ATP Sanitation Monitoring
Technical Document
Hygiena Test Device Yeast Detection
Purpose
In food and beverage manufacturing, the presence of undesired yeasts on processing equipment can introduce
product spoilage issues or undesirable off-flavors (i.e., in beer and wine production.) An important part of a thorough
cleaning process is real-time monitoring of cleaning effectiveness. Food and beverage manufacturers around the
world use the Hygiena ATP sanitation monitoring system to verify cleaning processes have worked correctly and
microbial and other residues have been effectively removed from surfaces. Because ATP systems detect Adenosine
Triphosphate (ATP) they cannot differentiate between microbial cells and other organic matter. This should not matter
to the person in charge of sanitation, because the main goal is to remove all organic residues from surfaces during
the cleaning process. However, the detectability of specific organisms at low levels, such as yeasts, by the Hygiena
ATP system is important to food and beverage processors handling yeasts which may become troublesome if left
undetected. The purpose of this study is to demonstrate the detectability of a variety of yeasts in the absence of a
food matrix in a range of Hygiena test devices. (It is important to note that yeasts do not exist in isolate and in a food
manufacturing plant as yeasts will always be found within a food matrix.) This study will demonstrate Hygiena ATP
test devices effectively detect a variety of yeasts at low levels with little variation in results.
Methods
All yeast were grown in PDB (Potato Dextrose Broth) at 37 °C and 30 °C for 72 hours and then sub-cultured onto PDA
(Potato Dextrose agar) to check purity. The yeasts were grown again in PDB before being diluted into MRD (Maximum
Recovery Diluent). There was no need to wash the yeast in saline prior to measurement as it is more practical to
measure the background ATP (non-yeast ATP) rather than try to remove it and stress the yeast in the process. Yeasts
in the field would never be washed of background (free) ATP, so this method is most practical. Background ATP (nonyeast ATP) was detected using a proprietary internal quality control test device which detects non-microbial ATP.
Yeasts used in this experiment:
1. Saccharomyces cerevisiae
2. Candida albicans
3. Zygosaccharomyces bailii
4. Candida parasilosis
5. Rhodotorula rubra
The following Hygiena devices were tested:
1. SuperSnap High-Sensitivity Surface ATP
2. UltraSnap Surface ATP
3. AquaSnap Total Water ATP
The dilution series used was as follows:
1. Neat culture
2. -1 dilution
3. -2 dilution
4. -3 dilution
5. -4 dilution
6. -5 dilution
7. -6 dilution
From each dilution 10µL was added directly to the swab bud or 100µL added to the dipper. Each device was activated
and read in the EnSURE Monitoring System. Five replicates were run from each dilution. Reference plates were also
done using the method of Miles and Mizra by inoculating each dilution as 10µL spots onto PDA to exactly estimate the
count per 10µL and also to estimate from the M&M method the CV% of the inoculation method independently of the
RLU results.
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Technical Document
Results
Yeast 1: Saccharomyces cerevisiae
Starting overnight culture = 2e7 yeast per mL (25,500,000)
Table 1: Average Saccharomyces cerevisiae detection in Hygiena test devices (RLU) (n=5)
SuperSnap UltraSnap AquaSnap Non-yeast ATP
Neat
9235
7969
9888
245
-1
7966
5312
9790
42
-2
2486
765
7648
8
-3
183
77
1116
1
-4
28
23
246
0
-5
0
0
12
0
Neat CV%
1
9
1
17
-1 CV%
3
8
2
11
-2 CV%
9
6
4
11
-3 CV%
9
10
13
149
-4 CV%
8
21
7
NA
-5 CV%
NA
NA
18
NA
Table 2: Plate Counts for Saccharomyces cerevisiae (CFU)
Dilution
Rep 1
Rep 2
Rep 3
Neat
TNTC
TNTC
TNTC
-1
TNTC
TNTC
TNTC
-2
TNTC
TNTC
TNTC
-3
255
241
274
-4
21
22
27
-5
1
2
2
Rep 4
TNTC
TNTC
TNTC
291
21
2
Rep 5
TNTC
TNTC
TNTC
243
24
2
Average / CFU Limit
NA
NA
NA
261 (<500 CFU)
23 (<50 CFU)
2 (<10 CFU)
Table 3: Limit of Detection for Saccharomyces cerevisiae (CFU)
