FOOD BIOLOGICAL CONTAMINANTS

High-Sensitivity Dry Rehydratable Film Method for

Enumeration of Coliforms in Dairy Products: Collaborative Study
CURIALE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO. 3, 1997
MICHAEL S. CURIALE, VIDHYA GANGAR, and ARMANDO DONORIO
Silliker Laboratories Group, Inc., Corporate Research Center, 160 Armory Dr, South Holland, IL 60473
SONYA GAMBREL-LENARZ and J. SUE MCALLISTER
3M Center 270-3N-04, St. Paul, MN 55144
Collaborators: B. Bailey; A.M. Bednar; B. Bowen; D. Brown; M. Bulthaus; J. Cash; M. Cirigliano; M. Cox; A. Donorio; O.F.
David; J. Fraser; K. Frye; V. Gangar; S. Gambrel-Lenarz; J. Hanlin; T. Helbig; J. Johnson; K. Jost-Keating; L. Kora; R.
Koeritzer; S. Kozlowski; M. Kraemer; S. Lally; B. Lambeth; K. Lawlor; V. Lewandowski; S. Lopez; S. McDonald; S.
McIntyre; M. Naq; M. Pierson; J. Reinhard; D. Richter; L. Saunders; P. Simpson; L. Smoot; M.S. Tong; D. Warburton; H.
Williams; A. Wilson-Perry; J. Yuan

A dry-film coliform count plate that is inoculated

with 5 mL sample was compared with the Violet
Red Bile Agar plate method in a collaborative study
by 18 laboratories. Products analyzed were 2%
milk, chocolate milk, cream, vanilla ice cream, cottage cheese, and cheese. Collaborators tested
blind duplicate uninoculated samples and samples
inoculated at low, medium, and high level. Significantly (P < 0.05) higher numbers of coliforms were
recovered by the dry-film method from 2% milk
samples at the 3 inoculum levels, the chocolate
milk at the low- and high-inoculum levels, and the
cream at the high-inoculum level. Significantly
higher counts were obtained by the agar method
for cottage cheese samples at the low-inoculum
level. The repeatability standard deviation for the
dry-film method was significantly higher for the
high-inoculum level chocolate milk sample and the
medium-inoculum level cottage cheese. The same
statistic was significantly higher for the agar
method at all 3 inoculum levels in the 2% milk and
the medium-inoculum level cream. The high-sensitivity dry rehydratable film method for enumeration
of coliforms in dairy products has been adopted
first action by AOAC INTERNATIONAL.

airy products are monitored routinely for coliform bacteria. The presence of these organisms indicates possible postprocessing contamination (1). The traditional

Submitted for publication August 25, 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
(1996) J. AOAC Int. 79, 42A, and Official Methods Board Actions (1996)
The Referee, March issue.

method for detection and enumeration is the Violet Red Bile

Agar (VRBA) plating method (1). The sensitivity of this
method is as low as 1 coliform colony-forming unit per gram
(cfu/g) by direct plating of liquid samples or by spreading
10 mL of a 1:10 dilution across 3 plates for other product types.
Another test method is the Petrifilm Coliform Count (PCC)
method (2). This method is as productive as the VRBA method
for low coliform levels. The PCC plate is hydrated with 1 mL
sample. To accommodate 10 mL sample, as used in the VRBA
method, 10 plates are required. This is impractical. The Petrifilm High-Sensitivity Coliform Count (PHSCC) plate was developed to handle larger sample volumes. The plate receives
5 mL of a sample dilution to achieve a sensitivity of 1 coliform
cfu/g when a 1:5 dilution is used, 2 cfu/g when a 1:10 dilution
is used, or 0.2 cfu/g when fluid milk is plated directly.
In a precollaborative trial of 20 dairy foods inoculated with
coliforms at 3 levels, the PHSCC method proved to be as efficient as the VRBA method (Curiale, unpublished data).
Collaborative Study

Test Products
The 6 products selected for the study2% milk, chocolate
milk, cream, vanilla ice cream, cottage cheese, and cheddar
cheeserepresent dairy product types commonly analyzed for
coliforms. These products were purchased at local retail outlets.
Each product consisted of a single production lot. All products
retained for the study had a coliform count of <1 cfu/g.

Preparation of Test Samples

A different coliform strain was selected for each product.
The organisms were obtained from the Silliker Research Laboratory (SLR) collection, where each is stored in 15% glycerol
at 70C. Citrobacter freundii SLR581, Enterobacter aerogenes SLR41, Klebsiella oxytoca SLR591, Klebsiella pneumonia SLR613, Escherichia coli SLR57, and Enterobacter agglomerans SLR584 were used for 2% milk, chocolate milk,

