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BioLaz®
Real-Time Microbial Monitor Response to Cleanroom
Cleaning Agents Provided by Shield Medicare
There are many cleaning agents that can be used
within a pharmaceutical cleanroom. Here we report
whether Klercide cleaning agents used in a simulated
Class A cleanroom environment could potentially
cause false positive Bio-Counts in the BioLaz Real-
Time Microbial Monitor, a sensor that detects
presence of microbiological organisms through the
use of fluorescence.
Figure 1. BioLaz Real-Time Microbial Monitor
Two different testing methods were conducted on
the chemicals.
 Those supplied in original packaging of a spray
bottle were tested as a spray bottle as well as
having the chemical nebulized.
 Those only in a liquid container were nebulized to
create a mist that was measured. Liquids were not
directly injected into the BioLaz Real-Time
Microbial Monitor.
The following cleaning agents were tested:
 Klercide-CR®
Sterile Biocide A
 Klercide-CR Sterile Filtered Biocide B
 Klercide-CR Sterile Filtered Biocide C
 Klercide-CR Sterile Biocide D
 Klercide-CR Sterile Biocide E
 Klercide-CR Sterile Filtered Biocide F
 Klercide 70/30®
Sterile IPA
 Klercide 70/30 Sterile Denatured Ethanol
Methods
Environmental Testing Chamber
To ensure a consistent environment, all testing was
performed inside an environmental testing chamber
that utilized a HEPA filter to recreate a Grade A
environment. During periods of manually
administering the spray the chamber door was
opened to allow delivery of the mist. This human
interaction was done in a manner to minimize the
amount of ambient air from the test laboratory
introduced into the test chamber.
Chemicals Supplied in a Trigger Spray Bottle
Prior to testing and prior to exposure to each cleaning
agent, the BioLaz Real-Time Microbial Monitor was
allowed to establish a zero count baseline value
inside the testing chamber. A mist generated from
one and two sprays was manually introduced into the
testing chamber. This mist was allowed to be drawn
into the BioLaz Real-Time Microbial Monitor by
normal sampling methods, i.e., the sample was drawn
in from the surrounding air that contained the cleaning
agent mist and the filtered chamber air. It was
important not to spray directly into the inlet as this is
an aerosol device and not designed to be exposed to
liquid ingress.
A total of two tests were run for each chemical
sprayed.
The same battery of tests was performed
simultaneously with a Lasair®
III Particle Counter in
addition to the BioLaz Real-Time Microbial Monitor.
The testing method was the same as described
above. This testing was done to assure droplets
generated by the spray were of a proper size and
quantity to be measured by the BioLaz Real-Time
Microbial Monitor.
Figure 2. Lasair III Aerosol Particle Counter

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Figure 3. HEPA filtered test chamber for manual
administration of mist
All Chemicals
Prior to testing and prior to exposure to each
cleaning agent, the BioLaz Real-Time Microbial
Monitor was allowed to establish a zero count
baseline value inside the test chamber by running a
sample of nebulized deionized (DI) water.
An aseptic nebulizer was filled with either the
cleaning agent or DI water and a fine mist was
sprayed into the filter chamber. This mist was
allowed to be drawn into the BioLaz Real-Time
Microbial Monitor by normal sampling methods, i.e.,
the sample was drawn in from the surrounding air
containing the cleaning agent mist and the filtered
chamber air. It was important not to spray directly
into the inlet, as this is an aerosol device and not
designed to be exposed to liquid ingress.
Each test consisted of a three-minute sample of DI
water followed by a ten-minute sample of the
chemical.
Chemicals in spray bottles were tested with the
chemical nebulized in addition to testing with the
spray container.
The chamber door remained closed at all times
when the nebulizing process occurred.
Figure 4. HEPA filtered test chamber for nebulized
administration of mist
Results
The DI water showed no Bio-Counts for any tests.
During periods when the cleaning agent mist was
generated, the non-biological channel and the
biological channel were monitored with the BioLaz
Real-Time Microbial Monitor. This compared the
liquid droplets being generated and sampled to
those that were recognized due to their fluorescence
as biological particles.
