A Comparative Evaluation of the Aerobic Procedure for Lactic Acid ...
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Page 1 This report presents the analytical results for comparison of the Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M ™ Petrifilm ™ Aerobic Count (AC) Plates to agars used in The Compendium of Methods for the Microbiological Examination of Foods, Chapter 19 and ISO 15214: Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of mesophilic lactic acid bacteria—Colony-count technique. All analyses were conducted at Q Laboratories, Inc. (Cincinnati, OH). 3M Petrifilm AC Plates and Aerobic Lactic Bacteria Broth were provided by 3M (Minneapolis, MN). Q Laboratories, Inc. is an independent company and is not affiliated with, nor a subsidiary of 3M. Q Laboratories, Inc. was contracted to perform an independent study by and for 3M and the data generated by Q Laboratories, Inc. during the execution of the study does not represent an endorsement of any product(s) by Q Laboratories, Inc. A Comparative Evaluation of the Aerobic Procedure for Lactic Acid Bacteria with 3M ™ Petrifilm ™ Aerobic Count Plates with Two Reference Methods for the Enumeration of Lactic Acid Bacteria in Food and Environmental Surfaces Kiel Fisher, Erin Crowley, Patrick Bird, Megan Boyle, Katherine Goetz, M. Joseph Benzinger Jr., Mark Juenger, Travis Huffman, James Agin, David Goins Q Laboratories, Inc. 1400 Harrison Avenue Cincinnati, OH 45214 A validation study of the Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M ™ Petrifilm ™ Aerobic Count (AC) Plates for the enumeration and detection of lactic acid bacteria was conducted at Q Laboratories, Inc., Cincinnati, Ohio. The PLAB counts were compared to the counts obtained by the agars used in two reference methods: The Compendium of Methods for the Microbiological Examination of Foods, Chapter 19 and ISO 15214: Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of mesophilic lactic acid bacteria—Colony-count technique. The Compendium reference agar, All Purpose Tween agar plus 2% sucrose and bromocresol purple (APTS) and the ISO reference agar, deMan, Rogasa, Sharpe plus bromocresol purple (MRS) were used in the in the analysis of both food and environmental swab samples. Four food matrices and one environmental surface were used in the comparative evaluation: salad dressings, ready to eat (RTE) salads, RTE meats, raw meats and stainless steel surfaces. For those matrices that were inoculated, various brands of each matrix were spiked with Lactobacillus fermentum ATCC 14931 or Weissella viridescens ATCC 12706 at two target recovery levels on 3M Petrifilm AC Plates: a low level of 1–15 CFU/ plate and a medium level of 15–75 CFU/ plate. Natural lactic acid bacteria contamination was recovered in five brands of RTE meat and in all brands of raw meat. A paired t-test, comparing the PLAB procedure and the reference methods at varying time points, was conducted at the 95% confidence level. Statistically comparable mean log values were determined for salad dressings, RTE salads, RTE meats, and stainless steel surfaces. The PLAB procedure demonstrated reliability as a rapid and selective quantitative method for the enumeration of lactic acid producing bacteria. June 24, 2011
Transcript of A Comparative Evaluation of the Aerobic Procedure for Lactic Acid ...
Page 1
This report presents the analytical results for comparison of the Aerobic Procedure for Lactic Acid Bacteria (PLAB) with
3M™ Petrifilm™ Aerobic Count (AC) Plates to agars used in The Compendium of Methods for the Microbiological Examination
of Foods, Chapter 19 and ISO 15214: Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of
mesophilic lactic acid bacteria—Colony-count technique. All analyses were conducted at Q Laboratories, Inc. (Cincinnati, OH).
3M Petrifilm AC Plates and Aerobic Lactic Bacteria Broth were provided by 3M (Minneapolis, MN).
Q Laboratories, Inc. is an independent company and is not affiliated with, nor a subsidiary of 3M. Q Laboratories, Inc. was contracted to perform an independent study by and for 3M and the data generated by Q Laboratories, Inc. during the execution of the study does not represent an endorsement of any product(s) by Q Laboratories, Inc.
A Comparative Evaluation of the Aerobic Procedure for Lactic Acid Bacteria with 3M™ Petrifilm™ Aerobic Count Plates with Two Reference Methods for the Enumeration of Lactic Acid
Bacteria in Food and Environmental Surfaces
Kiel Fisher, Erin Crowley, Patrick Bird, Megan Boyle, Katherine Goetz, M. Joseph Benzinger Jr., Mark Juenger, Travis Huffman, James Agin, David Goins
Q Laboratories, Inc. 1400 Harrison Avenue Cincinnati, OH 45214
A validation study of the Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M™ Petrifilm™ Aerobic Count (AC) Plates for the
enumeration and detection of lactic acid bacteria was conducted at Q Laboratories, Inc., Cincinnati, Ohio. The PLAB counts were
compared to the counts obtained by the agars used in two reference methods: The Compendium of Methods for the Microbiological
Examination of Foods, Chapter 19 and ISO 15214: Microbiology of food and animal feeding stuffs—Horizontal method for the
enumeration of mesophilic lactic acid bacteria—Colony-count technique. The Compendium reference agar, All Purpose Tween agar
plus 2% sucrose and bromocresol purple (APTS) and the ISO reference agar, deMan, Rogasa, Sharpe plus bromocresol purple (MRS)
were used in the in the analysis of both food and environmental swab samples. Four food matrices and one environmental surface
were used in the comparative evaluation: salad dressings, ready to eat (RTE) salads, RTE meats, raw meats and stainless steel
surfaces. For those matrices that were inoculated, various brands of each matrix were spiked with Lactobacillus fermentum ATCC
14931 or Weissella viridescens ATCC 12706 at two target recovery levels on 3M Petrifilm AC Plates: a low level of 1–15 CFU/ plate and
a medium level of 15–75 CFU/ plate. Natural lactic acid bacteria contamination was recovered in five brands of RTE meat and in all
brands of raw meat. A paired t-test, comparing the PLAB procedure and the reference methods at varying time points, was conducted
at the 95% confidence level. Statistically comparable mean log values were determined for salad dressings, RTE salads, RTE meats,
and stainless steel surfaces. The PLAB procedure demonstrated reliability as a rapid and selective quantitative method for the
enumeration of lactic acid producing bacteria.
June 24, 2011
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Materials and Methods
The methodology for this study was followed as outlined in the protocol: Comparison of a 3M™ Petrifilm™ Aerobic Count Plate
Method with Three Methods for the Enumeration of Lactic Acid Bacteria in Food and Environmental Samples, provided by 3M.
The study objective was to evaluate the performance of the Aerobic Procedure for Lactic Acid Bacteria with 3M™ Petrifilm™
Aerobic Count (AC) Plates to the ISO and Compendium Reference Methods for detecting and enumerating lactic acid bacteria.
The following matrices were analyzed: salad dressings, ready to eat (RTE) salads, RTE meats, raw meats, and stainless steel
surfaces. A detailed list of the brands used in the food analysis is presented in Table A.
Culture Preparation and Inoculation of Test PortionsReplicates of each matrix varied by food or surface type and were inoculated with Lactobacillus fermentum ATCC 14931 or
Weissella viridescens ATCC 12706 at two target recovery levels on 3M Petrifilm AC Plates: a low level of 1–15 colony forming
units (CFU)/plate and a medium level of 15–75 CFU/plate. To prepare salad dressings and RTE salads for inoculation, the
suspensions of L. fermentum and W. viridescens were prepared in deMan, Rogasa, Sharpe broth (MRS) with 1.0% glucose. The
L. fermentum inoculum was incubated aerobically for 24 ± 2 hours at 35 ± 2°C and the W. viridescens inoculum was incubated
microaerophilically for 24 ± 2 hours at 35 ± 2°C. Twenty-five 1g sample portions were inoculated, mixed thoroughly, and held
for 48 ± 6 hours at 10°C to allow the organism to equilibrate within the matrix. For the analysis of RTE meats, L. fermentum
and W. viridescens inocula were prepared in MRS broth and incubated for 24 ± 2 hours at 35 ± 2°C. Twenty-five gram sample
portions were inoculated, mixed thoroughly, and held in a CO2 atmosphere to simulate packaging conditions for 48 ± 6 hours at
10°C. Following the equilibration step for all food matrices, 25 ± 1g test portions were diluted 1:10 with 0.1% peptone water.