cream, ice cream, cottage cheese, and cheddar cheese, respectively. Test samples were prepared by inoculating them with a
wet suspension of cells. Cells were indirectly stressed in certain
products by the conditions of product storage or product acidity.
Working cultures were maintained on Eosin Methylene
Blue agar plates (BBL), and the inoculum was cultivated in
nutrient broth (NB; BBL) incubated at 25C. Stationary-phase
cultures stored overnight at 4C were diluted with Butterfields
phosphate diluent for inoculation of test products.
(a) 2% milk, chocolate milk, and cream.Approximately
4800 g of each product was obtained and mixed thoroughly by
shaking. The mixed product was divided into 4 equal portions,
the first 3 portions were inoculated, and the fourth portion
served as uninoculated control. The inoculum was added directly to the samples, and the inoculated sample was mixed
thoroughly by shaking. Target inoculation levels differed by powers of 10: 110 cells/mL for low inoculum, 11100 cells/mL for
medium inoculum, and 1011000 cells for high inoculum.
(b) Vanilla ice cream.Several one-half gallon containers
of ice cream were thawed and combined by mixing with a
mixer. The product was divided into 4 portions and inoculated
according to method specified above.
(c) Cottage cheese.Several quart containers of cottage
cheese were combined and mixed thoroughly with a mixer. The
product was divided into 4 portions and inoculated according
to method specified above.
(d) Cheddar cheese.High-moisture cheese was grated
and wet inoculated with a coliform culture for each of the specified inoculation levels.

reported as below a specific value were used in statistical analysis by converting them to the value without the less than sign.
Repeatability (sr) and reproducibility (sR) standard deviations,
relative standard deviations of repeatability (RSDr) and reproducibility (RSDR), and Cochran and Grubbs tests for outliers
were performed according to AOAC procedures (4). Outliers
were excluded from analysis. Correlated repeatability variances
were compared by a Pitman-Morgan style analysis (5).
996.02 Coliform Count in Dairy Products,
High-Sensitivity Dry Rehydratable Film Method
First Action 1996
(Applicable to determination of coliform count in dairy
products.)
Caution: See Appendix for Laboratory Safety for Safe
Handling of Microorganisms.
Method Performance:
See Table 996.02 for method performance data.

A. Principle
Method uses bacterial culture plates of dry medium and cold
H2O-soluble gel. Undiluted or diluted samples are added to
plates at 5.0 mL/plate. Pressure, when applied to plastic
spreader placed on overlay film, spreads samples over ca
62 cm2 growth area. Gelling agent is allowed to solidify, plates
are incubated, and coliforms are counted.

B. Apparatus and Reagent

Distribution of Samples to Collaborators
Inoculated and uninoculated portions were divided into ca
60 g samples. Each 60 g sample was divided in half to form
duplicate subsamples. For each product to be analyzed, duplicate subsamples of the uninoculated control and of each of the
3 inoculation levels were prepared for each collaborator. Samples were packaged in leak-proof containers and identified by
product date, analysis date, and sample code. The sample code
consisted of a number from 1 to 8 for each laboratory.
Samples were shipped by overnight carrier. Milk, cream,
cottage cheese, and cheese were packed with ice packs to maintain a temperature between 4 and 7C during shipment. Ice
cream was shipped frozen. One product type was shipped each
week of the study.

Microbiological Analysis of Test Samples

Each collaborator received 8 samples of each product to be
tested. Each sample consisted of 25 to 30 g material, sufficient
product for a single analysis by the PHSCC and VRBA methods. The methods were described in detail in an instruction
manual sent to each collaborating laboratory.

Data Analysis
Colony counts and cfu/g were recorded on data sheets provided by the Associate Referee. Procedures described by Peeler
and Maturin (3) were used to calculate cfu/g. The logarithms of
the plate count were used for all statistical calculations. Counts

(a) High-sensitivity coliform count plates.Plates contain

violet red bile nutrients conforming to APHA standards as
given in Compendium of Methods for Microbiological Examination of Foods, 3rd ed., 1992 (American Public Health Association, 1015 18th St, NW, Washington, DC), cold H2O-soluble
gelling agent, and 2,3,5-triphenyltetrazolium chloride indicator. Petrifilm High-Sensitivity Coliform Count (available from
Medical-Surgical Division/3M, St. Paul, MN 55144), or
equivalent product producing similar method performance statistics, meets these specifications.
(b) Plastic spreader.Provided with Petrifilm plates, designed to spread sample evenly over plate growth area.
(c) Pipets.Calibrated for bacteriological use. Automatic
pipet to deliver 5.0 mL may be used.
(d) Colony counter.Standard apparatus, Quebec model
preferred or one providing equivalent magnification (1.5) and
visibility (darkfield with backlight).
(e) Dilution water.Prepare stock solution as follows: Dissolve 34 g KH2PO4 in 500 mL H2O, adjust to pH 7.2 with 1N
NaOH (ca 175 mL), and dilute to 1 L with H2O. Prepare buffered H2O for dilutions by diluting 1.25 mL stock solution to 1 L
with boiled and cooled H2O. Autoclave 15 min at 121C.
(f) Stomacher.

C. Preparation of Test Sample

Specified dilutions are for maximum sensitivity. Higher dilutions may be plated as needed. Do not use diluents contain-

ing citrate or thiosulfate. Mix all dilutions by shaking 25

through 30 cm arc in 7 s. Stomach or blend solid samples
2 min to homogenize.
(a) Cream, half-and-half, condensed milk, egg nog, cottage
cheese, and related products.Make 1:5 dilution
(24.75 g/99 mL dilution H2O). Plate 5 mL on dry-film coliform
count plate. Colony count on plate is count/g.
(b) Sour cream and dipsProceed as in (a). After dilution,
adjust pH to 6.67.2 with 1N NaOH (ca 0.1 mL/g sample). Colony count on plate is count/g.
(c) Butter, margarine, and cheese.Make 1:10 dilution
(11 g/99 mL dilution H2O) with diluent prewarmed to 40
45C. Plate 5 mL on dry-film coliform count plate. Multiply
colony count by 2 to obtain count/g. Do not use citrate buffer
to homogenize sample.
(d) Buttermilk and yogurt.Proceed as in (c). After dilution, adjust pH to 6.67.2 with 1N NaOH (ca 0.1 mL/g sample).
Colony count on plate is count/g.
(e) Ice cream, sherbet, frozen yogurt, and mixes.Proceed
as in (a). Colony count on plate is count/g.
(f) Chocolate milk.Proceed as in (a). Colony count on
plate is count/g.
(g) Whole milk, 2% milk, 1% milk, and skim milk.Plate
5 mL on dry-film coliform count plate. Multiply colony count
by 0.2 to obtain count/g.

