103

4.6 10

1.9 10

1.2 103

4.6 103

5.9 103

4.4 10
9.3 10

1.2 10

1.8 10

2.0 10

1.4 10

9.5 10

5.6 10

1.0 102

1.3 104

4.0 103

8.6 103

6.9 103

3.5 103

1.5 104

1.3 104

3.5 103

7.7 103

16

4.1 10

8.7 103

17

4.5 103

4.7 103

1.2 104

1.6 104

4.0 103

7.0 103

2.6 103

4.3 103

4.6 103

6.4 103

2.2 103

3.2 103

18

5.5 10

4.4 10

1.1 10

1.0 10

3.4 10

1.1 10

6.6 10

4.4 10

1.0 10

1.8 10

3.7 10

1.3 104

19
20
a
b
c
d
e

5.1 103e
1.1 10

7.0 103e

1.6 104

3.0 104

5.3 103

5.5 10

1.8 10

2.4 10

6.0 10

1.5 10

1.3 10

2.7 10

1.5 104

3.3 10

1.0 10

2.7 10

4.5 10

4.0 10

2.5 10

4.6 103

6.6 10

1.0 10

3.3 10

4.3 10

6.3 10

4.8 10

4.3 103

1.7 10

5.3 10

6.2 10

15

2.1 10

1.3 10

3.0 10

3.5 10

1.3 10

14

2.8 10

4.0 103

3.1 10

1.1 10

4.7 10

1.0 104e

2.1 103

3.5 103

9.6 103

9.8 103

2.2 103

4.1 103

5.3 10

3.9 10

8.6 10

1.8 10

2.1 10

7.5 10

7.4 103

Letter in parentheses following the sample number refers to the lot from which the sample was drawn.
Statistical outlier for reference method. Data excluded from statistical analysis.
Estimated count. Counts on one or more pour plates fall outside the specified counting range of 10150 colonies.
Statistical outlier for test method. Data excluded from statistical analysis.
Estimated count due to spreader.

Table 4. Collaborative study results for yeast and mold counts in flour/meal by test and reference methods
Results of reference method, count/g
1 (B)a

Laboratory

2 (A)

3 (C)

4 (A)

8.7 102b

Results of test method, count/g


5 (B)

6 (C)

1 (B)

2 (A)

3 (C)

4 (A)

3.3 104

2.7 103

4.0 10

2.8 104

1.5 103

1.8 103

4.8 10

3.8 10

3.0 10

1.9 104

4.4 103

2.8 10

2.5 10

3.1 104

3.1 103

5.0 103

1.1 10

3.4 104

3.6 103

3.0 103

3.8 103

8.9 104

3.3 103

9.0 103

3.6 103

3.0 102

10

4.2 10

11

9.9 104

9.3 103

3.8 103

12

5.4 10

3.5 10

3.3 10

13

8.4 104

5.1 103

14

15
16

3.5 10

8.3 104
b

4.3 10

4c

3.1 10

3.4 10
4.1 10

2.7 10

3.4 103
2.8 10

18

2.1 10

3.9 10

20
a
b
c

5.0 104c

4.0 103

3.7 10

3.3 10

3.9 10
4.1 10

2b

1.6 105

1.3 103

2.2 104

1.8 103

1.2 103

3.2 103

1.7 104

2.2 103

4.9 103

3.5 10

2.4 103
b

5.7 10

2b

4.2 10

2.4 104
b

3.2 10

5b

4.0 10

2.3 103
b

1.6 10

2.3 103

4.4 104

2.6 10

3.2 10

3.2 103

6.1 104

3.2 10
4.0 10

6.0 104
b

4.0 10

2b

7.7 10
5.9 10

8.5 104
4.3 10

8.3 10
2.5 10
1.5 10

1.0 10

5.7 104

1.3 105

b,c

1.8 10

3b

4.0 103
3.4 10

5.5 10

4c

2.5 103b

2.6 103

1.0 105

3.5 10

4.4 10

3.2 10

6.3 102b
4.2 10

1.7 103c

9.6 104

2.8 10

6.3 10

1.6 103

2.5 10

2b

7.7 102b

3.4 103
2.4 10

4b

2.7 102b

3b

3.3 102

6 (C)