If necessary, subsequent dilutions were made in 90mL of 0.1% peptone water to achieve the target recovery levels on PLAB.
Additionally, naturally contaminated brands of both RTE and raw meats were diluted in 0.1% peptone water until target recovery
levels were achieved.
Table A. Matrix Details
Identification Brand
Salad Dressings RTE Salads
Caesar 1 Cardini’s Caesar Dressing Mustard Potato 1 Meijer Markets Mustard Potato Salad
Caesar 2 Newman’s Own Caesar Dressing Mustard Potato 2 Grandma’s Mustard Potato Salad
LoFat Caesar 1 Cardini’s Light Caesar Dressing Mustard Potato 3 Kroger Mustard Potato Salad
LoFat Caesar 2 Ken’s Steak House Light Caesar Dressing American Potato 1 Grandma’s Potato Salad with Egg
Ranch 1 Kroger Creamy Ranch Dressing American Potato 2 Meijer Markets American Potato Salad
Ranch 2 Ken’s Steak House Ranch Dressing American Potato 3 Kroger American Potato Salad
LoFat Ranch 1 Kroger Lite Creamy Ranch Dressing Macaroni 1 Kroger Macaroni Salad
LoFat Ranch 2 Kraft Light Ranch Reduced Fat Dressing Macaroni 2 Meijer Macaroni Salad
Catalina 1 Kraft Classic Catalina Dressing Plus Dip Macaroni 3 Grandmas’ Macaroni Salad
Catalina 2 Kroger California French Dressing Bean 1 Kroger Baked Beans
Blue Cheese 1 Kraft Roka Brand Blue Cheese Dressing Bean 2 Gordon Food Services Baked Beans
Blue Cheese 2 Kroger Chunky Blue Cheese Dressing Bean 3 Meijer Baked Beans
Blue Cheese 3 Ken’s Steak House Chunky Blue Cheese Dressing Coleslaw 1 Meijer Cole Slaw
Zesty Italian 1 Kroger Zesty Italian Dressing Coleslaw 2 Kroger Cole Slaw
Zesty Italian 2 Kraft Zesty Italian Dressing Coleslaw 3 Grandma’s Cole Slaw
RTE Meats Raw Meats
O/R Turkey 1 Sara Lee Fresh Ideas Oven Roasted Turkey Breast Ground Turkey 1 Honeysuckle White Ground Turkey 93/7
O/R Turkey 2 Kroger Deli Thin Sliced Oven Roasted Turkey Breast Ground Turkey 2 Kroger Ground Turkey 85/15
Roast Beef 1 Kroger Deli Thin Sliced Roast Beef Chicken 1 Tyson All Natural Chicken Breast Tenderloins 85/15
Roast Beef 2 Private Selection Homestyle Slow Roasted Roast Beef Chicken 2 Perdue Fresh Ground Chicken
O/R Chicken 1 Private Selection Homestyle Slow Roasted Chicken Breast Ground Beef 1 Kroger Ground Sirloin 90/10
O/R Chicken 2 Sara Lee Fresh Ideas Oven Roasted Chicken Breast Ground Beef 2 Laura’s Lean Ground Beef 92/8
Smoked Ham 1 Private Selection Smokehouse Hickory Ham Steak 1 Kroger Beef Top Sirloin
Smoked Ham 2 Kroger Deli Think Sliced Smoked Ham Steak 2 Kroger Beef Bottom Round Steak Choice
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For the analysis of stainless steel environmental surfaces, sixteen 1" x 1" surface areas per organism were inoculated with a
dilution of a L. fermentum or W. viridescens broth culture. The L. fermentum inoculum was prepared using Brain Heart Infusion
Broth and incubated aerobically for 24 ± 2 hours at 35 ± 2°C. The W. viridescens inoculum was prepared in MRS and incubated
microaerophilically for 24 ± 2 hours at 35 ± 2°C. The inoculated surfaces were left to dry at ambient temperature for 18 ± 2
hours prior to sampling. For each organism, eight surface areas were sampled using environmental swabs pre-moistened with
Dey-Engley Neutralizing broth (D/E) and eight surface areas were sampled using environmental swabs pre-moistened with
Letheen Broth (LB). The surface areas were sampled in both horizontal and vertical motions. After sampling, the D/E swabs
were placed into a test tube containing 9mL of D/E and the LB swabs were placed into a test tube containing 9mL of LB.
Subsequent dilutions of these samples were prepared in 9mL test tubes of either D/E or LB to achieve the target recovery levels
on 3M™ Petrifilm™ Aerobic Count (AC) Plates.
Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M Petrifilm AC Plates Method
For the PLAB procedure, a 2.0mL aliquot of sample was transferred to a test tube containing 2.0mL of Lactic Bacteria Broth.
After vortexing the solution, a 1.0mL aliquot (0.5mL of sample and 0.5mL of Lactic Broth) was plated onto the 3M Petrifilm
AC Plate. PLAB plates were assayed in quadruplicate and incubated aerobically at 35 ± 2°C for food samples and 25 ± 2°C
for environmental samples for up to 72 ± 3 hours. Halos (acid zones) with and without associated colonies on PLAB plates
were enumerated after 24 ± 2 hours, 48 ± 3 hours, and 72 ± 3 hours of incubation. The presence of colonies with associated gas
bubbles were recorded after 72 ± 3 hours of incubation. For those RTE and raw meat samples that were naturally contaminated,
isolated colonies recovered from the PLAB plates were identified by transferring to tryptic soy agar (TSA) and incubated for 24
± 2 hours at 35 ± 2°C. Final organism identifications were achieved by the VITEK Anaerobe Identification (ANI) method.
Compendial and ISO Reference Methods
For the Compendial method procedure using All Purpose Tween agar plus 2% sucrose and bromocresol purple (APTS), 1.0mL
sample aliquots were pour plated in duplicate and incubated aerobically at 25 ± 2°C for up to 72 ± 3 hours. In addition to APTS
pour plates, salad dressing and stainless steel environmental samples were spread plated onto APTS using 0.25mL sample
aliquots. For the ISO reference method procedure using MRS plus bromocresol purple, 1.0mL sample aliquots were pour plated
in duplicate and incubated anaerobically at 35 ± 2°C. Two sets of MRS pour plates were prepared, one set incubated for 48 ± 3
hours and the other set incubated for 72 ± 3 hours. For each reference agar, colonies with and without halos were enumerated
after 48 ± 3 hours and 72 ± 3 hours. After the 72 hour enumeration, a representative colony from each sample plate was
transferred to MRS broth containing a Durham tube to detect gas production and was incubated at 35 ± 2°C for 5 to 7 days.