D. Analysis
Place dry-film coliform count plate on a flat surface. Lift top
film and inoculate 5 mL sample onto center of film base. Carefully place top film down on inoculum. Distribute sample over
prescribed growth area with downward pressure in center of
plastic spreader. Leave plate undisturbed 25 min to allow gel to
solidify. Place plates in incubator in horizontal position, clear side up,
in stacks not exceeding 20 units. Incubate plates 24 2 h at 32C.
Count plates promptly after incubation period. Magnifier
illuminator may be used to facilitate counting. Coliforms appear as red colonies that have one or more gas bubbles associated with them (within 1 colony diameter). Count all colonies
in countable range (15150 colonies). Red colonies without
gas bubbles are not counted as coliform organisms.
To calculate coliform count, multiply total number of coliform colonies/plate (or average number of colonies/plate, if
counting duplicate plates of same dilution) by appropriate dilution factor. When counting colonies on duplicate plates of consecutive dilutions, calculate mean number of colonies for each
dilution before determining average bacterial count.
Estimated counts can be made on plates with >150 colonies
and should be reported as estimated counts. In making such
counts, circular growth area can be considered to contain ca
62 cm2. To isolate colonies for further identification, lift top
film and pick colony from gel.
Ref.: J. AOAC Int. 80, 505516(1997)
Results and Discussion
Eighteen laboratories participated in the study (Table 1).
The laboratories decided which of the 6 food products they

would analyze on the basis of interest and available time. Each

of the 6 foods was analyzed in at least 15 laboratories. The
logarithms of the coliform counts for the test samples are presented in Tables 2 through 7, and the repeatability and reproducibility statistics for the methods are shown in Table 8. None
of the laboratories reported difficulties with either test method
or test materials, other than those cited under each product in
the following discussion.

2% Milk
Milk samples were sent to 15 laboratories for analysis (Table 2). Coliforms were not detected in the control sample. The
lower values from the HSPCC method (<0.698) compared with
the VRBA method (<0.000) are due to the different sensitivities
of the methods. A 5-fold greater volume of milk was plated for
the HSPCC method. Thus upon direct plating, the sensitivity of
the HSPCC method was <0.2 cfu/g, whereas the sensitivity of
the VRBA method was <1 cfu/g.
Significantly higher mean log counts were obtained for all
inoculated samples with the HSPCC method (Table 8). The sr
values of the VRBA method were significantly higher for all
inoculum levels, indicating the method was more variable than
the HSPCC method. RSDr values ranged from 2.9 to 8.4% and
from 6.9 to 30.3% for the HSPCC and VRBA methods, respectively. The sR values were higher for the VRBA method, and
the RSDR values ranged from 4.5 to 14.9% for the HSPCC
method, and from 8.5 to 36.7% for the VRBA method. RSDr
and RSDR values were inversely related to the inoculum level.
The highest variability was observed at the low-inoculum level
(110 cells/mL), and the lowest variability occurred at the
high-inoculum level (1011000 cells/mL).

Chocolate Milk
Chocolate milk samples were sent to 16 laboratories for
analysis (Table 3). Laboratory 13 reported a deviation in plating the low- and medium-inoculum level inoculated samples
by the VRBA method. Consequently, the data were disqualified
for comparison.
Significantly higher mean log counts were obtained for the
low- and high-level inoculum samples by the HSPCC method
(Table 8). The sr value of the HSPCC method was significantly
higher for the high-inoculum level compared with the VRBA
method. RSDr values ranged from 4.4 to 39.4% and from 2.1
to 35.1% for the HPSCC and VRBA methods, respectively. The
sR values were higher for the VRBA method, and the RSDR
values ranged from 4.5 to 39.4% and from 6.6 to 58.3% for the
HPSCC and VRBA methods, respectively. RSDr and RSDR
values were inversely related to the inoculum level.