Table 1. Nebulized Samples: Summary of Counts*
BioLaz 1 ft
3
/min
Normalized
Total
Bio-
Counts
Total
Particles
> 0.5 µm
Klercide-CR
Sterile Biocide A (Lot A Bottle #1)
nebulized
0.1 387.0
Sterile Filtered Biocide B
nebulized
0.1 1,565.5
Sterile Filtered Biocide C
nebulized
0 1.3
Sterile Biocide D
nebulized
0.1 40.6
Sterile Biocide E
nebulized
0 645.4
Sterile Filtered Biocide F
nebulized
0 0.5
Klercide 70/30
Sterile IPA
nebulized
0 0.2
Sterile Denatured Ethanol
nebulized
0 0
DI water
nebulized
0 10.1
* Normalized counts may result in partial particle count calculation
Klercide-CR Sterile Biocide A exhibited high biological
counts when using the trigger spray, however these
counts were not seen when the nebulizer was used.
Additional testing was performed using a second
bottle of the same lot (Lot A) as well as a bottle from a
second lot (Lot B). Again, high biological counts were
seen when using the trigger spray but not when the
nebulizer was used.
Additional testing was performed to determine if the
trigger spray bottle containing the Klercide-CR Sterile
Biocide A could be contributing to the fluorescence
seen in the previous testing. Three spray bottles were
purchased and filled with DI water. Testing was
performed as before utilizing one and two sprays of
the DI water from each spray bottle. The DI water was
then discarded and Klercide-CR Sterile Biocide A was
poured into the appropriate spray bottle. Testing was
performed as before utilizing one and two sprays of
the Klercide-CR Sterile Biocide A. Biological counts
were not seen when using the purchased spray
bottles containing Klercide-CR Sterile Biocide A.

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Table 2. Summary of Sprayed Samples*
BioLaz 1 ft
3
/min
Normalized
Total
Bio-
Counts
Total
Particles
> 0.5 µm
Klercide-CR Sterile Biocide A (Lot A Bottle #1)
1 spray 0 122.0
1 spray repeated 18.0 389.0
2 sprays 75.0 908.0
2 sprays repeated 32.5 614.5
Klercide-CR Sterile Filtered Biocide B
1 spray 0 44
1 spray repeated 0 34
2 sprays 1 58
2 sprays repeated 0 33
Klercide-CR Sterile Filtered Biocide C
1 spray 0 23.0
1 spray repeated 0 34.0
2 sprays 0 92.0
2 sprays repeated 0 24.5
Klercide-CR Sterile Biocide E
1 spray 0 40
1 spray repeated 0 46
2 sprays 1 145
2 sprays repeated 0 146
Klercide-CR Sterile Filtered Biocide F
1 spray 0 11
1 spray repeated 0 36
2 sprays 0 66
2 sprays repeated 0 38
Klercide 70/30 Sterile IPA
1 spray 0 29
1 spray repeated 0 35
2 sprays 0 29
2 sprays repeated 0 20
Klercide 70/30 Sterile Denatured Ethanol
1 spray 2 42
1 spray repeated 0 24
2 sprays 3 197
2 sprays repeated 0 31
* Normalized counts may result in partial particle count calculation

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Table 3. Details of Sprayed Samples*
BioLaz 1 ft3
/min
Normalized
Lasair III 1 ft
3
/min
Normalized
Total
Bio-
Counts
Total
Particles
> 0.5 µm
Total
Particles
0.3 µm
Total
Particles
0.5 µm
Total
Particles
1 µm
Total
Particles
5 µm
Total
Particles
10 µm
Total
Particles
25 µm
Klercide-CR Sterile Biocide A (Lot A Bottle #1)
1 spray 7.8 466.2 49,896 52,722 128,610 8,292 8,046 2,922
1 spray repeated 11.7 252.5 38,979 35,271 110,178 7,857 7,155 2,316
2 sprays 132.1 3,760.7 64,350 64,074 64,074 15,666 17,922 7,788