Results and Discussion
For the statistical analysis, a paired t-test for two sample means was calculated to analyze statistical differences between the PLAB
counts and reference agar counts at varying time points. The PLAB and reference agar time points used in the statistical analyses
are listed in Table B. The t-tests were calculated using counts across all brands and organisms for each inoculation level within the
matrices. Two procedures were used to obtain the values used in the statistical comparison. For the first method (Method 1), one
PLAB replicate from each brand and organism was compared to one reference agar replicate from each brand and organism. The
PLAB counts were converted to a count per milliliter by multiplying the 0.5mL value by a factor of two. The count per milliliter
values were converted to a Log10 values and compared to single reference agar counts converted to Log10 values in the t-test. For
the second method (Method 2), all four PLAB replicates and both reference agar replicates were used in the t-test calculation. Two
3M Petrifilm AC Plate replicates were added together and the second two 3M Petrifilm AC Plate replicates were added together
to produce two count per milliliter values. The two counts calculated values were converted to Log10 values and averaged. The
Page 4
reference agar counts were converted to Log10 values and averaged. The averaged Log10 values from the PLAB method were
compared to averaged Log10 values from each of the reference agars.
Salad Dressings
For the 24 hour time point, colonies with associated halos were observed on the low level W. viridescens PLAB plates and
medium level W. viridescens PLAB plates had complete yellowing of the agar after 24 hours. The L. fermentum low and medium
level PLAB plates had no colonies or acid zones at 24 hours. Statistically comparable log values were observed comparing
48 hour 3M™ Petrifilm™ Aerobic Count (AC) Plate counts vs. 48 hour APTS pour plate counts at both levels using both
statistical procedures. A detailed list of t-test results for salad dressings is presented in Table 4 of Appendix 1.
RTE Salads
For the 24 hour time point, colonies with associated halos were observed on the low level W. viridescens PLAB plates. Medium
level L. fermentum and W. viridescens PLAB plates were completely yellow after 48 hours. At the 48 and 72 hour time point,
PLAB plates with colonies had associated halos and gas bubbles. Statistically comparable log values were observed comparing
72 hour 3M Petrifilm AC Plate counts vs. 72 hour MRS counts at the medium inoculation level using both statistical procedures.
Statistically comparable log values were observed comparing 72 hour 3M Petrifilm AC Plate counts vs. 72 hour APTS pour plate
counts at the medium inoculation level using t-test Method 2. A detailed list of t-test results for RTE salads is presented in Table
8 of Appendix 1.
RTE Meats
Colonies with associated halos were observed on PLAB plates from the naturally contaminated Private Selection Smokehouse
Hickory Ham. All low level PLAB plates had colonies with associated halos and gas production at the 48 hour time point. All
medium level PLAB plates were completely yellow at the 48 hour time point and had evidence of gas production. Statistically
comparable log values were calculated for the medium level 48 hour 3M Petrifilm AC Plate counts vs. 48 hour APTS pour plate
counts, 48 hour 3M Petrifilm AC Plate counts vs. 72 hour APTS pour plate counts, 72 hour 3M Petrifilm AC Plate counts vs.
72 hour APTS pour plate counts, and 72 hour 3M Petrifilm AC Plate counts vs. 72 hour MRS counts time points using both
t-test methods. L. acidophilus at an acceptable confidence level, L. jensenii at a very good confidence level, and L. cellobiosus
at an excellent confidence level were identified using the VITEK ANI method. A detailed list of t-test results for RTE meats is
presented in Table 11 of Appendix 1.
Raw Meats
At the 24 hour time point, PLAB plates were completely yellow with no associated colonies. At the 48 hour time point, PLAB
plates had colonies with associated gas production. APTS and MRS were overgrown with bacillus and an accurate enumeration
of lactic organisms could not be completed. L. acidophilus, L. jensenii, and L. casei were identified using the VITEK ANI
method at a low discrimination confidence level. A detailed list of PLAB counts for raw meats is presented in Table 12 of
Appendix 1. Due to overgrowth of the competing bacillus, no statistical comparisons were conducted for the raw meats.
Table B.
Method and Time Points Used in Statistical Analysis
48 Hour 3M™ Petrifilm™ AC Plate Counts vs. 48 Hour APTS Pour Plate Counts
48 Hour 3M™ Petrifilm™ AC Plate Counts vs. 72 Hour APTS Pour Plate Counts
48 Hour 3M™ Petrifilm™ AC Plate Counts vs. 72 Hour APTS Pour Plate Counts
72 Hour 3M™ Petrifilm™ AC Plate Counts vs. 72 Hour MRS Counts
72 Hour 3M™ Petrifilm™ AC Plate Counts vs. 72 Hour MRS Counts
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Stainless Steel Environmentals
Colonies with associated acid zones and gas production were observed on LAB plates at the 48 hour time period and complete
yellowing of the agars occurred at the 72 hour time point. Statistically comparable log values were calculated for the 48 hour
3M™ Petrifilm™ Aerobic Count (AC) Plate counts vs. 48 hour APTS pour plate counts comparison using both t-test methods. A
detailed list of t-test results for stainless steel environmental surface swabs is presented in Table 17 of Appendix 1.
Observations
The method comparison results obtained in the evaluation demonstrate the reliability of the PLAB procedure as an easy-to-use
method for the enumeration and detection of lactic acid organisms in a variety of foods and on stainless steel environmental
surfaces. The method requires a simple plating procedure and eliminates the lengthy gas production confirmation procedure
used in the reference methods. The PLAB method reduces the cost of sample preparation by eliminating the need for producing
and sterilizing reference method agars and eliminates costly materials needed to create and maintain an anaerobic environment
for MRS. The initial appearance of acid zones on PLAB is an accurate early indicator of the presence of lactic acid organisms
as acid zones were always observed surrounding typical red colonies. Colony associated gas production was observed on each
PLAB plate which is a distinct advantage over the reference procedures. The low inoculum level PLAB plates produced distinct
zones and colonies sooner than the medium level PLAB plates. It was often observed that the medium level did not have distinct
acid zones, instead the agar turned completely yellow and colonies were faint. When enumerating lactic organisms from matrices
with a high volume of non-target bacteria, the PLAB plates were easier to accurately enumerate compared to APTS. APTS often
had overgrowth of bacillus and other non- target bacteria, which made enumerating colonies with acid zones challenging. This
gives PLAB an advantage as only true lactic acid producing organisms and no bacillus was observed on 3M Petrifilm AC Plates.
3M and Petrifilm are trademarks of 3M.
Page 6
APPENDIX 1
Page 7
Tabl
e 1.
