1286 Alles et al.: Journal of AOAC International Vol. 98, No.
5, 2015
FOOD BIOLOGICAL CONTAMINANTS
Validation of a Minor Modification to the Soleris® Direct
Yeast and Mold Vial and Selective Supplement
Modification to AOAC Performance Tested MethodSM No. 051301
Abstract
Here we describe results of a study to validate minor reagent
formulation changes to the Soleris Direct Yeast and Mold
(DYM) automated growth-based method for semi-quantitative
detection of yeast and mold in food products. In order to
reduce the maximum concentration of the selective agent
chloramphenicol in the Soleris reagents, chloramphenicol was
removed from the selective supplement and added to the vial
growth medium itself. Therefore, both the vial medium and
supplement have been reformulated in an alternative version of
the method. A probability of detection (POD) statistical model
was used to compare Soleris results at multiple test thresholds
(dilutions) with plate counts determined using the U.S. Food
and Drug Administration Bacteriological Analytical Manual
dilution plating procedure. Three matrixes were tested; yogurt,
tomato juice, and cocoa powder. POD analysis showed that the
percentage of positive Soleris tests at various test thresholds
were within the limits predicted by the reference method plate
counts for all matrixes evaluated. Real-time stability data on
three manufactured lots showed that the modified Soleris vial
and supplement are stable for at a minimum of 10 months when
stored at 2–8°C. In sum, results presented here demonstrate that
the modifications to the Soleris DYM vial and supplement do
not impact method performance. The modified Soleris DYM
method can be used as an accurate alternative to conventional
dilution plating procedures for semi-quantitative determination
of yeast and mold at threshold levels, while saving as much as
3 days in analysis time.
Participants
Method Authors
Susan Alles, Susan McDougal, Oscar Caballero1,
Mark Mozola, and Jennifer Rice
Neogen Corp., 620 Lesher Pl, Lansing, MI 48912
Submitting Company
Neogen Corp., 620 Lesher Pl, Lansing, MI 48912
Reviewer
Yi Chen
U.S. Food and Drug Administration, Center for Food Safety
and Applied Nutrition, 5100 Paint Branch Pkwy, College Park,
MD 20740
Submitted for publication May 4, 2015.
The method was independently tested, evaluated, and certified by
the AOAC Research Institute as a Performance Tested MethodSM. See
http://www.aoac.org/testkits/steps.html for information on certification.
1
Corresponding author’s e-mail: ocaballero@neogen.com
DOI: 10.5740/jaoacint 15109
Scope of Method
Target organisms.—Yeasts and molds.
Matrixes.—Nonfat dry milk, ice cream mix, salad dressing,
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yogurt, dried fruit, orange juice concentrate, tomato juice, saw
palmetto powder, corn flour, cocoa powder, cocoa butter, cocoa
liquor, dry pet food, and black pepper.
Summary of validated performance claims.—Statistically
equivalent to the U.S. Food and Drug Administration
Bacteriological Analytical Manual (FDA/BAM), Chapter 18,
dilution plating method (1) for semi-quantitative determination
of yeasts and molds in foods at threshold levels using a
probability of detection (POD) model.
Definitions
(a) POD (obs).—Observed POD for the test method;
number of test method positive results divided by the number of
portions tested, with 95% confidence interval.
(b) POD (pred).—POD for the test method predicted from
the reference method result. POD (pred.) = 1 – e–c, where e is
the base of the natural logarithm (value = 2.7183) and c is the
number of input CFU per test based on the reference method
result.
(c) Pass/Fail result.—If the POD (pred.) falls within the
lower and upper 95% confidence limits of the POD (obs), the
result is “Pass,” otherwise “Fail.”
(d) Soleris test threshold.—The detection limit of the Soleris
method determined by the volume and dilution of test sample
homogenate added to the Soleris vial. For example, a threshold
of >100 CFU/g represents 1 mL of a 1:100 dilution added to the
vial, equivalent to 0.01 g of original test sample.
Principle
Soleris DYM is a growth-based, automated method with an
optical endpoint (2). A dilution of the test sample homogenate
is inoculated directly into the test vial. The prepared vial is then
placed in the Soleris instrument set at the appropriate incubation
temperature. As yeasts and molds grow and metabolize, carbon
dioxide is generated, which diffuses from the growth medium
through a gas-permeable layer and into the indicator portion of
the Soleris vial. Dissolved carbon dioxide leads to the formation
of carbonic acid, reducing the pH and resulting in a change in
color of the chemical indicator over time. The color is monitored
by the instrument and a change in color of a certain magnitude,
determined by the Soleris software, is indicative of a positive
test result. The Soleris instrument contains temperature-
controlled incubation chambers and photodiode-based optical
detection devices for measurement of color changes in the
bottom indicator portion of the vial.
 Alles et al.: Journal of AOAC International Vol. 98, No. 5, 2015 1287
The Soleris DYM test is used in a “dilute-to-specification”
or threshold manner, in which the result is positive or negative
around a desired cutoff (in CFU/g) determined by the dilution