2 sprays repeated 132.1 1,639.5 86,406 92,388 283,020 23,022 36 6,198
Klercide-CR Sterile Biocide A (Lot A Bottle #2)
1 spray 42.7 505.0 25,110.0 23,532.0 42,816.0 5,406.0 948.0 153.0
1 spray repeated 93.2 1,274.3 41,364.0 41,346.0 74,727.0 5,706.0 2,154.0 1,092.0
2 sprays 334.1 3,605.3 27,492.0 27,489.0 49,659.0 49,659.0 1,935.0 1,257.0
2 sprays repeated 336.4 2,796.4 68,227.8 69,567.6 130,719.6 13,375.8 3,223.8 2,415.6
Klercide-CR Sterile Biocide A (Lot B)
1 spray 233.1 2,898.2 37,380.0 36,654.0 53,064 3,726.0 432 168
1 spray repeated 101.0 1,274.3 35,184.0 33,468.0 47,766 3,768.0 738 210
2 sprays 784.8 12,804.9 120,240.0 138,804.0 224,532 12,222.0 1,092 558
2 sprays repeated 459.9 6,210.6 58,215.6 64,375.8 97,392 5,503.8 1,200 864
Klercide-CR Sterile Biocide B
1 spray 0 357.4 36,462 24,912 173,640 28,020 11,760 3,288
1 spray repeated 0 528.4 46,590 41,136 417,414 36,882 10,710 2,682
2 sprays 0 2,292.2 66,942 54,552 296,898 53,634 20,238 6,942
2 sprays repeated 15.5 955.7 53,976 37,128 308,124 62,622 19,068 6,168
Klercide-CR Sterile Biocide C
1 spray 0 139.9 20,328 11,976 3,150 2,448 2,310 960
1 spray repeated 0 38.9 19,770 10,260 5,346 2,958 2,352 1,056
2 sprays 7.8 46.6 47,250 26,562 8,994 5,226 4,488 1,374
2 sprays repeated 0 233.1 48,126 36,270 28,692 6,612 5,550 2,172
Klercide-CR Sterile Biocide E
1 spray 0 62.2 13,782 12,342 25,902 4,914 2,142 612
1 spray repeated 0 116.6 18,510 20,358 36,936 7,272 2,502 978
2 sprays 7.8 1,010.1 34,488 33,078 63,906 12,030 4,170 1,848
2 sprays repeated 7.8 520.6 36,000 36,666 69,810 12,306 5,502 2,904
Klercide-CR Sterile Filtered Biocide F
1 spray 0 85.5 57,126 71,148 89,022 4,080 7,506 1,650
1 spray repeated 0 69.9 71,748 84,240 98,706 4,230 7,902 3,570
2 sprays 0 567.2 90,432 97,398 161,412 13,350 22,392 5,808
2 sprays repeated 23.3 1,429.7 95,658 105,930 137,148 6,000 11,994 5,850
Klercide 70/30 Sterile IPA
1 spray 0 69.9 83,886 58,734 25,878 1,974 3,198 966
1 spray repeated 0 38.9 69,036 44,544 16,782 1,236 1,806 594
2 sprays 0 116.6 105,666 60,684 22,152 2,328 4,902 2,832
2 sprays repeated 0 124.3 89,478 52,590 19,308 2,850 6,204 4,416
Klercide 70/30 Sterile Denatured Ethanol
1 spray 0 132.1 71,094.0 49,812.0 31,338.00 1,596.0 2,352 1,884
1 spray repeated 0 7.8 15,502.5 10,414.5 5,737.56 250.5 474 327
2 sprays 0 38.9 65,544.0 44,640.0 25,338.00 3,414.0 6,528 3,354
2 sprays repeated 0 85.5 58,140.0 37,410.0 20,226.00 2,628.0 4,986 2,838
* Normalized counts may result in partial particle count calculation

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Table 4. Details of Nebulized Samples*
BioLaz 1 ft
3
/min
Normalized
Lasair III 1 ft
3
/min
Normalized
Total
Bio-
Counts
Total
Particles
> 0.5 µm
Total
Particles
0.3 µm
Total
Particles
0.5 µm
Total
Particles
1 µm
Total
Particles
5 µm
Total
Particles
10 µm
Total
Particles
25 µm
Klercide-CR Sterile Biocide A (Lot A Bottle #1)
Nebulized 0 0 2,212,418 491,731 3,784.2 0.6 0 0
Klercide-CR Sterile Biocide A (Lot B)
Nebulized 0 0 2,522,100 781,561 20,766.0 0 0 0
* Normalized counts may result in partial particle count calculation
Table 5. Details of Additional Testing of Biocide A in New Bottles*
BioLaz 1 ft
3
/min
Normalized
Lasair III 1 ft
3
/min
Normalized
Total Bio-
Counts
Total
Particles
> 0.5 µm
Total
Particles
0.3 µm
Total
Particles
0.5 µm
Total