3.3 103

9.3 102c

1.1 10

2.2 103

19

4.3 10

1.8 104

4.0 102b

17

4.9 10

5 (B)

3b

1.0 103b
8.0 10

2b

7.0 102b,c

2.9 10

3c

4.5 10

1.6 10

4.2 10

1.5 10

3.3 10

2.6 104

1.4 103

1.6 103

2.2 103

2.4 104

2.6 103

6.0 10

3.3 10

2.8 10

4.0 10

7.5 10

3.3 104

1.1 104

1.4 103

6.0 102

2.9 103

2.4 104

8.0 102

1.8 10

1.1 10

8.0 10

2.4 10

1.7 10

1.5 103

2.6 104

2.8 103

3.3 103

3.1 103

2.1 104

3.3 103

4.8 103

2.2 103

2.0 104

1.3 103

1.4 103

5.8 10
3.2 10

2.8 10
1.9 10

1.0 10
4.0 10

3.4 10
3.1 10

7.2 10
2.3 10

6.1 104

4.2 103

3.7 103

2.3 104

6.2 104

1.6 103

1.6 10

2.2 10

1.1 10

7.0 10

2.0 10

1.0 103

4.9 104

1.7 103

2.6 103

1.8 103

5.5 104

5.6 103

6.2 103

1.9 10

6.0 10

9.0 10

1.5 10

2.9 10

1.1 105

2.3 103

5.0 103

3.4 103

7.8 104

6.5 103

4.4 103

9.1 10

6.0 10

4.3 10

1.9 10

8.6 10

4.7 102b

2.3 104

2.8 103

7.6 103

1.6 103

1.0 105

1.0 103

3.1 10

2.8 10

2.1 10

1.6 10

5.9 10

3.5 10

9.4 103

7.7 102b

1.7 105

1.8 103

1.3 103

1.8 103

6.3 104

5.1 103

2.2 10

3.3 10

1.0 10

1.0 10

1.1 10

6.3 10

3.2 103

Letter in parentheses following the sample number refers to the lot from which the sample was drawn.
Estimated count. Counts on one or more pour plates fall outside the specified counting range of 10150 colonies.
Estimated count due to spreader.

Table 5. Collaborative study results for yeast and mold counts in orange juice by test and reference methods
Results of reference method, count/mL
1 (A)a

Laboratory

2 (B)

3 (A)

1.1 103

2.5 105

1.1 10

4.2 10

4 (C)

9.0 102c
1.2 10

6 (C)

2.3 104

3.3 105

4.7 105

1.0 103

3.9 106

1.0 103

2.9 106

1.2 107

1.8 106

3.6 106

9.5 10

3.9 10

1.4 10

1.2 10

3.4 10

2.0 10

3.5 10

2.8 10

9.6 102

1.1 107

1.3 103

1.2 106

7.6 106

2.2 106

1.2 10

1.4 10

5.0 10

3.9 10

1.0 10

7.1 10

9.3 10

2.4 10

5.6 10

6.6 10

9.2 10

6.2 105

>1.5 106

5.5 105

>1.5 106

2.1 105

4.9 105

4.4 10

3.5 10

3.0 10

3.3 10

2.0 10

2.0 103

3.0 107

2.3 103

2.8 106

2.1 107

2.5 106

9.5 102

2.1 107

1.4 103

11

9.1 10

12
13
h

14

2.4 103
c

1.2 10

3c

N/A

>1.5 10

2.1 107
2.1 10

2.9 107
c

9.3 10

5c

1.2 10

1.6 103c
2.9 10

1.1 103

2.6 105

1.2 103

8.1 105

1.5 107

5.3 105

1.0 10

2.0 10

2.6 10

3.7 10

1.6 10

5.2 106

3.0 106

2.2 107

3.7 106

>1.0 108

1.7 10

>1.0 10

1.2 10

>1.0 10

>1.0 10

1.7 104

7.2 105

3.2 105

1.8 103

1.8 107

9.1 102

2.3 106

1.9 107

3.1 106

7c

2.2 106

1.2 10

3.2 10

1.1 10

6.9 10

1.9 10

2.5 10

1.2 10

5.0 10

5.1 102

2.7 105

2.6 107

2.0 104

1.5 10

1.8 10

3.5 10

1.5 10

2.1 10

3.1 10

1.6 10

2.0 10

1.1 10

1.0 105

17

2.5 10

18

3.6 103

3.4 107

2.8 103

19

1.5 10

8.1 10

1.1 10

20

3.6 103

3.4 10

1.6 103
c

1.5 10

4.8 105
3

1.2 106

2.0 107

>1.5 10

3.1 107

1.3 10

5c

1.4 103

1.8 103

2.4 105
c

2.7 105

16

3.2 10

>1.5 10

5.0 107

1.2 10

6.1 10

2.6 105

N/A
5

>1.5 10

2.1 103

N/A
3

15

5 (B)