and volume of sample homogenate added to the vial. It is
assumed that 1 CFU introduced into the Soleris vial will lead
to a positive result.
General Information
Standard reference methods for detection and enumeration
of yeasts and molds in foods include the FDA/BAM dilution
plating technique and similar methods (1). These methods
typically require 5 days to obtain results. The Soleris DYM
method, through sensitive determination of metabolic activity
during microbial growth, produces results within 48 h for
most foods. This method was validated for a variety of foods:
nonfat dry milk, ice cream mix, salad dressing, yogurt, dried
fruit, orange juice concentrate, tomato juice, saw palmetto
powder, corn flour, cocoa powder, cocoa liquor, cocoa butter,
dry pet food and black pepper, and granted Performance Tested
Method℠ (PTM) status (PTM No. 051301; 3). The selective
supplement used with the vial contains chlortetracycline and
chloramphenicol to inhibit growth of bacteria. To present
users with an option in which the maximum concentration of
chloramphenicol in any reagent is reduced, an alternative vial/
supplement combination was developed. In this alternative
formulation, chloramphenicol is included in the vial medium,
with the supplement now containing only chlortetracycline.
This alternative vial/supplement formulation is the subject of
the current validation study.
Materials and Methods
Test Kit Information
(a) Test name.—Soleris® Direct Yeast and Mold, 9 mL
(b) Cat. No.—DYM-109 (vial and growth medium without
chloramphenicol), DYM-109C (vial and growth medium with
chloramphenicol).
(c) Ordering information.—Inside the United States.—
Neogen Corp., 620 Lesher Pl, Lansing, MI 48912, Tel: 517-
372-9200, Fax: 517-372-0108, Web site: www.neogen.com.
Outside the United States.—Contact above office for local
distributor information.
Supplies and Reagents
Supplies and reagents available from Neogen Corp.:
(a) Yeast and Mold Supplement (Product no. YI-110, for use
with vial DYM-109; or, product no. YI-110C, for use with vial
DYM-109C).—Four vials/package, 10 mL each. Store at 2–8°C.
(b) Soleris 32 (product no. BSX32) or Soleris 128 (product
no. BSX128) instrument.—Containing one or four temperature-
controlled (15–60° ± 0.5°C) incubator drawers, respectively,
with 32 locations in each drawer. Each test location contains
an LED-based optical sensor for measurement of changes
in absorbance over time. The system includes a dedicated
computer, monitor, and software.
(c) Soleris DYM vial.—Sterile growth medium (9 mL) in
a plastic vial, in boxes of 100 vials. One test sample per vial.
Store at 2–8°C. Requires use of the Soleris instrument.
Other required supplies:
(a) Stomacher®-type bags.—With filter (product No. 6827).
(b) Balance.—For weighing samples, minimum 100 g
capacity, ±0.1 g.
(c) Micropipettor and tips.—100–1000 µL.
(d) 200-proof ethanol.
Safety Precautions
Use of this test should be restricted to individuals with
appropriate laboratory training in microbiology. Reagents are
for laboratory use only. All pipetting transfers should be made
using either a disposable pipet and pipetting aid or micropipette
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with disposable tips. Refer to the Material Safety Data Sheet
available from Neogen Corp. for more information. Used Soleris
vials, sample homogenates and dilutions, and pipets or pipet
tips should be handled and disposed of as potentially infectious
material. The preferred method for disposal of contaminated
materials is autoclaving. Items that cannot be autoclaved may
be decontaminated by treatment with a disinfectant solution, for
example 10% bleach, followed by rinsing with water.
Preparation of Yeast and Mold Supplement
(a) For DYM-109 vial.—To one vial of Yeast and Mold
Supplement (YI-110), add 1.0 mL of 200-proof ethanol. Wet
powder well. Then add 9 mL sterile deionized water. Mix well.
Store in refrigerator up to 7 days after rehydration.
(b) For DYM-109C vial.—To one vial of Yeast and Mold
Supplement (YI-110C), add 10 mL sterile deionized water. Mix
well. Store in refrigerator up to 7 days after rehydration.
Sample Preparation
(a) Prepare a 1:10 sample homogenate by adding 225 mL
0.1% peptone water to 25 g food sample. Homogenize the
sample in a Stomacher for 2 min.
(b) Prepare dilutions of the sample homogenate to achieve
the desired test threshold, e.g., for >10 CFU/g, use sample
homogenate as is; for >100 CFU/g, prepare further 1:10 dilution
in 0.1% peptone water; for >1000 CFU/g, prepare further 1:100
dilution, etc.
(c) Add rehydrated Yeast and Mold Supplement to each vial,
0.60 mL for products containing a bacterial starter culture or
0.15 mL for all other foods.
(d) Add 1.0 mL of sample homogenate or dilution to the
Soleris vial. Cap the vial tightly and invert three times to mix.
Back off the vial cap to allow air exchange.
(e) Proceed to Soleris analysis.
General Preparation
The test should be performed under normal laboratory
conditions with respect to humidity, temperature, lighting, etc.
Soleris vials should not be used beyond their expiration date.
Soleris Analysis
Note: The Soleris system requires installation and user
training. Both are provided by Neogen Corp.
1288 Alles et al.: Journal of AOAC International Vol. 98, No. 5, 2015

