dilution series hong kong - emsd
TRANSCRIPT
Disinfection ExperimentVWMs GmbH - Vienna Water Monitoring
Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse
17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail:
[email protected], www.v-w-m.at
Contents
INTRODUCTION 1
Introduction 1 1st Dilution Series 2
ColiMinder® Results 2 Lab Results 3 Correlation between Lab Results and ColiMinder Readings 3
Preparation of the 2nd Dilution Series 4 Preparation of Samples 4
Results of 2nd Dilution Series 5 Results of 3nd Dilution Series 6 Conclusion 6
HYPOTHESIS WHY THE LAB RESULTS DO NOT REPRODUCE THE DILUTION FACTORS 7
Background 7 A 50% dilution using autoclaved sample: 9
ColiMinder Measurement Parameter 10
Hong Kong October 2019
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
ColiMinder® - Rapid Microbiological Measurements
Figure 1: ColiMinder “Finn” at Stone Cutters Island Sewage Treatment Works.
Introduction
A ColiMinder® is a fully automated measurement device used to measure the microbiological contamination of
water.
A ColiMinder® (with device name Finn) has been purchased by the DSD and installed at Stone Cutters Island Sewage
Treatment Works (SCISTW) on 4th December 2017. Since this time the ColiMinder is monitoring the sample stream
producing 24 measurements per day.
In preparation of the IWA ASPIRE Conference a dilution experiment has been planned in order to evaluate the
performance of the technology across the full measurement range. Thankfully, DSD has provided its ColiMinder to
perform these experiments.
As the accuracy and reproducibility of the ColiMinder as well as its measurement range and especially the
correlation between lab results and ColiMinder results is of great interest, a dilution series has been produced and
measured 3 times by the ColiMinder.
The same samples have been sent to a lab in order to compare the ColiMinder results to the classic culture-based
lab results.
Introduction
In order to proof the accuracy, reproducibility and the linearity of the ColiMinder® results across the measurement
range, a dilution series has been produced using a sample from a wastewater treatment plan effluent stream.
This sample has been diluted in 8 steps from 100% sample down to 0.1%.
Therefore, the results of the measurement of the diluted sample should be a fraction - corresponding to the dilution
percentage - of the result for the undiluted sample. From the result of the undiluted (100%) sample, a theoretical
value was calculated for each dilution.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
1st Dilution Series A lab has been commissioned to produce a dilution series using autoclaved
sample to dilute the original sample.
10 samples have been prepared: 100%, 50%, 20%, 10%, 5%, 2%, 1%, 0.5%,
0.1%, 0.0%.
In order to validate the correlation with conventional laboratory results, the
diluted samples were split into two parts each. Of the 500 ml prepared from
each of the 10 samples, 450 ml samples were sent to an independent
laboratory and the remaining 50 ml were measured by ColiMinder. The
ColiMinder performed 3 measurements of each sample.
ColiMinder® Results The ColiMinder® measurement have been produced fully automated using the 10-fold multisampling module.
Linearity
FIGURE 2 LEFT: TABLE OF THEORETICAL VALUE (CALCULATED FROM THE 100% RESULTS DOWN) AND MEASURED VALUE (AVERAGE FROM 3
MEASUREMENTS) RIGHT: CORRELATION BETWEEN THEORETICAL VALUE AND MEASURED VALUE
The measurement results of the dilution series proof that the measurement system is performing extremely linear
across the full measurement range.
Reproducibility
Each of the samples has been measured 3 times in order to evaluate the reproducibility of the results.
FIGURE 3 RESULTS TABLE FROM 3 MEASUREMENTS PER DILUTION
The results show that the variation of results is far below 5% for the most part of the series. Only at very low
contaminations the variation is higher. This is most likely to be explained by the fact that these low contaminated
samples can be easily influenced by a minimal carry over from highly contaminated samples, when measured in the
same apparatus.