Particles
1 µm
Total
Particles
5 µm
Total
Particles
10 µm
Total
Particles
25 µm
Bottle A1 DI Water
1 spray 0 15.5 11,736 2,688 3,480 2,796 7,452 3,168
1 spray repeated 15.5 186.5 8,412 2,268 3,144 2,424 6,072 2,472
2 sprays 25.9 56.9 4,584 848 4,536 3,044 63,800 2,264
2 sprays repeated 0 0 8,088 1,764 7,668 6,816 14,352 5,016
Biocide A Lot A Bottle #1
1 spray 0 0 63,564 62,256 108,192 6,864 2,592 1,548
1 spray repeated 0 0 66,300 59,640 138,168 10,152 7,488 3,264
2 sprays 0 0 93,048 82,932 259,800 19,884 15,864 6,792
2 sprays repeated 0 0 103,848 98,748 294,420 28,980 33,660 9,888
Bottle A2 DI Water
1 spray 0 15.5 5,940 1,656 2,484 1,476 4,524 2,244
1 spray repeated 0 46.6 3,564 852 648 456 1,116 804
2 sprays 15.5 31.1 21,756 4,332 18,732 11,424 26,628 9,972
2 sprays repeated 15.5 77.7 1,416 300 84 0 0 0
Biocide A Lot A Bottle #2
1 spray 0 0 43,092 35,844 143,484 9,300 9,252 4,164
1 spray repeated 0 0 28,212 23,736 32,304 4,296 4,824 2,520
2 sprays 0 0 59,088 50,388 139,056 13,980 14,280 5,784
2 sprays repeated 0 0 80,472 73,632 167,676 11,976 7,296 3,768
Bottle B DI Water
1 spray 15.5 15.5 7,188 2,124 2,784 2,688 5,340 1,368
1 spray repeated 0 15.5 1,212 468 24 36 12 0
2 sprays 0 0 9,120 2,268 3,684 2,388 5,004 2,940
2 sprays repeated 0 0 32,820 9,780 13,020 9,600 22,080 11,640
Biocide A Lot B1
1 spray 0 0 12,864 9,312 20,208 2,472 456 540
1 spray repeated 0 0 23,748 18,732 54,276 4,572 4,488 2,052
2 sprays 0 0 53,088 49,392 129,756 11,196 8,148 2,784
2 sprays repeated 0 0 56,592 43,764 102,756 9,324 4,320 1,500
* Normalized counts may result in partial particle count calculation
Conclusion
For chemicals the BioLaz Real-Time Microbial Monitor
did not indicate Bio-Counts when subjected to the
mists of the cleaning agents listed in the test. However
if external contamination such as certain brighteners
are used, there may be fluorescence characteristics.
This is why it is important to test under production
conditions to determine what baseline conditions are.
Additionally, the system was able to establish a zero
count baseline level at the end of each exposure
period, indicating there was no damage or residual
effect to the instrument. Care should be taken when
using Klercide-CR® Sterile Biocide A in the provided
trigger spray bottle as it appears to exhibit
fluorescence as measured by the BioLaz Real-Time
Microbial Monitor, possibly due to materials in the
container, not the chemical.

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_________________________________________________________________________________________________________________
Author: Elizabeth Bennett
ebennett@pmeasuring.com
303.443.7100 x221
Elizabeth joined Particle Measuring Systems in 2012 as a Microbiology Application Scientist. She has a B.S.
in Microbiology from Colorado State University.
BioLaz®
and Lasair®
are registered trademarks of Particle Measuring Systems, Inc.
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© 2013 Particle Measuring Systems. All rights reserved.
Reproduction or translation of any part of this work without the permission of the copyright owner is unlawful. Requests for permission or
further information should be addressed to Particle Measuring Systems, Inc. at 1-800-238-1801
Technical Paper 52
5/16/2013