SuperSnap UltraSnap AquaSnap
LoD Yeast CFU
<50
<50
<10
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Yeast 2: Candida albicans
Starting overnight culture = 2e7 yeast per mL (23,660,000)
Table 4: Average Candida albicans detection in Hygiena test devices (RLU) (n=5)
SuperSnap UltraSnap AquaSnap Non-yeast ATP
Neat
9083
8068
9891
257
-1
7664
6864
9374
74
-2
2838
996
8265
8
-3
290
95
1146
0
-4
91
30
133
0
-5
1
5
9
0
Neat CV%
2
3
1
3
-1 CV%
2
2
4
20
-2 CV%
10
4
3
10
-3 CV%
8
5
13
NA
-4 CV%
6
11
15
NA
-5 CV%
37
16
8
NA
Table 5: Plate Counts for Candida albicans (CFU)
Dilution
Rep 1
Rep 2
Neat
TNTC
TNTC
-1
TNTC
TNTC
-2
TNTC
TNTC
-3
201
250
-4
24
27
-5
4
2
Rep 3
TNTC
TNTC
TNTC
266
21
1
Rep 4
TNTC
TNTC
TNTC
191
31
1
Rep 5
TNTC
TNTC
TNTC
275
16
2
Average / CFU Limit
NA
NA
NA
237 (<250 CFU)
24 (<50 CFU)
2 (<10 CFU)
Table 6: Limit of Detection for Candida albicans (CFU)
SuperSnap UltraSnap AquaSnap
LoD Yeast CFU
<50
<50
<10
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Yeast 3: Zygosaccharomyces bailii
Starting overnight culture = 1e6 yeast per mL (1,280,000)
Table 7: Average Zygosaccharomyces bailii detection in Hygiena test devices (RLU) (n=5)
SuperSnap UltraSnap AquaSnap Non-yeast ATP
Neat
6928
7892
9917
659
-1
1718
2993
9400
174
-2
243
263
4023
19
-3
34
27
480
1
-4
4
5
47
0
-5
0
0
5
0
Neat CV%
5
2
1
7
-1 CV%
7
4
3
10
-2 CV%
8
11
5
14
-3 CV%
7
19
14
91
-4 CV%
15
32
10
NA
-5 CV%
NA
NA
9
NA
Table 8: Plate Counts for Zygosaccharomyces bailii (CFU)
Dilution
Rep 1
Rep 2
Rep 3
Neat
TNTC
TNTC
TNTC
-1
TNTC
TNTC
TNTC
-2
142
112
128
-3
11
25
18
-4
0
0
2
-5
0
1
0
Rep 4
TNTC
TNTC
114
14
3
0
Rep 5
TNTC
TNTC
144
19
1
0
Average / CFU Limit
NA
NA
128 (<250 CFU)
17 (<50 CFU)
1 (<10 CFU)
0 (<10 CFU)
Table 9: Limit of Detection for Zygosaccharomyces bailii (CFU)
SuperSnap UltraSnap AquaSnap
LoD Yeast CFU
<50
<50
<10
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Yeast 4: Candida parasilosis
Starting overnight culture = 3e7 yeast per mL (3,124,000)
Table 10: Average Candida parasilosis detection in Hygiena test devices (RLU) (n=5)
SuperSnap UltraSnap AquaSnap Non-yeast ATP
Neat
8874
8351
9999
129
-1
8122
4875
9999
25
-2
3124
478
8114
7
-3
284
58
897
1
-4
33
12
142
0
-5
0
0
8
0
Neat CV%
2
11
2
11
-1 CV%
4
5
5
9
-2 CV%
4
6
5
14
-3 CV%
8
8
12
121
-4 CV%
9
15
9
NA
-5 CV%
NA
NA
14
NA
Table 11: Plate Counts for Candida parasilosis (CFU)
Dilution
Rep 1
Rep 2
Rep 3
Neat
TNTC
TNTC
TNTC
-1
TNTC
TNTC
TNTC
-2
TNTC
TNTC
TNTC
-3
312
347
325
-4
28
24
31
-5
2
3
3
Rep 4
TNTC
TNTC
TNTC
284
18
1
Rep 5
TNTC
TNTC
TNTC
294
27
1
Average / CFU Limit
NA
NA
NA
312 (<500 CFU)
26 (<50 CFU)
2 (<10 CFU)
Table 12: Limit of Detection for Candida parasilosis (CFU)
SuperSnap UltraSnap AquaSnap
LoD Yeast CFU
<50
<50
<10
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Yeast 5: Rhodotorula rubra
Starting overnight culture = 1e7 yeast per mL (1,130,000)
Table 13: Average Rhodotorula rubra detection in Hygiena test devices (RLU) (n=5)
SuperSnap UltraSnap AquaSnap Non-yeast ATP
Neat
8712
7894
8989
88
-1
5841
5111
8478
11
-2
2811
1421
7133
4
-3
198
88
1011
0
-4
21
4
114
0
-5
0
0
5
0
Neat CV%
2
1
1
1
-1 CV%
4
4
4
8
-2 CV%
4
4
6
10
-3 CV%
8
5
10
88
-4 CV%
11
12
19
NA
-5 CV%
NA
NA
14
NA
Table 14: Plate Counts for Rhodotorula rubra (RLU)
Dilution
Rep 1
Rep 2
Rep 3
Neat
TNTC
TNTC
TNTC
-1
TNTC
TNTC
TNTC
-2
TNTC
TNTC
TNTC
-3
112
147
125
-4
14
16
15
-5
0
0
0
Rep 4
TNTC
TNTC
TNTC
84
14
1
Rep 5
TNTC
TNTC
TNTC
94
17
0
Average / CFU Limit
NA
NA
NA
113 (<250 CFU)
15 (<50 CFU)
0 (<10 CFU)
Table 15: Limit of Detection for Rhodotorula rubra (CFU)
SuperSnap UltraSnap AquaSnap
LoD Yeast CFU
<50
<50
<10
Conclusion
In general, SuperSnap and UltraSnap Surfaces ATP test devices detect approximately <50 CFU/swab yeasts.
AquaSnap Total Water ATP test devices detect approximately <10 CFU/mL yeasts. Variation of results at these low
levels was minimal with CV% ranges as follows: SuperSnap (6-11%), UltraSnap (12-21%), and AquaSnap (8-18%).
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