Sal
ad D
ress
ing
Resu
lts —
Lac
toba
cillu
s fe
rmen
tum
Dres
sing
Aero
bic
Proc
edur
e fo
r Lac
tic A
cid
Bact
eria
with
3M
™ P
etrifi
lm™
Aer
obic
Cou
nt P
late
s (0
.5m
L)AP
TS (0
.25m
L)AP
TS (1
.0m
L)M
RS (1
.0m
L)
24 H
our
48 H
our*
72 H
our*
48 H
our
72 H
our
48 H
our
72 H
our
48 H
our
72 H
our
Repl
icat
e
AB
CD
AB
CD
AB
CD
AB
AB
AB
AB
AB
AB
Low Level
Caes
ar 1
00
00
00
00
00
00
00
00
00
31
00
10
Caes
ar 2
00
00
00
00
22
60
00
01
00
84
913
4931
LoFa
t Cae
sar 1
00
00
00
00
00
10
00
00
00
30
00
11
LoFa
t Cae
sar2
0
00
00
00
00
10
00
00
00
00
11
34
6
Ranc
h 1
00
00
00
03
20
05
00
00
00
32
2316
3635
Ranc
h 2
00
00
10
00
20
01
00
00
00
14
00
46
LoFa
t Ran
ch 1
00
00
43
33
159
96
00
00
00
21
1212
3237
LoFa
t Ran
ch 2
00
00
1721
2420
4544
4250
00
1310
00
5040
2030
123
117
Cata
lina
10
00
00
00
00
00
00
00
00
00
11
21
2
Cata
lina
20
00
00
00
00
02
00
00
00
00
14
420
21
Blue
Che
ese
10
00
00
00
01
00
10
00
00
01
10
07
3
Blue
Che
ese
20
00
00
00
00
31
10
00
00
01
32
11
3
Blue
Che
ese
30
00
00
00
01
00
00
00
00
04
40
03
4
Zest
y Ita
lian
10
00
00
00
01
01
00
00
00
00
01
04
0
Zest
y Ita
lian
20
00
00
00
00
00
00
00
00
01
31
312
13
Medium Level
Caes
ar 1
00
00
00
00
00
00
00
00
00
13
41
58
Caes
ar 2
00
00
31
14
1322
1711
00
44
00
2021
1826
3846
LoFa
t Cae
sar 1
00
00
01
00
11
12
00
11
00
69
46
711
LoFa
t Cae
sar 2
00
00
15
27
68
78
00
24
00
1826
1722
1930
Ranc
h 1
00
00
2119
2117
5436
5758
00
1623
00
110
100
9910
616
615
9
Ranc
h 2
00
00
11
10
42
54
00
01
00
1113
1718
2826
LoFa
t Ran
ch 1
00
00
2316
2011
6357
5939
00
2218
00
116
119
143
156
152
150
LoFa
t Ran
ch 2
00
00
6769
8380
9390
8796
00
3023
00
216
177
289
301
317
306
Cata
lina
10
00
00
00
02
22
10
00
10
010
1920
1815
15
Cata
lina
20
00
00
00
00
20
20
00
00
013
1116
1216
21
Blue
Che
ese
10
00
01
10
11
12
10
01
00
08
619
2128
27
Blue
Che
ese
20
00
01
61
34
94
90
02
20
011
1313
1926
21
Blue
Che
ese
30
00
02
32
03
66
40
00
00
025
2621
3029
26
Zest
y Ita
lian
10
00
00
11
06
82
40
00
00
020
1725
2233
33
Zest
y Ita
lian
20
00
00
10
03
20
30
00
00
09
710
817
10
* Aci
d zo
nes
Page 8
Tabl
e 2.
Sal
ad D
ress
ing
Resu
lts —
Wei
ssel
la v
iride
scen
s
Dres
sing
Aero
bic
Proc
edur
e fo
r Lac
tic A
cid
Bact
eria
with
3M
™ P
etrifi
lm™
Aer
obic
Cou
nt P
late
s (0
.5m
L)AP
TS (0
.25m
L)AP
TS (1
.0m
L)M
RS (1
.0m
L)
24 H
our
48 H
our*
72 H
our*
48 H
our
72 H
our
48 H
our
72 H
our
48 H
our
72 H
our
Repl
icat
e
AB
CD
AB
CD
AB
CD
AB
AB
AB
AB
AB
AB
Low Level
Caes
ar 1
00
00
00
00
00
00
00
00
00
00
00
00
Caes
ar 2
00
00
00
00
00
00
00
00
12
63
12
00
LoFa
t Cae
sar 1
00
00
10
00
10
00
00
00
22
22
21
00
LoFa
t Cae
sar2
0
00
00
00
00
00
00
00
03
63
71
21
1
Ranc
h 1
00
00
10
12
10
12
13
13
1714
1817
1110
109
Ranc
h 2
30
67
41
810
51
911
35
37
1923
1923
2017
2931
LoFa
t Ran
ch 1
88
610
2012
1620
2112
1620
85
85
4250
4451
3943
5046
LoFa
t Ran
ch 2
79
88
3625
3529
3625
3529
1217
1217
5247
5549
1816
1114
Cata
lina
10
10
00
10
00
10
00
00
00
10
20
20
0
Cata
lina
24
48
828
4545
4028
4545
4020
1923
2189
8689
8670
7378
66
Blue
Che
ese
13
20
06
82
16
82
11
21
429
2229
2426
2420
19
Blue
Che
ese
25
43
611
1011
811
1211
84
54
527
2828
3120
1636
37
Blue
Che
ese
34
44
49
514
89
514
82
22
240
4041
4427
2029
40
Zest
y Ita
lian
11
20
12
30
22
30
20
00
04
44
47
85
4
Zest
y Ita
lian
20
00
00
00
00
00
00
00
04
34
33
41
3
Medium Level
Caes
ar 1
00
00
00
00
00
00
00
00
00
00
00
00
Caes
ar 2
00
00
00
00
00
00
00
00
79
711
96
66
LoFa
t Cae
sar 1
00
00
00
00
00
00
00
00
910
910
106
21
LoFa
t Cae
sar 2
00
00
00
00
00
00
00
00
1817
2021
127
109
Ranc
h 1
00
00
12
51
35
63
00
1317
6973
6973
4349
4856
Ranc
h 2
00
00
6263
6653
6565
6758
2723
3127
111
120
115
126
8193
8810
1
LoFa
t Ran
ch 1
00
00
120
113
134
120
123
117
136
123
6284
6489
226
219
231
226
200
188
206
221
LoFa
t Ran
ch 2
00
00
135
154
187
167
135
154
187
169
8993
8993
251
266
254
270
189
186
177
159
Cata
lina
10
00
02
43
33
43
30
00
08
88
811
912
13
Cata
lina
20
00
016
520
021
321
017
120
321
321
011
112
111
712
657
051
357
051
312
112
612
811
9
Blue
Che
ese
10
00
033
2921
1834
3224
189
49
4NA
NANA
NA8
86
11
Blue
Che
ese
20
00
045
6571
6945
6672
7137
3838
3814
616
615
116
816
618
013
915
8
Blue
Che
ese
30
00
060
7282
9560
7384
9842
4742
4826
625
527
026
020
022
121
622
9
Zest
y Ita
lian
10
00
025
1527
2525
1728
2515
2017
2157
6257
6448
6659
72
Zest
y Ita
lian
20
00
00
00
03
20
30
00
016
1316
1315
1819
13
* Aci
d zo
nes
Page 9
Tabl
e 3.
Sal
ad D
ress
ing
Gas
Prod
uctio
n Re
sults
Sala
d Dr
essi
ng
Lact
obac
illus
ferm
entu
mW
eiss
ella
viri
desc
ens
72 H
our 3
M™
Pet
rifilm
™
Aero
bic
Coun
t Pla
te72
Hou
r AP
TS S
prea
d Pl
ate
72 H
our
APTS
Pou
r Pla
te72
Hou
r M
RS72
Hou
r 3M
™ P
etrifi
lm™
Ae
robi
c Co
unt P
late
72 H
our
APTS
Spr
ead
Plat
e72
Hou
r AP
TS P
our P
late
72 H
our
MRS
Caes
ar 1
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Caes
ar 2
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
LoFa
t Cae
sar 1
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
LoFa
t Cae
sar 2
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Ranc
h 1
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Ranc
h 2
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
LoFa
t Ran
ch 1
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
LoFa
t Ran
ch 2
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Cata
lina
1Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Cata
lina
2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Blue
Che
ese
1Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Blue
Che
ese
2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Blue
Che
ese
3Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Zest
y Ita
lian
1Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Zest
y Ita
lian
2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Tabl
e 4.
Sal
ad D
ress
ing
T-Te
st R
esul
ts
Com
paris
on
Met
hod
1 —
0.5
x 2
Rep
licat
esM
etho
d 2
— A
naly
sis
of 4
Rep
licat
es
3M™
Pet
rifilm
™ A
erob
ic
Coun
t Pla
te M
ean
Ref M
ean
P-Va
lue
3M™
Pet
rifilm
™ A
erob
ic
Coun
t Pla
te M
ean
Ref M
ean
P-Va
lue
Low Inoculum
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 4
8 ho
ur A
PTS
0.36
730.
4759
0.28
870.
4128
0.48
230.
4190
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.36
730.
6696
0.00
100.
4128
0.66
990.