Table 1. Comparative testing results and probability of detection calculations for the Soleris Direct Yeast and Mold method
Soleris result Observed PODf
Reference plate Soleris test Predicted No. vials No. vials
a b c d e g
Food type Inoculum count, CFU/g threshold, CFU/g CFU/vial POD positive tested LCL POD UCL Interpretation
Yogurt Candida famata >10 000 7.1 1.00 20 20 0.83 1.00 1.00 Pass
h 5
ATCC 60229 7.1 × 10 >100 000 7.1 0.99 20 20 0.83 1.00 1.00 Pass

>1 000 000 0.71 0.51 7 20 0.18 0.35 0.57 Pass

<10 >10 0 0 0 20 0 0 0.17 Pass

Tomato juice Penicillium citrinum >10 000 77 1.00 20 20 0.83 1.00 1.00 Pass
5
ATCC 34375 7.7 × 10 >100 000 7.7 1.00 20 20 0.83 1.00 1.00 Pass

>1 000 000 0.77 0.54 7 20 0.18 0.35 0.57 Pass

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<10 >10 0 0 0 20 0 0 0.52 Pass

Cocoa powder Aspergillus tamarii >100 000 39 1.00 20 20 0.83 1.00 1.00 Pass
6
ATCC 26950 3.9 × 10 >1 000 000 3.9 0.98 20 20 0.83 1.00 1.00 Pass

>10 000 000 0.39 0.32 10 20 0.30 0.50 0.70 Pass

<10 >10 0.1 0.1 0 20 0 0 0.17 Pass

a
Direct plating method: Bacteriological Analytical Manual, chapter 18, dilution plating method (1).
b
Dilute-to-specification approach. Positive result expected if organism concentration in test dilution is greater than Soleris test threshold.
c
CFU per Soleris vial based on reference method plate count.

