1. Hygiena SUPERSNAP
ATP and PRO-Clean
Protein Swab Testing

2. Hygiena SUPERSNAP ATP Swabs and
Luminometer
• . The SuperSnap swabs allows for detection of concentrations of ATP
from 1 -1000 ppm and emits light directly proportional to the
concentrateion swabbed by virtue of the Luciferase/ Luciferin
reagent.
• The ATP luminometer (Figure 1) measures light generated by the
Luciferase/ Luciferin system and reports in Relative Light Units (RLU);
the higher the concentration of ATP the higher the RLU.

3. Hygiena SUPERSNAP ATP Swab and
Luminometer
Figure1: Hygenia SuerpSnap swab

4. Hygeian PRO-Clean Protein Swab
• The PRO-Clean Rapid Protein Residue test (Figure 2) is a colorimetric
test utilizing the BCA assay which turns from green to purple if
protein is present in the sample; green indicating a negative protein
sample and purple for a positive protein sample.

5. Hygiena PRO-Clean Protein Swab
Figure 2: Hygenia Pro-Clean Protein swab Figure 3: The colorimetric indicator on the PRO-Clean swab

6. METHODS

7. Methods: Stainless Steel Strips
• To test the efficacy of the Hygiena swabs using the stainless steel
strips as a surface to inoculate, four sample sets were prepared: a
control, a 5.0% Horse blood, 0.5% Horse blood and Bovine serum
albumin set. Each set consisted of 25 stainless steel strips which were
sonicated with house deionized water for 20 minutes.
• 10 µl were used to inoculate each strip with the exception of the
control group. The blood solutions and BSA were allowed to dry for
20 minutes and the first readings were taken denoted as day 0.
• Readings were taken every 7 days for 14 days.

8. Methods: Lumbar RAPIDCLEAN Ronguers
• Using Lumbar RAPDICLEAN Ronguers as a substrate to inoculate, five
samples sets were prepared: the four sets originally prepared in the
stainless steel strips model with the addition of a modified version of
nelson soil (15 ml egg yolk, 15ml Horse blood, and 300 mg of Hog
musin).
• Eight areas, as shown in Figure 4, were selected to inoculate with the
four protein samples (1 control group).
• The same protocol was followed for the Lumbar rongeurs as for the
stainless steel strips with the exception of swab readings be obtained
at day 0 only.

9. Lumbar RAPIDCLEAN Ronguers
Figure 4: Two hard to clean (HTC) and two Easy to clean (ETC)
areas were selected to be inoculated for swab analysis. The two
hard to clean areas are indentations in the device.

10. Methods: micro BCA assay Standard curve
• In order to determine the protein absorbance and concentration of
the four samples the micro BCA assay kit was used to obtain a
standard curve using Ultraviolet visible spectrophotometry.
• The protocol provided by Pierce for the micro BCA assay standard
curve was followed in order to receive delivery counts of protein
concentrations of the blood samples used to inoculate the stainless
steel strips and the lumbar RAPIDCLEAN Rongeurs.
• The samples were prepared for mass spec analysis by aliquoting 10µl
in to 990µl of water and mixing with the working reagent provided by
the micro BCA assay.

11. RESULTS

12. Results: ATP and protein residue readings
from Stainless Steel Strips
DAY 0 ATP RLU PROTEIN SWAB
-control 0 -
Control 1, 2, 3 -, -, -
5.0% Horse Blood 3920, 3576, 3448 +++, +++, +++
0.5% Horse Blood 1708, 1904, 2366 +, +, -
BSA 4, 4, 3 ++, ++, ++
DAY 7 ATP RLU PROTEIN SWAB
-control 0 -
Control 2, 2, 10 -, -, -
5.0% Horse Blood 2835, 2782, 3412 +++, +++, +++
0.5% Horse Blood 751, 1155, 1683 +,+,+
BSA 35, 6, 10 ++, ++, ++
DAY 14 ATP RLU PROTEIN SWAB
-control 0 -
control 5, 12, 7 -, -, -
5.0% Horse Blood 2838, 3321, 1422 +++, +++, +++
Table 1: Chart showing the
data obtained from the
stainless steel strips when
inoculated with 5.0%, 0.5%
Horse blood and Bovine
Serum Albumin. The
negatives and positives
correlate with the color
indicator on the PRO-Clean
swab. A (-) is green, (+) is
grey, (++) is light purple and
(+++) is purple and the color
change indicates protein
concentration from no
protein concentration to a
high protein concentration.