Cream
Cream samples were sent to 17 laboratories for analysis (Table 4). Laboratory 12 did not complete the coliform confirmation procedure for the low- and medium-level inoculated samples for the VRBA method. Consequently, the data were
disqualified for comparison.
Significantly higher mean log counts were obtained for the
high-level inoculated samples with the HSPCC method (Ta-

ble 8). The sr value of the VRBA method was significantly

higher for the medium-inoculum level, indicating the method
was more variable than the HSPCC method. The RSDr values
ranged from 2.8 to 39.9% and from 2.1 to 41.9% for the
HSPCC and VRBA methods, respectively. The sR values were
higher for the VRBA method at the medium- and high-inoculum levels and lower at the low-inoculum level, in comparison
with the HSPCC method. RSDR values ranged from 3.5 to
39.9% and from 4.1 to 41.9% for the HPSCC and VRBA methods, respectively. RSDr and RSDR values were inversely related to the inoculum level.
The high-sensitivity dry-film coliform count procedure was
as productive as or more productive than the agar method. For
15 of 17 comparisons, the mean coliform count was higher by
the dry-film procedure. The difference was significant (P < 0.05)
for 6 comparisons. For 2% milk, the mean coliform count was
significantly higher and the repeatability was better at all 3 inoculum levels by the dry-film method. Differences for the other
study products and inoculation levels did not follow a pattern,
indicating the dry-film method is a suitable replacement for the
agar plate method.

Ice Cream
Vanilla ice cream samples were sent to 16 laboratories for
analysis (Table 5). Laboratory 6 did not receive a sufficient
quantity of a control sample to complete the analyses according
to method instructions. Consequently, the data were disqualified for comparison.
Higher mean log counts for the medium- and high-level inoculated samples were obtained with the HSPCC method than
with the VRBA method (Table 8). The sr values for the VRBA
method did not differ significantly from those for the HSPCC
method. RSDr values ranged from 4.6 to 138.1% and from 5.0
to 108.3% for the HPSCC and VRBA methods, respectively.
The sR values were higher for the VRBA method at the low-inoculum level and lower at the medium- and high-inoculum levels, in comparison with the HSPCC method. RSDR values
ranged from 9.0 to 141.2% and from 8.7 to 147.1% for the
HPSCC and VRBA methods, respectively. RSDr and RSDR
values were inversely related to the inoculum level.

Cottage Cheese
Cottage cheese samples were sent to 18 laboratories for
analysis (Table 6). Results for the HSPCC analysis of the lowlevel samples included a statistical outlier according to the Cochran test. These results (laboratory 9) were disqualified for
comparison.
Significantly higher mean log counts were obtained for the
low-inoculum samples with the VRBA method (Table 8). The
sr value for the HSPCC method was significantly higher for the
medium-inoculum level, indicating a greater degree of variability in comparison with the VRBA method. RSDr values
ranged from 3.4 to 9.8% and from 2.7 to 5.9% for the HPSCC
and VRBA methods, respectively. The sR values were higher
for the HSPCC method for all inoculum levels. The RSDR values ranged from 3.7 to 13.0% and from 3.0 to 6.3% for the

HPSCC and VRBA methods, respectively. RSDr and RSDR

values were inversely related to the inoculum level.

Cheddar Cheese
Cheddar cheese samples were sent to 15 laboratories for
analysis (Table 7). Mean log counts and repeatability standard
deviations for the medium- and high-level inoculated samples
were not significantly different for the comparison of HSPCC
and VRBA methods (Table 8). The very low numbers of coliforms isolated from the low inoculum samples and the differences in sensitivity precluded statistical comparisons of the
means and sr values. RSDr values ranged from 2.3 to 17.7% and
from 3.1 to 416.0% for the HPSCC and VRBA methods, respectively. The sR values were higher for the VRBA method at
the low- and high-inoculum levels and lower at the medium-inoculum level when compared with the HSPCC method. RSDR
values ranged from 7.2 to 17.7% and from 9.5 to 416.0% for
the HPSCC and VRBA methods, respectively. High RSDr and
RSDR values for the VRBA method at the low inoculation level
reflect the high count obtained by one collaborator. Because
few coliforms were recovered by either method from these
samples, statistical evaluation of low-level samples did not represent a realistic measure of method performance. RSDr and
RSDR values were inversely related to the inoculum level.
Recommendation
On the basis of the results, it is recommended that the highsensitivity dry rehydratable film method for enumeration of
coliforms in dairy products be adopted first action.
Acknowledgments
The assistance of the following individuals is gratefully acknowledged:
David Brown and Shirley Kozlowski, Cornell University,
Ithaca, NY
Tom Helbig, Bud Lambeth, John Reinhard, and Phil Simpson, Mid-America Dairyman, Springfield, MO
Barb Bailey and Judy Fraser, Hagen-Dazs, Minneapolis,
MN
Mary Bulthaus and Julie Johnson, Dairy Quality Control
Institute, St. Paul, MN
Bruce Bowen and Don Warburton, Health and Welfare Canada, Bureau of Microbial Hazards, Ottawa, Ontario, Canada
Robert Koeritzer, Vickie Lewandowski, and Lisa Saunders,
Land OLakes, Inc., Minneapolis, MN
K. Lawlor, Merle Pierson, and H. Williams, Virginia Tech,
Blacksburg, VA
Maria Kraemer, Sara Lopez, Sheri McIntyre, and Malini
Naq, Dole, San Jose, CA
Adele M. Bednar and Mike Cirigliano, Unilever/Lipton,
Inc., Englewood, NJ
Les Smoot and M.S. Tong, Nestle USA Quality Assurance
Laboratory, Dublin, OH
Sandy McDonald, 3M Company, St. Paul, MN
Jim Cash and Orlando F. David, Borden, Inc., Gadsten, AL