Probability of detection (predicted): Based on reference method plate count (POD = 1 – e–c).
d

e
Detection times within 48 h indicate a positive result at the test threshold selected.
f
Probability of detection (observed): Fraction of Soleris results positive. LCL and UCL are 95% lower and upper confidence limits, respectively.
g
Test for equivalence of reference plate count and Soleris results. For “Pass”, POD (pred) must lie with POD (obs) LCL-UCL range.
h
American Type Culture Collection, Manassas, VA.

(a) In the Soleris software menu.—Select an incubator Internal Validation Studies

drawer. The incubation temperature should be set to 28 ± 0.5°C
for all foods.
(b) On the sample position grid.—Select the test (DYM) and
drag/drop to the selected sample position.
(c) On the sample queue screen.—Enter the following:
Sample identification number, and, if desired, production lot
number, plant information, and user name.
(d) Place the inoculated Soleris vials into the selected drawer
locations.
(e) Once all the samples are entered.—Select and click Start.
(f) Test samples will incubate for 48 h. A detection curve will
be generated in real time. The Soleris software will indicate a
positive test result. Positive results will generally be indicated in
less than 48 h. Note: If testing cocoa butter, set the test duration
to 60 h.
Interpretation of Results
(a) Negative criterion.—Tests producing no detection within
48 h are considered negative at the test threshold selected.
(b) Positive criterion.—Detection times within 48 h verified
by visual confirmation of indicator color change (blue-green to
yellow-green or yellow) indicates a positive result at the test
threshold selected.
Comparative Testing of Inoculated Foods
The Soleris DYM method was compared to the FDA/BAM
directing plating technique (1) for all foods tested. Inoculation
of foods was required in all three cases to achieve sufficiently
high levels for testing at multiple threshold levels. Inoculation
of test products was performed using pure cultures from the
American Type Culture Collection (Manassas, VA; Table 1).
Preparation of inocula.—Molds were grown on potato
dextrose agar plates for 5 days at 25°C or until plates were
completely dry. Candida famata was grown in tryptic soy
broth with yeast extract for 24–48 h at 30°C. Procedures for
preparation of mold inocula were adapted from those described
by Entis and Lerner (4).
Low-moisture product (cocoa powder).—Cocoa powder was
inoculated with Aspergillus tamarii. The mold culture plate was
placed in a plastic bag with approximately 1 kg of test product
and shaken. After equilibration for a minimum of 14 days at
room temperature, the mold level was determined by plate count
and the seeded product diluted with additional test product, if
necessary, to achieve desired levels.
High-moisture products (yogurt and tomato juice).—Yogurt
was inoculated with an appropriate dilution of an overnight
culture of Candida famata. Tomato juice was inoculated with
Penicillium citrinum. Growth was harvested from a culture
plate and a spore suspension was prepared in buffer (Maximum
Recovery Diluent or equivalent). The spore titer was determined
using a hemocytometer. An aliquot of an appropriate dilution
 Alles et al.: Journal of AOAC International Vol. 98, No. 5, 2015 1289
of the spore preparation was added to the product and mixed
thoroughly. Inoculated products were held for 48–72 h at 2–8°C
for equilibration of the inoculum prior to testing.
Sample preparation.—For each food type, a 1:10 sample
homogenate (25 g plus 225 mL buffer) was prepared in
accordance with the reference method. Further dilutions were
prepared for Soleris testing at three or more test thresholds and
for the reference method plate count. Soleris test thresholds
were chosen based on an estimate of the microbial load of the
test sample and with the goal of achieving fractional positive
results for at least one level, i.e., 5–15 positive results out of
20 replicate assays at a particular dilution. This ensures that the
Soleris method has been challenged at an inoculation level of
approximately 1 CFU/vial. A homogenate was also prepared
from uninoculated test material, and this homogenate was tested
at the >10 CFU/g threshold level.
Soleris testing.—For the Soleris method, 20 vials were
inoculated with 1 mL of dilution for each test threshold.