13. Results: ATP and protein residue readings
from Lumbar RAPIDCLEAN Rongeurs
CONTROL ATP RLU PROTEIN SWAB
Easy to Clean 0, 0 -, -
Hard to Clean 0, 0 -, -
5.0% Horse Blood ATP RLU PROTEIN SWAB
Easy to Clean 3097, 2396 +++, +++
Hard to Clean 1180, 1410 +++, +++
0.5% Horse Blood ATP RLU PROTEIN SWAB
Easy to Clean 2026, 2499 +++,+++
Hard to Clean 900, 27 +,+
BSA ATP RLU PROTEIN SWAB
Easy to Clean 6, 14 -, -
Hard to Clean 7, 10 -,-
Nelson Soil ATP RLU PROTEIN SWAB
Easy to Clean 6008, 5887 +++, +++
Hard to clean 2467, 3014 +++, +++
Table 2: Results
from the ATP and
protein swabbing of
the Lumbar
Rongeurs.
The negatives and
positives correlate
with the color
indicator on the
PRO-Clean swab.
A (-) is green, (+)
is grey, (++) is light
purple and (+++)
is purple and the
color change
indicates protein
concentration
from no protein
concentration to a
high protein
concentration.

14. Results: Protein absorbance and
concentrations from stainless steel strips
Sample (10µl) A @562nm Protein conc. µg/ml
5.0% Horse Blood 2.041
2.207
2.240
110.581
125.100
128.087
0.5% Horse Blood 0.181
0.151
0.170
5.280
4.445
4.971
BSA 0.885
0.930
0.942
32.741
35.010
35.625
Table 3: Protein absorbance and concentration of the horse
blood samples and the BSA sample on day 0 of inoculation of
the stainless steel strips.

15. Results: Protein absorbance and concentrations
from Lumbar RAPIDLCLEAN Ronguers
Sample (10µl) A @ 562nm Protein conc. µg/ml
5.0% Horse Blood 2.813
2.691
2.412
88.883
84.589
74.890
0.5% Horse Blood 0.308
0.348
0.264
7.231
8.427
5.919
BSA 0.825
0.948
0.924
22.572
26.787
26.037
Nelson Soil 3.806
3.845
3.825
125.049
114.986
125.762
Table 4: Protein absorbance and concentration of horse blood
samples, BSA, and Nelson soil after inoculation of the Lumbar
Rongeurs.

16. Conclusion
• Both the Hygiena SuperSnap ATP swabs and PRO-Clean Rapid Protein
Test swabs are both effective at detecting ATP and protein
concentrations after exposure to air for a period of time.
• The results obtained from the two tests indicate that the Hygiena
SuperSnap ATP swabs seem to be more sensitive, and the sensitivity
for both swabs may be determined by the surface area swabbed.
• This study allowed the efficacy of both swabs to be tested indicating
that both can be used to determine whether surface has been
cleaned thoroughly and can be applicable to a hospital or food
industry setting as well as testing loaner medical kits for any microbial
contamination after use.

17. CRUDE PROBIOTIC
BACTERIAL LYSATE
ACTIVITY TESTING
USING THE BacT/ALERT
SYSTEM

18. Nutraceutix Crude Bacterial Probiotic lysates
and Powder Lysates from Biocare
• The crude bacterial probiotic lysates prepared by JJSA from cultrues
provided by Nutraceutix were exposed to 10kgy of electron beam
radiation.
• The prepared lysates were transferred from sterile bags to sterile
45ml Falcon tubes and stored at -70°C.
• The Powder Lysates were sent from Biocare Coppenhagen.

19. Nutraceutix Crude Probioitc Lysates
Table 1: Only lysates
1,2,4,6,9,10,13,14, and
16 were tested using
the BacT/ALERT
system.

20. Biocare Powder Lysates

21. BacT/ALERT system

22. BacT/ALERT system

23. METHODS and
METHODOLOGY

24. Methodology
• With traditional detection methodslooking for ZOI, 10µl of 10^8 of
S.aureus was added into a top agar 7ml (TSB with 0.7% agar) then
poured onto a TSA plate.
• 50µl of the tested lysates were deposited onto the S.aureus
inoculated plates.
• With the Biocare lysates there was a milky deposit on the plate,
making it diffuclt to obtain ZOI. This was corrected using the
BacT/ALERT system which can detect bacterial growth in turbidity.