John Hanlin, Althea Wilson-Perry, and James Yuan, Campbell Soup Co., Camden, NJ
Kathy Jost-Keating, Silliker Laboratories of New Jersey,
Garwood, NJ
Lori Kora, J.R. Simplot, Caldwell, ID
Dawn Richter, Ore-Ida Foods, Ontario, OR
Mary Cox, Kerry Frye, and Stephan Lally, Crowley Foods,
Inc., Arkport, NY
References
(1) Christen, G.L., Davidson, P.M., McAllister, J.S., & Roth,
L.A. (1992) in Standard Methods for the Examination of

(2)
(3)

(4)
(5)

Dairy Products, American Public Health Association, Washington, DC, pp. 247270
Curiale, M.S., Fahey, P., Fox, T.L., & McAllister, J.S. (1989)
J. Assoc. Off. Anal. Chem. 72, 312318
Peeler, J.T., & Maturin, L.J. (1992) in Bacteriological Analytical Manual 7th Ed., AOAC INTERNATIONAL,
Arlington, VA, pp. 1726
Statistics reference (1989) J. Assoc. Off. Anal. Chem. 72,
694704
Snedecor, G.W., & Cochran, W.G. (1980) Statistical Methods, 7th Ed., Iowa State University Press, Ames, IA

Table 996.02. Method performance for determination of coliforms in dairy products by high-sensitivity dry
rehydratable film method
Product

Level

Mean log,
cfu/g

sr

sR

2% milk

Low
Medium
High
Low
Medium
High
Low
Medium
High
Low
Medium
High
Low
Medium
High
Low
Medium
High

0.681
1.576
2.437
0.612
1.637
2.602
0.638
1.661
2.679
0.147
1.098
1.899
1.420
2.410
3.361
0.311
1.633
3.122

0.058
0.067
0.071
0.241
0.088
0.114
0.255
0.046
0.081
0.203
0.146
0.087
0.139
0.112
0.115
0.055
0.105
0.071

0.101
0.082
0.109
0.241
0.090
0.116
0.255
0.071
0.095
0.208
0.178
0.170
0.185
0.131
0.125
0.055
0.180
0.226

Chocolate milk

Cream

Vanilla ice cream

Cottage cheese

Cheddar cheese

a
b

r = 2.8 sr.
R = 2.8 sR.

RSDr, %

RSDR,%

8.4
4.3
2.9
39.4
5.4
4.4
39.9
2.8
3.0
138.1
13.3
4.6
9.8
4.7
3.4
17.7
6.4
2.3

14.9
5.2
4.5
39.4
5.5
4.5
39.9
4.3
3.5
141.2
16.2
9.0
13.0
5.4
3.7
17.7
11.0
7.2

ra

Rb

0.162
0.188
0.199
0.675
0.246
0.319
0.714
0.129
0.227
0.568
0.409
0.244
0.389
0.314
0.322
0.154
0.294
0.199

0.283
0.230
0.305
0.675
0.252
0.325
0.714
0.199
0.266
0.582
0.498
0.476
0.518
0.367
0.35
0.154
0.504
0.633

Table 1. Participation in collaborative study by

laboratories
2% Chocolate
Vanilla ice Cottage
Laboratory Milk
milk
Cream cream
cheese

Cheddar
cheese
spread

1
2

+
+

+
+

+
+

+
+

+
+

+
+

3
4

+
+

+
+

+
+

+
+

+
+

5
6
7
8
9
10
11
12
13
14
15
16

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

17

18

Table 2. Analysis of 2% milk by high-sensitivity dry-film (HBPCC) and Violet Red Bile Agar (VRBA) procedures
Control
HSPCC
Collaborator
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

Low inoculum
VRBA

HSPCC

Medium inoculum
VRBA

HSPCC

High inoculum

VRBA

HSPCC

VRBA

No. 7

No. 8

No. 7

No. 8

No. 1

No. 2

No. 1

No. 2

No. 3

No. 4

No. 3

No. 4

No. 5

No. 6

No. 5

No. 6

<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699

<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699
<0.699

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

0.602
0.699
0.602
0.602
0.778
0.699
0.602
0.699
0.699
0.699
0.602
0.699
0.699
0.602
0.954

0.477
0.602
0.778
0.602
0.778
0.778
0.602
0.699
0.699
0.778
0.602
0.602
0.602
0.699
0.903

0.699
0.477
0.477
0.000
0.477
0.477
0.301
0.477
0.699
0.699
0.699
0.699
0.477
0.699
0.477

0.477
0.845
0.000
0.301
0.477
0.699
0.699
0.602
0.699
0.699
0.602
0.699
0.477
0.477
0.602

1.640
1.480
1.560
1.460
1.460
1.660
1.640
1.560
1.680
1.560
1.580
1.410
1.680
1.570
1.530

1.650
1.560
1.680
1.410
1.660
1.600
1.560
1.700
1.620
1.480
1.660
1.510
1.560
1.610
1.560

1.320
1.360
1.110
0.954
1.230
1.570
1.230
1.320
1.110
1.320
1.320
1.360
1.080
1.510
1.040

1.230
1.540
1.540
1.340
1.300
1.480
1.260
1.200
1.450
1.460
1.460
1.080
1.110
1.610
1.000

2.410
2.380
2.340
2.200
2.480
2.480
2.680
2.560
2.320
2.340
2.480
2.400
2.480
2.340
2.450

2.400
2.430
2.430
2.230
2.640
2.430
2.490
2.510
2.320
2.570
2.580
2.480
2.450
2.380
2.430