1.2 106

4 (C)

9.1 105

1.8 103

3 (A)

1.4 106

10

2 (B)

1.3 103

1.1 10

1 (A)

1.5 106

6 (C)

1.0 103

5 (B)

Results of test method, count/mL

3c

3.5 103

1.0 10

5c

1.3 105
c

6.0 10

4c

4.3 105
c

1.0 10

5c

1.3 105

N/A

N/A
5

1.2 106

5.4 10
c

1.3 10

7c

3.1 107
7.1 10

1.7 107

5.7 10

4c

2.9 105c
3.3 10

6.7 105
c

1.2 10

5c

7.2 105

7.3 107c

3.2 103

2.4 107

5.3 103

1.1 107

8.2 106

1.7 105

1.7 10

1.1 10

1.3 10

8.0 10

2.0 10

1.0 103

1.7 107

1.0 103

2.3 106

1.7 107

2.3 106

1.8 106

6.7 10

1.0 10

8.2 10

2.1 10

3.3 10

2.5 103

2.9 107

8.9 102

3.1 106

2.8 107

4.8 106

1.6 10

4.2 10

1.6 10

8.2 10

2.7 10

2.3 106

1.7 107

7.3 105

8.6 102

1.7 107

1.0 103

5.6 105

Letter in parentheses following the sample number refers to the lot from which the sample was drawn.
Statistical outlier for reference method. Data excluded from statistical analysis. Collaborator also reported a scheduling deviation. Samples were initiated on December 13, 1994, instead of
December 7, 1994.
Estimated count. Counts on one or more pour plates fall outside the specified counting range of 10150 colonies or number of positive squares on filter exceeded 1520 (>95% of squares positive).
Statistical outlier for both methods. Data excluded from statistical analysis.
Statistical outlier for test method. Data excluded from statistical analysis.
Samples 1 and 3 are statistical outliers for reference method. Data from these 2 samples excluded from statistical analysis.
Samples 2, 4, 5, and 6 are indeterminately high for both methods. Data from these 4 samples excluded from statistical analysis.
N/A, valid counts not available for reference method. Collaborator used a single pipette to transfer all dilutions into Petri dishes and proceeded from lowest dilution to highest dilution, resulting in
carryover of significant inoculum from each dilution to the next higher. Counts on lowest dilution were too numerous to count on 5 of 6 samples, and results of higher dilutions were compromised by
this technique error. Data excluded from statistical analysis.

Table 6. Collaborative study results for yeast and mold counts in yogurt by test and reference methods a
Results of reference method, count/g
1 (A)b

Laboratory
c

2.6 10

7d

2.2 106
d

1.8 10

7d

2.5 107d

2.3 10

7d

>1.5 107

9.9 106

1.2 107

3.7 10

9.7 10

2.0 10

5.0 10

1.2 107
d

1.8 107d

>1.5 10

2.1 10

12
13

16
17
18
19
20

5.3 106
d

2.1 10

>1.5 10

1.6 107d
1.5 10

7d

1.6 107d

1.8 10

3.3 10

1.6 107d

2.2 10

7d

7d

2.0 107d

1.9 10

1.5 10

1.3 107

>1.5 10

1.6 107d

2.6 10

9.9 106
9.5 10

5.5 105

2.7 10

>1.5 10

1.6 107d
1.2 10

7d

9.8 106

2.2 10

1.3 107

2.2 10
1.4 10

2.3 107d

1.5 107d

2.4 10

1 (A)

7.7 106
2.2 10

7d

2 (B)

>1.0 107
d

8.0 10

6d

3 (C)