26. Methods: Inoculation of BacT/ALERT bottles
and treatment with the Lysates
• A Quanti-Cult of Staphylococcus aureus was grown in A BacT/ALERT
bottle overnight in the BacT/ALERT system at 35°C.
• The overnight culture was then serially diluted to 10⁻⁴, where the
10⁻² dilution was used to inoculate the BacT/AELRT bottles treated
with the bacterial lysates.
• The Biocare powder lysates were weighed out to 0.4 grams and then
asepcitcally transferred from sterile microcentrifuge tubes to the
BacT/ALERT bottles.
• For the Crude Probiotic Bacterial Lysates from Nutraceutix, 0.4ml was
used to treat the BacT/ALERT bottles.

27. Methods: Inoculation of BacT/ALERT bottles
and treatment with the Lysates
• Five samples sets were prepared.
• Two samples sets with 0.4ml and 0.4g treated with the Crude Probiotic
Bacterial Lysate and the Powder Lysate respectively, Sample sets 1A and 1B.
• Four sample sample sets with 2ml and 2.0g treated with the Crude Porbioitc
Bacterial Lysate and the Powder Lysate respectively, Sample sets 2A, 2B, 3A
and 3B.
• One sample set of 2.0g of Powder lysate without any inoculum, sample set
4A.
• The sample sets were incubated in the BacT/Alert system for 7 days in order to
asses the absence of bacterial growth or time of detection of bacterial growth,
with the exception of sample set3A. Sample set 3A, was left to be agitated by
the BacT/ALERT system in order to effectively dissolve the powder lysate.

28. Sample Set 1A: Biocare Lysates 0.4g
Sample (Set 1A) Time to detection (hours)
BL Negative control No Growth
S. aureus 10⁻¹ 0.17
S. aureus 10⁻² 0.29
S. aureus 10⁻³ 0.44
S. aureus 10⁻⁴ 0.67
BL #1 0.29
BL #2 0.28
BL #3 0.29
BL #4 0.29
BL #5 0.27
BL #6 0.27
BL #7 0.28
BL #8 0.27
BL #9 0.29

29. Sample set 1B: Nutraceutix Lysates 0.4ml
Sample (Set 1B) Time to detection (hours)
BL Negative control No Growth
S. aureus 10⁻¹ 0.17
S. aureus 10⁻² 0.29
S. aureus 10⁻³ 0.44
S. aureus 10⁻⁴ 0.67
CPBL #1 0.28
CPBL #2 0.27
CPBL #4 0.27
CPBL #6 0.28
CPBL #9 0.28
CPBL #10 0.28
CPBL #13 0.29
CPBL #14 0.27
CPBL #16 0.33

30. Sample set 2A: Biocare Lysates 2.0g
Sample Time to detection (hours, Set 2A)
BL Negative control No Growth
S. aureus 10⁻¹ 0.18
S. aureus 10⁻² 0.28
S. aureus 10⁻³ 0.41
S. aureus 10⁻⁴ 0.68
BL #1 0.52
BL #2 0.48
BL #3 0.47
BL #4 0.44
BL #5 0.47
BL #6 0.44
BL #7 0.47
BL #8 0.45
BL #9 0.44
Time to detection (hours, Set 3A)
No Growth
0.19
0.29
0.43
0.61
0.46
0.41
0.46
0.43
0.39
0.39
0.46
0.36
0.40

31. Sample Set 3B: Nutraceutix Lysates 2ml
Sample
BL Negative control
S. aureus 10⁻¹
S. aureus 10⁻²
S. aureus 10⁻³
S. aureus 10⁻⁴
CPBL #1
CPBL #2
CPBL #4
CPBL #6
CPBL #9
CPBL #10
CPBL #13
CPBL #14
CPBL #16
Time to detection (hours, 2B)
No Growth
0.18
0.28
0.41
0.68
0.51
0.40
0.43
0.52
0.40
0.55
0.69
0.37
1.00
Time to detection (hours, Set 3B)
No Growth
0.20
0.33
0.46
0.59
1.27
0.74
0.72
0.75
0.83
0.97
1.42
0.39
0.82