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 (non-
yeast 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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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 Rep 4 Rep 5 Average / CFU Limit
Neat TNTC TNTC TNTC TNTC TNTC NA
-1 TNTC TNTC TNTC TNTC TNTC NA
-2 TNTC TNTC TNTC TNTC TNTC NA
-3 255 241 274 291 243 261 (<500 CFU)
-4 21 22 27 21 24 23 (<50 CFU)
-5 1 2 2 2 2 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 Rep 3 Rep 4 Rep 5 Average / CFU Limit
Neat TNTC TNTC TNTC TNTC TNTC NA
-1 TNTC TNTC TNTC TNTC TNTC NA
-2 TNTC TNTC TNTC TNTC TNTC NA
-3 201 250 266 191 275 237 (<250 CFU)
-4 24 27 21 31 16 24 (<50 CFU)
-5 4 2 1 1 2 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 Rep 4 Rep 5 Average / CFU Limit
Neat TNTC TNTC TNTC TNTC TNTC NA
-1 TNTC TNTC TNTC TNTC TNTC NA
-2 142 112 128 114 144 128 (<250 CFU)
-3 11 25 18 14 19 17 (<50 CFU)
-4 0 0 2 3 1 1 (<10 CFU)
-5 0 1 0 0 0 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 Rep 4 Rep 5 Average / CFU Limit
Neat TNTC TNTC TNTC TNTC TNTC NA
-1 TNTC TNTC TNTC TNTC TNTC NA
-2 TNTC TNTC TNTC TNTC TNTC NA
-3 312 347 325 284 294 312 (<500 CFU)
-4 28 24 31 18 27 26 (<50 CFU)
-5 2 3 3 1 1 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 Rep 4 Rep 5 Average / CFU Limit
Neat TNTC TNTC TNTC TNTC TNTC NA
-1 TNTC TNTC TNTC TNTC TNTC NA
-2 TNTC TNTC TNTC TNTC TNTC NA
-3 112 147 125 84 94 113 (<250 CFU)
-4 14 16 15 14 17 15 (<50 CFU)
-5 0 0 0 1 0 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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