0003
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS0.
4355
0.66
960.
0148
0.47
520.
6699
0.02
90
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.36
730.
8375
0.00
010.
4128
0.82
650.
0001
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS0.
4355
0.83
750.
0002
0.47
520.
8265
0.00
03
Medium Inoculum
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 4
8 ho
ur A
PTS
0.91
830.
7680
0.34
420.
8984
0.76
570.
3911
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.91
831.
4198
0.00
020.
8984
1.40
200.
0001
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
1190
1.41
980.
0075
1.15
711.
4020
0.01
59
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.91
831.
4655
0.00
000.
8984
1.47
170.
0000
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
1190
1.46
550.
0010
1.15
711.
4717
0.00
04
Page 10
Tabl
e 5.
RTE
Sal
ad R
esul
ts —
Lac
toba
cillu
s fe
rmen
tum
Dres
sing
Aero
bic
Proc
edur
e fo
r Lac
tic A
cid
Bact
eria
with
3M
™ P
etrifi
lm™
Aer
obic
Cou
nt P
late
s (0
.5m
L)AP
TS (1
.0m
L)M
RS (1
.0m
L)
24 H
our*
48 H
our*
72 H
our*
48 H
our
72 H
our
48 H
our
72 H
our
Repl
icat
e
AB
CD
AB
CD
AB
CD
AB
AB
AB
AB
Low Level
Mus
tard
Pot
ato
15
47
16
68
76
88
718
2120
2715
1916
19
Mus
tard
Pot
ato
23
62
45
88
96
88
919
1920
2118
1514
20
Mus
tard
Pot
ato
33
44
38
810
98
810
919
3220
3717
1921
18
Amer
ican
Pot
ato
13
57
29
810
79
1110
736
2939
3019
2328
23
Amer
ican
Pot
ato
23
48
48
1212
88
1213
822
1724
1822
1624
21
Amer
ican
Pot
ato
34
26
34
810
84
811
812
2519
2923
1219
26
Mac
aron
i 15
44
89
69
99
69
1017
2524
2617
2020
16
Mac
aron
i 23
711
88
915
118
915
1256
4663
5520
2727
21
Mac
aron
i 33
23
25
45
76
55
830
2731
3019
2822
24
Bean
16
43
411
55
911
55
925
3127
3220
2421
21
Bean
27
67
513
710
1513
710
1718
1919
2118
2718
20
Bean
33
64
714
911
1214
911
1226
3230
3432
2226
20
Cole
slaw
13
56
64
67
84
78
821
1928
2216
2221
20
Cole
slaw
24
63
49
78
79
78
718
2119
2219
2623
28
Cole
slaw
37
21
211
98
1011
1010
1018
2822
2920
2016
19
Medium Level
Mus
tard
Pot
ato
122
1926
2876
7166
7076
7169
7121
022
621
723
712
814
014
113
6
Mus
tard
Pot
ato
241
4030
2986
8081
6686
8281
7029
429
130
329
718
619
916
817
9
Mus
tard
Pot
ato
336
3326
3162
7170
7862
7170
7828
525
928
827
316
016
614
114
7
Amer
ican
Pot
ato
140
3627
2974
8996
9077
9196
9028
726
729
727
521
122
620
019
7
Amer
ican
Pot
ato
229
2431
3066
7074
7066
7074
7222
426
326
923
220
620
118
719
9
Amer
ican
Pot
ato
333
2826
2088
7981
8088
8081
8125
722
926
623
718
820
116
618
0
Mac
aron
i 136
4240
2779
7866
6979
7866
6919
621
922
319
819
619
118
817
2
Mac
aron
i 244
3936
4166
5871
7067
5973
7050
449
753
952
119
618
817
618
9
Mac
aron
i 326
2420
1949
5955
6349
6056
6323
023
224
223
820
221
820
620
0
Bean
111
1721
2064
4951
5465
4954
5522
420
122
624
218
615
820
118
6
Bean
236
3130
2679
8281
7682
8281
7721
619
120
122
315
516
114
617
2
Bean
319
2224
3466
6059
7166
6160
7223
617
820
824
517
719
216
118
9
Cole
slaw
131
2926
3059
4963
6659
5263
6617
921
022
119
012
010
810
910
0
Cole
slaw
236
3718
2281
8090
7781
8090
7718
219
520
620
514
616
612
914
6
Cole
slaw
320
2622
2366
6960
6167
7061
6417
720
120
918
211
010
611
911
1
* Aci
d zo
nes
Page 11
Tabl
e 6.
RTE
Sal
ad R
esul
ts —
Wei
ssel
la v
iride
scen
s
Dres
sing
Aero
bic
Proc
edur
e fo
r Lac
tic A
cid
Bact
eria
with
3M
™ P
etrifi
lm™
Aer
obic
Cou
nt P
late
s (0
.5m
L)AP
TS (1
.0m
L)M
RS (1
.0m
L)
24 H
our*
48 H
our*
72 H
our*
48 H
our
72 H
our
48 H
our
72 H
our
Repl
icat
e
AB
CD
AB
CD
AB
CD
AB
AB
AB
AB
Low Level
Mus
tard
Pot
ato
13
31
23
52
33
52
38
713
88
47
5
Mus
tard
Pot
ato
22
24
15
55
15
55
116
1216
1217
1318
20
Mus
tard
Pot
ato
33
42
15
77
25
77
217
2317
2513
1216
16
Amer
ican
Pot
ato
12
14
34
25
74
35
814
1414
1510
1010
11
Amer
ican
Pot
ato
21
22
53
46
103
46
1012
1312
1915
616
12
Amer
ican
Pot
ato
31
10
32
11
42
11
413
1413
149
1413
13
Mac
aron
i 11
10
11
32
21
32
24
24
24
32
2
Mac
aron
i 21
11
11
32
11
33
13
43
48
86
7
Mac
aron
i 30
12
30
44
10
44
15
55
53
55
5
Bean
17
11
28
42
38
42
320
1323
1316
1318
19
Bean
21
40
04
73
04
73
011
1014
109
108
7
Bean
32
21
14
46
54
46
526
2226
2213
2120
16
Cole
slaw
10
01
00
01
00
01
03
23
23
21
2
Cole
slaw
20
20
01
20
11
21
18
88
88
117
8
Cole
slaw
30
00
04
00
04
00
01
42
50
11
3
Medium Level
Mus
tard
Pot
ato
114
1626
2048
3245
3850
3245
3964
7764
7710
811
196
110
Mus
tard
Pot
ato
221
1926
1941
4140
3943
4440
3913
515
314
815
388
9195
96
Mus
tard
Pot
ato
320
2224
2249
5650
5150
5650
5116
115
818
115
876
8381
87
Amer
ican
Pot
ato
121
1616
2630
3338
4334
3338
4312
410
912
410
911
310
489
101
Amer
ican
Pot
ato
220
1916
1936
3841
4036
3841
4011
812
211
812
211
711
611
110
8
Amer
ican
Pot
ato
313
1719
1629
3635
2733
3635
2717
015
117
015
182
8780
77
Mac
aron
i 17
1210
926
2919
2026
3019
2155
6655
5622
2830
31
Mac
aron
i 212
169
1720
2630
1920
2830
1967
7867
8140
3642
31
Mac
aron
i 313
1518
2223
2934
3023
3134
3246
3653
3931
3630
32
Bean
118
1926
1929
3332
3629
3332
3614
316
115
216
162
6970
78
Bean
216
1313
1621
2933
4022
2933
4110
711
410
711
477
8176
88
Bean
318
2020
2519
2729
3019
2830
3220
418
520
019
111
510
811
010
1
Cole
slaw
16
313
66
613
146
613
1620
2321
2617
1310
19
Cole
slaw
211
1718
1613
2430
2813
2430
2897
8310
697
4240
4639
Cole
slaw
314
1411
1419
2121
2220
2223
2227
3035
3841
4540
40
* Aci
d zo
nes
Page 12
Tabl
e 7.