1.840
2.300
2.200
1.920
2.280
2.200
2.280
1.860
2.380
2.260
2.320
2.110
2.450
2.410
1.790

2.080
2.300
2.360
2.300
2.110
2.260
2.040
1.780
2.180
2.110
2.200
2.000
2.180
2.260
2.200

Table 3. Analysis of chocolate milk by high-sensitivity dry-film (HBPCC) and Violet Red Bile Agar (VRBA) procedures
Control
HSPCC
Collaborator
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
a

Low inoculum
VRBA

HSPCC

Medium inoculum
VRBA

HSPCC

High inoculum

VRBA

HSPCC

VRBA

No. 2

No. 8

No. 2

No. 8

No. 3

No. 6

No. 3

No. 6

No. 1

No. 5

No. 1

No. 5

No. 4

No. 7

No. 4

No. 7

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

0.903
0.699
0.602
0.778
<0.000
0.602
0.301
0.699
0.602
0.699
0.778
0.845
a a

0.477
0.845
0.903

0.602
0.602
0.477
0.477
0.845
0.301
0.699
0.477
0.699
0.301
0.699
0.845

0.602
0.301
0.699

0.602
0.477
0.477
<0.000
0.477
0.602
<0.000
0.699
0.000
0.699
0.602
0.602

0.477
0.699
0.477

0.602
0.477
0.000
0.301
0.699
0.301
0.000
0.477
0.000
0.699
0.845
0.602

0.778
0.477
0.778

1.520
1.760
1.670
1.540
1.630
1.710
1.620
1.610
1.450
1.600
1.660
1.480

1.620
1.660
1.540

1.750
1.620
1.660
1.720
1.720
1.660
1.810
1.770
1.560
1.490
1.740
1.620

1.640
1.670
1.610

1.610
1.560
1.460
1.260
1.680
1.510
1.380
1.690
1.080
1.630
1.580
1.620

1.510
1.320
1.430

1.540
1.660
1.700
1.590
1.450
1.530
1.360
1.760
1.110
1.710
1.860
1.680

1.510
1.410
1.430

2.490
2.600
2.630
2.450
2.720
2.600
2.560
2.720
2.570
2.540
2.790
2.660
2.950
2.600
2.690
2.490

2.600
2.650
2.570
2.590
2.490
2.630
2.540
2.810
2.410
2.580
2.730
2.510
2.460
2.620
2.540
2.480

2.530
2.600
2.530
2.360
2.490
2.510
2.320
2.580
2.040
2.460
2.620
2.640
2.300
2.460
2.400
2.280

2.410
2.600
2.400
2.260
2.360
2.540
2.300
2.660
2.000
2.430
2.630
2.600
2.380
2.460
2.430
2.380

= laboratory error.

Table 4. Analysis of cream by high-sensitivity dry-film and Violet Red Bile Agar (VRBA) procedures
Control
HSPCC
Collaborator
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
18
a

Low inoculum
VRBA

HSPCC

Medium inoculum
VRBA

HSPCC

High inoculum

VRBA

HSPCC

VRBA

No. 5

No. 6

No. 5

No. 6

No. 1

No. 8

No. 1

No. 8

No. 3

No. 7

No. 3

No. 7

No. 2

No. 4

No. 2

No. 4

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

0.778
0.699
0.602
0.699
0.845
0.699
0.699
0.903
0.602
0.699
<0.000
a a

0.903
0.903
0.602
<0.000
0.845

0.778
0.301
0.602
0.954
0.477
0.954
0.477
0.602
0.602
0.477
0.699

0.602
0.301
0.699
0.699
0.699

0.699
0.602
0.602
0.000
0.602
0.778
0.602
0.699
0.301
0.699
0.778

0.699
0.477
0.477
0.699
0.699

0.845
0.301
0.699
0.602
0.301
0.000
0.845
0.602
0.699
0.602
0.477

0.602
0.602
0.000
0.602
0.778

1.730
1.610
1.560
1.570
1.760
1.720
1.640
1.670
1.600
1.710
1.760

1.670
1.600
1.660
1.570
1.660

1.720
1.640
1.610
1.540
1.720
1.680
1.620
1.750
1.680
1.590
1.750

1.750
1.750
1.700
1.530
1.620

1.730
1.430
1.200
1.410
1.610
1.760
1.510
1.610
1.610
1.680
1.700

1.610
1.570
1.400
1.600
1.610

1.510
1.510
1.730
1.540
1.490
1.540
1.680
1.620
1.540
1.480
1.750

1.620
1.580
1.520
1.710
1.570

2.720
2.620
2.640
2.710
2.660
2.820
2.730
2.720
2.600
2.670
2.650
2.490
2.720
2.530
2.580
2.700
2.720

2.760
2.640
2.630
2.620
2.750
2.640
2.760
2.900
2.540
2.570
2.800
2.630
2.900
2.700
2.670
2.580
2.700

2.590
2.400
2.540
2.380
2.450
2.620
2.630
2.460
2.490
2.640
2.600
2.630
2.460
2.570
2.300
2.590
2.560

2.540
2.320
2.610
2.400
2.580
2.610
2.720
2.580
2.480
2.590
2.580
2.480
2.580
2.590
2.300
2.580
2.530

= incomplete analyses.