>1.0 107

1.2 106

>1.0 107

>1.0 107

>1.0 10

>1.0 107

2.2 10

1.1 10

2.8 10

8.8 10

7.4 107d
1.4 10

2.0 107d

>1.5 10

1.8 107d
1.7 10

1.7 106
d

3.2 10

7d

4.4 106
2.3 10

1.3 10

1.4 107
>1.5 10

2.1 107d

1.8 107d

2.7 10

7d

1.8 107d

2.2 107d

1.9 10

7d

1.3 107

7.5 106d
2.4 10

9.6 106d

1.7 105
>1.0 107

>1.0 10

>1.0 10

>1.0 107

7.4 10

5.8 10

6d

9.6 10

6d

>1.0 107

>1.0 107

>1.0 107

>1.0 107

>1.0 107

>1.0 107

>1.0 107

>1.0 10
2.2 10
>1.0 10

2.4 107d

1.1 10

1.6 10

6.0 10

>1.0 107

7d

7.8 106d

5.9 105

7.9 106

>1.0 107

>1.0 10

1.1 10

2.9 10

>1.0 107

6d

>1.0 107

8.5 106d

>1.0 10

>1.0 10

>1.0 107

>1.0 107

1.4 10

>1.0 10

>1.0 107

1.3 107

7d

1.5 10

N/A
7

7d

6 (C)

1.5 106

>1.0 107

5 (B)

1.0 107

4 (A)

>1.0 107

1.7 107d

7d

2.9 10

7d

N/A
7d

1.7 107d

7d

1.7 10

7d

6 (C)

4.3 106
d

N/A

1.4 107
d

3.4 106

1.3 10

6.8 10

7d

2.4 107d

1.1 10

1.3 107d

N/A
7d

1.8 107d
1.3 10

7.3 106

N/A

1.4 10

1.4 106

N/A
d

2.8 10

7d

1.7 107d

7d

15

2.1 107d

2.0 10

7d

1.7 107d

11

14

10

3.5 106
d

>1.5 107

5 (B)

2.6 107d

4 (A)

4.9 10

3 (C)

2 (B)

Results of test method, count/g

5.4 106d
7

1.8 10

2.9 106
>1.0 10

>1.0 10

>1.0 107
d

9.2 10

6d

4.7 106
>1.0 10

>1.0 10

>1.0 10

8.9 106d

>1.0 107

>1.0 107

1.4 106

3.2 106

3.5 106

1.2 10

3.1 106
>1.0 10

1.7 10
>1.0 10

>1.0 107

2.9 105

1.9 105

1.5 105

3.4 105

1.0 106

1.7 105

>1.0 107

>1.0 107

>1.0 107

>1.0 107

>1.0 10

>1.0 10

>1.0 107

>1.0 107

8.3 10

6d

8.0 10

6d

>1.0 107

>1.0 107

>1.0 10

4.5 10

6.4 106d

>1.0 107

>1.0 10

7.7 106
d

8.9 10

6d

>1.0 10
9.6 10

6d

>1.0 10
8.5 10

6d

9.2 106d

>1.0 107

>1.0 107

>1.0 107

>1.0 107

9.6 10

6d

>1.0 107

8.5 10

6d

4.3 106

9.2 10

6d

>1.0 107

7.6 106d

8.7 106d

Data set contains many samples for which the test method was indeterminately high (i.e., >1.0 107/g) and for which the reference gave either an indeterminately high value or else gave a
determinately high result in excess of 1.0 107/g. All such samples were excluded from statistical analysis.
Letter in parentheses following the sample number refers to the lot from which the sample was drawn.
Scheduling deviation. Samples initiated on December 13, 1994, instead of December 7, 1994. Data excluded from statistical analysis.
Estimated count. Counts on one or more pour plates fall outside the specified counting range of 10150 colonies or number of positive squares on filter exceeded 1520 (>95% of squares positive).
N/A, valid counts not available for reference method from this collaborator. Collaborator used a single pipette to transfer all dilutions into Petri dishes and proceeded from lowest dilution to highest
dilution, resulting in carryover of significant inoculum from each dilution to the next higher. Counts on the lowest dilution were too numerous to count on all 6 samples, and results of higher dilutions
were compromised by this technique error. Data excluded from statistical analysis.

Figure 986.32A. Filtration unit.

Figure 986.32B. Filtration unit clamp.