RTE
Sal
ad G
as P
rodu
ctio
n Re
sults
RTE
Sala
d
Lact
obac
illus
ferm
entu
mW
eiss
ella
viri
desc
ens
72 H
our 3
M™
Pet
rifilm
™
Aero
bic
Coun
t Pla
te72
Hou
r AP
TS P
our P
late
72 H
our
MRS
72 H
our 3
M™
Pet
rifilm
™
Aero
bic
Coun
t Pla
te72
Hou
r AP
TS P
our P
late
72 H
our
MRS
Mus
tard
Pot
ato
1Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Mus
tard
Pot
ato
2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Mus
tard
Pot
ato
3Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Amer
ican
Pot
ato
1Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Amer
ican
Pot
ato
2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Amer
ican
Pot
ato
3Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Mac
aron
i 1Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Mac
aron
i 2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Mac
aron
i 3Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Bean
1Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Bean
2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Bean
3Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Cole
slaw
1Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Cole
slaw
2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Cole
slaw
3Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
Tabl
e 8.
RTE
Sal
ad T
-Tes
t Res
ults
Com
paris
on
Met
hod
1 —
0.5
x 2
Rep
licat
esM
etho
d 2
— A
naly
sis
of 4
Rep
licat
es
3M™
Pet
rifilm
™ A
erob
ic
Coun
t Pla
te M
ean
Ref M
ean
P-Va
lue
3M™
Pet
rifilm
™ A
erob
ic
Coun
t Pla
te M
ean
Ref M
ean
P-Va
lue
Low Inoculum
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 4
8 ho
ur A
PTS
0.92
291.
1523
0.00
000.
9479
1.13
850.
0000
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.92
291.
1866
0.00
000.
9479
1.18
250.
0000
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS0.
9670
1.17
830.
0000
0.95
581.
1825
0.00
00
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.92
291.
0813
0.00
580.
9479
1.09
210.
0002
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS0.
9670
1.10
280.
0066
0.95
581.
0921
0.00
02
Medium Inoculum
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 4
8 ho
ur A
PTS
1.91
642.
1540
0.00
001.
9499
2.15
570.
0000
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
1.91
642.
1782
0.00
001.
9499
2.17
530.
0000
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
9515
2.17
820.
0000
1.95
712.
1753
0.00
00
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
1.91
641.
9965
0.00
541.
9499
1.98
860.
1109
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
9515
1.98
080.
2831
1.95
711.
9886
0.19
26
Page 13
Tabl
e 9.
RTE
Mea
t Res
ults
Mea
tOr
gani
sm
Aero
bic
Proc
edur
e fo
r Lac
tic A
cid
Bact
eria
with
3M
™ P
etrifi
lm™
Aer
obic
Cou
nt P
late
s (0
.5m
L)AP
TS (1
.0m
L)M
RS (1
.0m
L)
24 H
our*
48 H
our*
72 H
our*
48 H
our
72 H
our
72 H
our
Repl
icat
e
AB
CD
AB
CD
AB
CD
AB
AB
AB
Low Level
O/R
Turk
ey 1
Lact
obac
illus
00
00
10
01
21
11
43
76
87
O/R
Turk
ey 2
Lact
obac
illus
00
00
30
21
31
31
46
99
65
O/R
Chic
ken
2La
ctob
acill
us0
00
00
10
11
10
23
45
67
4
O/R
Turk
ey 1
Wei
ssel
la0
00
03
64
34
65
515
1217
1215
11
O/R
Turk
ey 2
Wei
ssel
la0
00
05
78
85
89
819
2221
2211
13
O/R
Chic
ken
2W
eiss
ella
00
00
69
1110
610
1312
1826
1831
1514
Smok
ed H
am 1
Natu
ral C
onta
min
atio
n11
67
511
66
1014
68
1069
5370
5635
32
Smok
ed H
am 2
Natu
ral C
onta
min
atio
n0
00
06
911
67
1111
880
7784
8017
17
Roas
t Bee
f 1Na
tura
l Con
tam
inat
ion
00
00
65
34
66
56
1518
1719
1517
Roas
t Bee
f 2Na
tura
l Con
tam
inat
ion
00
00
1011
87
1211
97
2023
2123
2835
O/R
Chic
ken
1Na
tura
l Con
tam
inat
ion
00
00
67
77
69
77
3530
4033
2520
Medium Level
O/R
Turk
ey 1
Lact
obac
illus
00
00
1521
2017
1624
2019
4650
4954
4639
O/R
Turk
ey 2
Lact
obac
illus
00
00
1822
2417
2022
2419
6160
6561
5451
O/R
Chic
ken
2La
ctob
acill
us0
00
010
1113
1012
1415
1028
3229
3226
26
O/R
Turk
ey 1
Wei
ssel
la0
00
011
1410
1711
1512
1038
3038
3126
24
O/R
Turk
ey 2
Wei
ssel
la0
00
021
2015
1921
2218
1941
4441
4430
29
O/R
Chic
ken
2W
eiss
ella
00
00
2117
1718
2420
1720
4637
4840
4140
Smok
ed H
am 1
Natu
ral C
onta
min
atio
n0
00
00
00
069
8889
9562
757
062
757
034
239
9
Smok
ed H
am 2
Natu
ral C
onta
min
atio
n0
00
066
8277
7566
8378
8079
879
879
879
812
011
2
Roas
t Bee
f 1Na
tura
l Con
tam
inat
ion
00
00
3945
5152
4145
5353
155
161
158
171
130
121
Roas
t Bee
f 2Na
tura
l Con
tam
inat
ion
00
00
6669
8180
7069
8380
177
185
184
191
135
139
O/R
Chic
ken
1Na
tura
l Con
tam
inat
ion
00
00
6050
4655
6055
4757
131
129
135
131
136
126
* Aci
d zo
nes
Page 14
Tabl
e 10
. RTE
Mea
t Gas
Pro
duct
ion
Resu
lts
RTE
Mea
t
Lact
obac
illus
ferm
entu
mW
eiss
ella
viri
desc
ens
72 H
our 3
M™
Pet
rifilm
™
Aero
bic
Coun
t Pla
te72
Hou
r AP
TS P
our P
late
72 H
our
MRS
72 H
our 3
M™
Pet
rifilm
™
Aero
bic
Coun
t Pla
te72
Hou
r AP
TS P
our P
late
72 H
our
MRS
O/R
Turk
ey 1
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
O/R
Turk
ey 2
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
O/R
Chic
ken
2Po
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
ePo
sitiv
e
RTE
Mea
tNa
tura
l Con
tam
inat
ion
Smok
ed H
am 1
Posi
tive
Posi
tive
Posi
tive
Smok
ed H
am 2
Posi
tive
Posi
tive
Posi
tive
Roas
t Bee
f 1Po
sitiv
ePo
sitiv
ePo
sitiv
e
Roas
t Bee
f 2Po
sitiv
ePo
sitiv
ePo
sitiv
e
O/R
Chic
ken
1Po
sitiv
ePo
sitiv
ePo
sitiv
e
Tabl
e 11
. RTE
Mea
t T-T
est R
esul
ts
Com
paris
on
Met
hod
1 —
0.5
x 2
Rep
licat
esM
etho
d 2
— A
naly
sis
of 4
Rep
licat
es
3M™
Pet
rifilm
™ A
erob
ic
Coun
t Pla
te M
ean
Ref M
ean
P-Va
lue
3M™
Pet
rifilm
™ A
erob
ic
Coun
t Pla
te M
ean
Ref M
ean
P-Va
lue
Low Inoculum
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 4
8 ho
ur A
PTS
0.88
741.