Table 5. Analysis of vanilla ice cream by high-sensitivity dry-film (HSPCC) and Violet Red Bile Agar (VRBA) procedures
Control
HSPCC
Collaborator
1
2
4
5
6
7
8
9
10
11
12
13
14
15
16
17
a

Low inoculum
VRBA

HSPCC

Medium inoculum
VRBA

HSPCC

High inoculum

VRBA

HSPCC

VRBA

No. 4

No. 5

No. 4

No. 5

No. 2

No. 7

No. 2

No. 7

No. 1

No. 8

No. 1

No. 8

No. 3

No. 6

No. 3

No. 6

<0.000
<0.000
<0.000
<0.000
a a

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
0.000
<0.000

<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

0.000
<0.000
<0.000
<0.000
0.000
0.301
<0.000
0.301
0.000
0.000
0.301
0.000
<0.000
<0.000
<0.000
0.000

0.477
0.000
0.301
0.301
<0.000
0.477
<0.000
<0.000
0.477
0.301
0.699
0.000
0.301
0.000
0.477
0.000

0.477
<0.000
<0.000
0.000
0.000
<0.000
<0.000
<0.000
0.602
0.000
0.477
<0.000
<0.000
<0.000
<0.000
<0.000

0.301
0.301
0.477
<0.000
0.477
0.000
<0.000
<0.000
0.699
0.477
0.602
0.477
0.000
0.000
0.000
<0.000

0.699
1.110
1.200
1.150
1.200
0.954
0.699
1.180
1.150
1.300
0.903
1.300
1.200
1.200
1.110
0.903

1.000
1.180
1.180
1.150
1.260
0.778
1.150
1.040
1.380
1.200
1.000
0.845
1.200
1.380
1.110
1.040

1.040
1.040
1.280
1.000
1.320
0.699
1.000
0.954
1.200
1.230
1.040
1.040
1.200
0.954
0.778
0.845

1.000
1.180
1.180
1.150
1.150
0.845
1.110
1.040
1.400
1.300
1.150
0.845
0.845
1.110
0.845
0.845

1.860
1.940
2.180
1.900
2.000
1.770
1.910
1.760
1.880
2.080
1.560
1.680
2.180
1.910
1.860
1.720

1.850
2.080
2.040
1.860
1.910
1.760
1.920
1.540
2.000
2.110
1.760
1.860
2.150
2.150
1.850
1.730

1.840
2.000
1.960
1.900
2.150
1.450
1.810
1.730
1.990
2.000
1.810
1.730
1.760
1.830
1.810
1.750

1.800
1.970
1.950
1.850
1.990
1.650
1.880
1.410
1.980
2.040
1.910
1.790
2.000
1.900
1.930
1.670

Incomplete analysis.

Table 6. Analysis of cottage cheese by the high-sensitivity dry-film (HSPCC) and Violet Red Bile Agar (VRBA) procedures
Control
HSPCC
Collaborator
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
a

Low inoculum
VRBA

HSPCC

Medium inoculum
VRBA

HSPCC

High inoculum

VRBA

HSPCC

VRBA

No. 2

No. 6

No. 2

No. 6

No. 1

No. 8

No. 1

No. 8

No. 3

No. 5

No. 3

No. 5

No. 4

No. 7

No. 4

No. 7

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

1.110
1.080
1.510
1.700
1.650
1.430
1.180
1.260
a a

1.510
1.380
1.280
1.600
1.490
1.530
1.560
1.460
1.530

1.400
0.903
1.640
1.380
1.540
1.560
1.540
1.510

1.200
1.380
1.230
1.610
1.200
1.430
1.530
1.520
1.460

1.680
1.360
1.570
1.460
1.730
1.520
1.610
1.510

1.540
1.510
1.490
1.430
1.510
1.400
1.620
1.540
1.620

1.610
1.360
1.490
1.570
1.410
1.360
1.680
1.640

1.480
1.430
1.580
1.560
1.480
1.530
1.410
1.510
1.600

2.430
2.400
2.490
2.480
2.360
2.530
2.300
2.260
2.520
2.180
2.410
2.360
2.540
2.530
2.400
2.560
2.450
2.610

2.400
2.260
2.410
2.450
2.570
2.510
2.630
2.340
2.520
2.080
2.400
2.180
2.430
2.340
2.480
2.280
2.180
2.480

2.320
2.380
2.340
2.480
2.450
2.340
2.430
2.410
2.320
2.410
2.460
2.460
2.320
2.360
2.410
2.360
2.180
2.450

2.460
2.400
2.380
2.380
2.410
2.340
2.460
2.380
2.300
2.180
2.320
2.410
2.400
2.340
2.360
2.280
2.340
2.430

3.460
3.150
3.280
3.360
3.410
3.340
3.510
3.300
3.480
3.400
3.340
3.380
3.430
3.460
3.570
3.340
3.150
3.450

3.520
3.180
3.520
3.300
3.360
3.480
3.510
3.320
3.510
3.260
3.340
3.230
3.230
3.280
3.110
3.540
3.180
3.300

2.940
3.280
3.180
3.180
3.200
3.180
3.180
3.300
2.830
3.280
3.180
3.200
3.300
3.180
3.300
3.040
3.000
3.280

3.180
3.340
3.230
3.180
3.180
3.360
3.200
3.040
3.180
3.110
3.260
3.180
3.280
3.150
3.180
2.940
3.180
3.200

= laboratory results (<1, 17, 32, 50) for low-inoculum level were Cochran outliers.