Duration of the Soleris test was 48 h for all foods.
Reference method plate counts.—The FDA/BAM dilution
plating method was performed using Dichloran rose bengal
chloramphenicol agar (or Dichloran 18% glycerol agar for
foods with aw < 0.95). The method was modified by plating
10 replicates of each dilution, rather than the specified triplicate
plating, in order to obtain a more accurate count. Standard
counting rules were followed, i.e., counts were based on plates
containing 30–300 colonies whenever possible.
Data analysis.—Results for each food type were analyzed
using a POD model. The predicted POD for the Soleris method
was calculated using the formula POD (pred.) = 1 – e–c, where
c = CFU/vial at a given test threshold based on the reference
method plate count result. The observed POD for the Soleris
method was calculated at each test threshold by dividing the
number of positive Soleris results by the number of portions
tested (20). The 95% confidence interval around POD (obs) was
calculated using a POD interval calculator (P. Wehling, personal
communication). If POD (pred) was within the confidence
interval of POD (obs), the Soleris and reference method results
were considered to be statistically not different.
Example:
Reference method result: 63 CFU/g
Soleris test threshold: >100 CFU/g
Soleris POD (pred.): 1 – e–0.63 = 0.467
Soleris POD (obs.): 9/20 = 0.450
Soleris POD (obs.) with confidence intervals: 0.258–0.658
Result: Pass
Results
Results are reported in Table 1. Reference method plate counts
for inoculated test products were 7.1 × 105 CFU/g for yogurt,
7.7 × 105 CFU/g for tomato juice, and 3.9 × 106 CFU/g for
cocoa powder. For all three foods, plate counts of uninoculated
product were <10 CFU/g.
All trials produced fractional positive results at one or more
test threshold levels with the Soleris method. Based on POD
analysis, the percentage of positive Soleris tests was within the
limits predicted by the reference method plate counts for all
three foods at all threshold levels tested.
Lot-to-Lot Consistency and Stability Testing
Real-time stability and lot-to-lot consistency testing were
conducted on three manufactured lots of the DYM-109C vial
and supplement. Results showed no evidence of a significant
increase in detection times with increasing age of the vials,
indicating that the vials are stable for at least 10 months from
date of manufacture when stored at the specified temperature
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of 2–8°C.
Discussion
The alternative vial and supplement formulation developed
for the Soleris DYM method produced expected results in
testing of three foods. In all cases the percentage of positive
Soleris results obtained was within the limits predicted by the
reference plating procedure. With the new vial/supplement
formulation, the maximum concentration of chloramphenicol
in any reagent is reduced compared to the original vial/
supplement formulation. The Soleris DYM method can be
used as an effective alternative to FDA/BAM reference plating
procedures for semi-quantitative determination of yeasts and
molds in foods. Use of the Soleris DYM method in a “dilute
to specification” mode allows users to match test thresholds
with product release specifications. Compared to direct plating
procedures, the Soleris method offers the advantages of
minimal labor and reduced analysis time; results are obtained in
48 h or less for most foods, compared to the 5 days required by
conventional plating methods.
Conclusions
It is recommended that the modification to the Soleris DYM
method to add the modified vial and supplement formulation
be approved for estimation of yeasts and molds at designated
threshold levels in nonfat dry milk, ice cream mix, salad
dressing, yogurt, dried fruit, orange juice concentrate, tomato
juice, saw palmetto powder, corn flour, cocoa powder, cocoa
liquor, cocoa butter, dry pet food, and black pepper.
References
(1) U.S. Food and Drug Administration (2001) Bacteriological
Analytical Manual Online, Chapter 18. http://www.fda.gov/
Food/FoodScienceResearch/LaboratoryMethods/ucm071435.
htm
(2) Neogen Corp. (2011) Soleris Operator’s Manual, Version 7
(3) Pereault, M., Alles, S., Caballero, O., Sarver, R., McDougal, S.,
Mozola, M., & Rice, J. (2014) J. AOAC Int 97, 1084–1091
(4) Entis, P. & Lerner, I. (1996) J. Food Prot. 59, 416–419