1959
0.00
150.
8646
1.20
700.
0003
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.88
741.
2937
0.00
030.
8646
1.29
380.
0002
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS0.
9814
1.29
370.
0008
0.98
321.
2938
0.00
03
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.88
741.
1144
0.00
470.
8646
1.13
520.
0127
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS0.
9814
1.11
440.
0017
0.98
321.
1352
0.02
74
Medium Inoculum
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 4
8 ho
ur A
PTS
1.58
821.
9921
0.12
781.
5815
1.99
630.
1186
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
1.58
822.
0044
0.11
581.
5815
2.00
820.
1077
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
7758
2.00
440.
0148
1.80
272.
0082
0.01
18
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
1.58
821.
8460
0.28
861.
5815
1.83
820.
2952
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
7758
1.84
600.
1480
1.80
271.
8382
0.37
81
Page 15
Tabl
e 12
. Raw
Mea
t Res
ults
Mea
tOr
gani
sm
Aero
bic
Proc
edur
e fo
r Lac
tic A
cid
Bact
eria
with
3M
™ P
etrifi
lm™
Aer
obic
Cou
nt P
late
s (0
.5m
L)AP
TS (1
.0m
L)M
RS (1
.0m
L)
24 H
our*
48 H
our*
72 H
our*
48 H
our
72 H
our
48 H
our
72 H
our
Repl
icat
e
AB
CD
AB
CD
AB
CD
AB
AB
AB
AB
Low Level
Grou
nd T
urke
y 1
NC0
00
05
34
45
35
6TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Grou
nd T
urke
y 2
NC0
00
013
1714
1519
1817
16TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Chic
ken
1NC
00
00
01
01
01
01
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
Chic
ken
2NC
00
00
54
41
54
53
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
Grou
nd B
eef 1
NC0
00
09
811
1211
913
15TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Grou
nd B
eef 2
NC0
00
04
36
75
46
9TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Stea
k 1
NC0
00
09
146
712
1718
15TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Stea
k 2
NC0
00
05
75
56
95
7TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Medium Level
Grou
nd T
urke
y 1
NC0
00
010
1012
1512
1213
15TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Grou
nd T
urke
y 2
NC0
00
041
3037
4047
3337
41TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Chic
ken
1NC
00
00
76
56
86
56
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
Chic
ken
2NC
00
00
1717
1513
3531
3940
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
Grou
nd B
eef 1
NC0
00
00
00
011
413
114
612
3TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Grou
nd B
eef 2
NC0
00
011
2017
2451
5355
46TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Stea
k 1
NC0
00
046
4137
3249
4140
32TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
Stea
k 2
NC0
00
027
2426
2830
3126
29TN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TCTN
TC
* Aci
d zo
nes
NC=
Nat
ural
Con
tam
inat
ion
TNTC
= T
o Nu
mer
ous
to C
ount
Tabl
e 13
. Raw
Mea
t Gas
Pro
duct
ion
Resu
lts
Raw
Mea
t
Natu
ral C
onta
min
atio
n
72 H
our 3
M™
Pet
rifilm
™ A
erob
ic C
ount
Pla
te72
Hou
r APT
S Po
ur P
late
72 H
our M
RS
Grou
nd T
urke
y 1
Posi
tive
Posi
tive
Posi
tive
Grou
nd T
urke
y 2
Posi
tive
Posi
tive
Posi
tive
Chic
ken
1Po
sitiv
ePo
sitiv
ePo
sitiv
e
Chic
ken
2Po
sitiv
ePo
sitiv
ePo
sitiv
e
Grou
nd B
eef 1
Posi
tive
Posi
tive
Posi
tive
Grou
nd B
eef 2
Posi
tive
Posi
tive
Posi
tive
Stea
k 1
Posi
tive
Posi
tive
Posi
tive
Stea
k 2
Posi
tive
Posi
tive
Posi
tive
Page 16
Tabl
e 14
. Sta
inle
ss S
teel
Env
ironm
enta
l Res
ults
— L
acto
baci
llus
ferm
entu
m
Sam
ple
Dilu
ent
Aero
bic
Proc
edur
e fo
r Lac
tic A
cid
Bact
eria
with
3M
™ P
etrifi
lm™
Aer
obic
Cou
nt P
late
s (0
.5m
L)AP
TS (0
.25m
L)AP
TS (1
.0m
L)M
RS (1
.0m
L)
24 H
our
48 H
our*
72 H
our*
48 H
our
72 H
our
48 H
our
72 H
our
48 H
our
72 H
our
Repl
icat
e
AB
CD
AB
CD
AB
CD
AB
AB
AB
AB
AB
AB
Low Level
Swab
1N
Buffe
r0
00
05
48
65
58
65
49
811
522
1115
1722
17
Swab
2N
Buffe
r0
00
07
911
137
911
138
817
76
622
2421
2231
40
Swab
3N
Buffe
r0
00
011
1012
1511
1112
154
78
1315
1629
3227
2527
25
Swab
4N
Buffe
r0
00
015
1614
1315
1615
1310
820
1611
1123
2220
1938
29
Swab
5N
Buffe
r0
00
09
138
1010
138
115
910
1711
1122
2130
3219
16
Swab
6N
Buffe
r0
00
07
75
67
75
63
45
87
715
1314
1411
13
Swab
7N
Buffe
r0
00
08
98
88
68
75
310
68
817
1618
2018
19
Swab
8N
Buffe
r0
00
04
47
75
47
73
35
64
58
1012
1116
19
Medium Level
Swab
1N
Buffe
r0
00
028
6060
6756
6059
6739
4039
4012
211
112
211
110
011
810
710
0
Swab
2N
Buffe
r0
00
020
8138
3940
4138
3919
1919
1966
8166
8190
9290
81
Swab
3N
Buffe
r0
00
017
3029
4033
3028
4022
2222
2281
7781
7791
7772
72
Swab
4N
Buffe
r0
00
014
3538
3028
3637
3216
1616
1649
5549
5550
5155
70
Swab
5N
Buffe
r0
00
030
5959
6360
5959
6328
2528
2510
699
106
9995
109
9199
Swab
6N
Buffe
r0
00
019
3945
3738
3945
3718
2218
2275
7975
7978
7758
71
Swab
7N
Buffe
r0
00
014
2925
2528
2925
2515
1715
1770
6370
6366
7560
72
Swab
8N
Buffe
r0
00
018
4050
5337
4050
5325
2725
2798
9698
9610
092
8877
Low Level
Swab
9Le
thee
n0
00
06
78
116
710
115
38
57
1013
2014
2015
15
Swab
10
Leth
een
00
00
1012
139
1013
139
66
1111
129
2419
1920
1611
Swab
11
Leth
een
00
00
812
1016
912
1016
89
1618
1313
2725
2622
2530
Swab
12
Leth
een
00
00
67
55
67
66
42
75
78
1517
1822
3124
Swab
13
Leth
een
00
00
1110
1210
1110
1510
76
1312
1616
3231
3330
3433
Swab
14
Leth
een
00
00
78
1818
87
810
65
119
97
1814
3019
2126
Swab
15
Leth
een
00
00
98
1110
98
1010
65
129
88
1717
2227
2423
Swab
16
Leth
een
00
00
1211
119
1211
119
55
1010
87
1613
1516
1920
Medium Level
Swab
9Le
thee
n0
00
050
3941
4450
3841
4425
2525
2576
7176
7166
6772
75
Swab
10
Leth
een
00
00
3335
3936
3334
3836
1415
1415
5952
5952
4849
5037
Swab
11
Leth
een
00
00
2825
2930
2825
2930
1517
1517
6971
6971
5666
7273
Swab
12
Leth
een
00
00
4143
4440
4143
4440