Table 7. Analysis of cheddar cheese by high-sensitivity dry-film (HSPCC) and Violet Red Bile Agar (VRBA) procedures
Control
HSPCC
Collaborator
1
2
3
4
6
7
8
9
10
11
12
13
14
15
18

Low inoculum
VRB

HSPCC

Medium inoculum
VRB

HSPCC

High inoculum
VRB

HSPCC

VRB

No. 2

No. 5

No. 2

No. 5

No. 1

No. 3

No. 1

No. 3

No. 4

No. 8

No. 4

No. 8

No. 6

No. 7

No. 6

No. 7

<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301

<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000

<0.301
<0.301
<0.301
<0.301
0.301
<0.301
<0.301
<0.301
0.301
<0.301
0.301
<0.301
<0.301
<0.301
<0.301

<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
<0.301
0.301
<0.301
0.602
0.301
<0.301
0.301
<0.301
<0.301

<0.000
<0.000
<0.000
0.000
<0.000
<0.000
<0.000
<0.000
<0.000
0.000
<0.000
<0.000
<0.000
<0.000
0.301

<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
1.680
<0.000
0.301
<0.000
<0.000
0.000
0.000
0.000

1.720
1.880
1.850
1.510
1.560
1.780
1.680
1.430
1.430
1.560
1.720
1.340
1.780
1.760
1.340

1.760
1.810
1.860
1.340
1.260
1.680
1.760
1.600
1.580
1.580
1.860
1.620
1.820
1.640
1.490

1.380
1.900
1.700
1.830
1.580
1.670
1.760
1.780
1.540
1.740
1.630
1.610
1.660
1.450
1.360

1.560
1.780
1.720
1.610
1.670
1.700
1.770
1.600
1.600
1.770
1.560
1.150
1.670
1.570
1.530

3.110
3.080
3.230
3.200
3.280
3.230
3.410
3.180
3.080
2.580
3.410
3.180
3.150
3.080
3.000

3.150
3.080
3.150
3.260
3.280
3.280
3.400
3.080
2.880
2.380
3.340
3.110
3.080
3.150
2.830

2.660
3.080
2.980
2.830
3.150
3.180
3.180
2.990
3.000
1.650
3.040
3.000
2.970
2.910
2.960

2.700
3.080
3.110
2.950
3.150
3.110
3.200
2.850
3.080
1.960
3.040
2.830
3.000
3.080
2.820

Table 8. Statistical analysis of collaborative results for comparison of high-sensitivity dry-film (HSPCC) and Violet Red Bile Agar (VRBA) coliform count methods
Mean log, cfu/g
Product
2% milk

Chocolate milk

Cream

Vanilla ice cream

Cottage cheese

Cheddar cheese

a
b
c

Inoculum
level

Low
Medium
High
Low
Medium
High
Low
Medium
High
Low
Medium
High
Low
Medium
High
Lowc
Medium
High

15
15
15
15
15
16
16
16
17
16
16
16
17
18
18
15
15
15

HSPCC
a

0.681a
1.576a
a
2.437a
a
0.612a
1.637
a
2.602a
0.638
1.661
a
2.679a
0.147
1.098
1.899
1.420
2.410
3.361
0.311
1.633
3.122
a

VRBA

HSPCC

0.540
1.296
2.165
0.464
1.521
2.436
0.562
1.576
2.529
0.168
1.050
1.851
a
1.524a
2.374
3.178
0.076
1.628
2.918

0.058
0.067
0.071
0.241
0.088
b
0.114b
0.255
0.046
0.081
0.203
0.146
0.087
0.139
b
0.112b
0.115
0.055
0.105
0.071

Significantly higher mean (P < 0.05).

Significantly higher sr (P < 0.05).
Too few positive samples for valid statistical comparison of methods.

RSDr, %

sr
VRBA
b

0.163b
0.147b
b
0.150b
0.163
0.106
0.052
0.235
b
0.127b
0.053
0.182
0.108
0.093
0.089
0.065
0.104
0.316
0.116
0.089
b

HSPCC
8.4
4.3
2.9
39.4
5.4
4.4
39.9
2.8
3.0
138.1
13.3
4.6
9.8
4.7
3.4
17.7
6.4
2.3

VRBA
30.3
11.3
6.9
35.1
7.0
2.1
41.9
8.1
2.1
108.3
10.3
5.0
5.9
2.7
3.3
416.0
7.1
3.1

RSDR, %

sR
HSPCC

VRBA

HSPCC

0.101
0.082
0.109
0.241
0.090
0.116
0.255
0.071
0.095
0.208
0.178
0.170
0.185
0.131
0.125
0.055
0.180
0.226

0.198
0.182
0.184
0.271
0.182
0.161
0.235
0.127
0.105
0.247
0.175
0.161
0.096
0.071
0.116
0.316
0.155
0.340

14.9
5.2
4.5
39.4
5.5
4.5
39.9
4.3
3.5
141.2
16.2
9.0
13.0
5.4
3.7
17.7
11.0
7.2

VRBA
36.7
14.1
8.5
58.3
11.9
6.6
41.9
8.1
4.1
147.1
16.7
8.7
6.3
3.0
3.6
416.0
9.5
11.6