2020
2020
8977
8977
8066
6953
Swab
13
Leth
een
00
00
3633
3532
3633
3532
1514
1514
5863
5863
8190
5973
Swab
14
Leth
een
00
00
3327
2526
3327
2526
1211
1211
4037
4037
7776
5550
Swab
15
Leth
een
00
00
2526
2630
2526
2630
1312
1312
6664
6664
7270
6955
Swab
16
Leth
een
00
00
3430
3039
3430
3039
1819
1819
5666
5666
6659
7170
* Aci
d zo
nes
Page 17
Tabl
e 15
. Sta
inle
ss S
teel
Env
ironm
enta
l Res
ults
— W
eiss
ella
viri
desc
ens
Sam
ple
Dilu
ent
Aero
bic
Proc
edur
e fo
r Lac
tic A
cid
Bact
eria
with
3M
™ P
etrifi
lm™
Aer
obic
Cou
nt P
late
s (0
.5m
L)AP
TS (0
.25m
L)AP
TS (1
.0m
L)M
RS (1
.0m
L)
24 H
our
48 H
our*
72 H
our*
48 H
our
72 H
our
48 H
our
72 H
our
48 H
our
72 H
our
Repl
icat
e
AB
CD
AB
CD
AB
CD
AB
AB
AB
AB
AB
AB
Low Level
Swab
1N
Buffe
r0
00
01
21
31
23
31
11
25
105
107
812
13
Swab
2N
Buffe
r0
00
04
41
25
41
20
00
05
65
66
910
19
Swab
3N
Buffe
r0
00
03
01
13
01
30
00
04
75
78
119
12
Swab
4N
Buffe
r0
00
00
01
10
02
10
00
212
1112
1215
1316
17
Swab
5N
Buffe
r0
00
01
00
01
00
03
33
310
410
46
611
10
Swab
6N
Buffe
r0
00
00
01
00
01
11
21
211
1311
1311
1110
12
Swab
7N
Buffe
r0
00
01
01
01
11
00
10
126
2226
2217
2123
19
Swab
8N
Buffe
r0
00
02
86
64
108
81
11
38
58
56
44
9
Medium Level
Swab
1N
Buffe
r0
00
00
820
170
1229
226
616
1649
5849
5831
3431
59
Swab
2N
Buffe
r0
00
09
107
711
108
80
00
023
2623
2722
1731
20
Swab
3N
Buffe
r0
00
013
1015
2115
1622
218
88
826
4326
4335
2735
27
Swab
4N
Buffe
r0
00
04
87
34
87
33
64
610
1911
1914
2215
27
Swab
5N
Buffe
r0
00
09
1213
1310
1315
142
52
621
2821
2933
3133
31
Swab
6N
Buffe
r0
00
011
79
1614
1013
161
41
417
917
922
2123
28
Swab
7N
Buffe
r0
00
015
911
1319
1014
132
46
424
1724
1728
3128
34
Swab
8N
Buffe
r0
00
03
13
23
25
20
00
119
59
514
1316
15
Low Level
Swab
9Le
thee
n0
00
00
08
20
19
31
21
311
1111
1316
1411
9
Swab
10
Leth
een
00
00
03
33
14
34
20
20
139
139
1111
78
Swab
11
Leth
een
00
00
01
24
01
45
11
11
137
137
2120
1415
Swab
12
Leth
een
00
00
03
66
15
139
10
10
1725
1725
1921
1922
Swab
13
Leth
een
00
00
07
1010
312
1314
33
35
3031
3231
2619
3130
Swab
14
Leth
een
00
00
04
54
08
58
01
02
1815
1816
1718
1724
Swab
15
Leth
een
00
00
114
107
218
1511
55
55
2122
2423
2124
2232
Swab
16
Leth
een
00
00
00
32
00
42
00
20
08
48
59
415
Medium Level
Swab
9Le
thee
n0
00
02
55
43
75
41
31
38
108
1010
1316
13
Swab
10
Leth
een
00
00
28
34
310
56
11
12
116
116
158
1510
Swab
11
Leth
een
00
00
014
1216
317
1319
54
54
1720
1720
2514
4733
Swab
12
Leth
een
00
00
00
113
50
145
34
34
1918
1919
117
1228
Swab
13
Leth
een
00
00
21
32
21
32
00
10
54
54
97
37
Swab
14
Leth
een
00
00
30
711
54
911
00
00
515
515
46
1213
Swab
15
Leth
een
00
00
20
31
42
42
01
01
1312
1312
106
1221
Swab
16
Leth
een
00
00
10
04
12
15
11
23
68
128
66
213
* Aci
d zo
nes
Page 18
Tabl
e 16
. Sta
inle
ss S
teel
Env
ironm
enta
l Gas
Pro
duct
ion
Resu
lts
Envi
ronm
enta
ls
Lact
obac
illus
ferm
entu
mW
eiss
ella
viri
desc
ens
72 H
our 3
M™
Pet
rifilm
™
Aero
bic
Coun
t Pla
te72
Hou
r AP
TS S
prea
d Pl
ate
72 H
our
APTS
Pou
r Pla
te72
Hou
r M
RS72
Hou
r 3M
™ P
etrifi
lm™
Ae
robi
c Co
unt P
late
72 H
our
APTS
Spr
ead
Plat
e72
Hou
r AP
TS P
our P
late
72 H
our
MRS
1 D/
E Ne
utra
lizin
g Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
2 D/
E Ne
utra
lizin
g Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
3 D/
E Ne
utra
lizin
g Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
4 D/
E Ne
utra
lizin
g Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
5 D/
E Ne
utra
lizin
g Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
6 D/
E Ne
utra
lizin
g Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
7 D/
E Ne
utra
lizin
g Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
8 D/
E Ne
utra
lizin
g Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
1 Le
thee
n Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
2 Le
thee
n Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
3 Le
thee
n Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
4 Le
thee
n Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
5 Le
thee
n Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
6 Le
thee
n Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
7 Le
thee
n Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
8 Le
thee
n Br
oth
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Posi
tive
Tabl
e 17
. Sta
inle
ss S
teel
Env
ironm
enta
l T-T
est R
esul
ts
Com
paris
on
Met
hod
1 —
0.5
x 2
Rep
licat
esM
etho
d 2
— A
naly
sis
of 4
Rep
licat
es
3M™
Pet
rifilm
™ A
erob
ic
Coun
t Pla
te M
ean
Ref M
ean
P-Va
lue
3M™
Pet
rifilm
™ A
erob
ic
Coun
t Pla
te M
ean
Ref M
ean
P-Va
lue
Low Inoculum
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 4
8 ho
ur A
PTS
0.98
120.
9772
0.96
20.
8932
0.98
160.
3253
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.98
121.
1636
0.00
570.
8932
1.15
730.
0005
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
0293
1.16
360.
0453
0.95
501.
1573
0.00
23
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.98
121.
2553
0.00
030.
8932
1.23
120.
0000
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
0293
1.25
530.
0012
0.95
501.
2312
0.00
01
Medium Inoculum
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 4
8 ho
ur A
PTS
0.98
121.
491
0.00
000.
8932
1.49
660.
0000
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.98
121.
5133
0.00
000.
8932
1.50
280.
0000
48 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
0293
1.50
170.
0000
0.95
501.
5028
0.00
00
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur A
PTS
0.98
121.
5245
0.00
000.
8932
1.55
030.
0000
72 h
our 3
M P
etrifi
lm A
erob
ic C
ount
Pla
te v
s. 7
2 ho
ur M
RS1.
0293
1.52
450.
0000
0.95
501.
5503
0.00
00
Page 19
3M and Petrifilm are trademarks of 3M. Used under license in Canada.70-2009-9494-8