FIGURE 4 SINGLE MEASUREMENT RESULTS OF 3 MEASUREMENT SERIES LEFT: LINEAR SCALE RIGHT: LOG SCALE
Dilution Series 1 Series 2 Series 3 Average Standard Deviation Percent
0.0% 7.38 0.59 3.98 3.40 85.2
0.1% 6.12 4.24 5.62 5.33 0.79 14.9
0.5% 20.07 19.04 17.64 18.92 1.00 5.3
1.0% 42.43 40.16 38.87 40.49 1.47 3.6
2.0% 88.32 84.03 84.70 85.68 1.88 2.2
5.0% 224.81 215.41 215.39 218.53 4.44 2.0
10% 436.51 425.20 414.71 425.47 8.90 2.1
20% 901.48 880.82 858.01 880.10 17.76 2.0
50% 2289.15 2254.99 2269.08 2271.07 14.02 0.6
100% 4484.67 4503.12 4430.95 4472.91 30.61 0.7
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Lab Results The respective theoretical value for each dilution has been calculated from the lab result of the 100% sample
applying the respective dilution factor.
FIGURE 5 LEFT: TABLE OF THEORETICAL VALUE (CALCULATED FROM THE 100% RESULTS DOWN) AND LAB RESULTS; RIGHT: CORRELATION BETWEEN
THEORETICAL VALUE AND MEASURED VALUE
The lab results do not show any useful accuracy, the blank control showed zero CFU and from the 10% dilution
upward the tendency is correct but the correlation with the actual ratio of bacterial content, produced by the
dilution, is not reproduced.
This test should be reproduced not in single tests per sample but in duplets or triplets.
Correlation between Lab Results and ColiMinder Readings
Since the actual dilution, respectively the reduction of contamination, produced by the diluting the original sample,
has not been reproduced by the lab results, it seems not meaningful to correlate both results.
Based on highes Value
0.0% 0 0
0.1% 930 110000
0.5% 4650 330000
1.0% 9300 580000
2.0% 18600 490000
5.0% 46500 560000
10% 93000 490000
20% 186000 690000
50% 465000 870000
100% 930000 930000
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Preparation of the 2nd Dilution Series
As in the first dilution series lab evaluation of the samples did not deliver any useful results a second dilution series
has been produced. This time the diluted samples have been sent in doublets to three independent labs for
evaluation.
Again, a lab has been commissioned to produce a dilution series from 100% sample down to 0.1% using autoclaved
sample (WWTP effluent) as dilution medium.
Preparation of Samples
In order to produce the dilution series 15 liters of sample have been collected from the WWTP discharge stream.
After 30 min of settling, the upper 11 liters (without sediments) have been used as raw sample.
9 liters of raw sample have been autoclaved to be used as diluent. The remaining 2 liters of raw sample have been
kept at 4°C until the dilutions where produced.
In order to also validate the correlation with traditional lab results the diluted samples have been split into parts.
• 1000ml of sample has been produced of each dilution.
• This 1000 ml have been split in 6 times 150ml to be sent to Labs.
• The remaining 100ml have been used for ColiMinder Measurements
60 bottles of sample have been used to supply three independent labs with 20 samples each -two samples of each
dilution.
Number of samples per Lab 2 10 1000 ml
Number of Labs 3
Volume Per Sample 150 ml Bottles to be sent to the Lab´s
ColiMinder Samples Volume 100 ml Nr Bottles of Volume
Requried Volume Per Sample 1000 ml 60 150 ml
Number of Samples 9 50 ml conical tubes ColiMinder
Control - Blank 1 10 50 ml
10 50 ml
Autoclaved Sample 8114 9000 9 litres 11%
Sample 1886 2000 2 litres 6%
Total Volume Sample 10000 11000 11 litres 10%
Sample Series Prepare 1000 ml per dilution
Dilution Percentage Autoclaved Sample Volume Sample ml
Control - Sample 0 0.0% 1000.0 0.0 ml
Sample 1 0.1% 999.0 1.0 ml
Sample 2 0.5% 995.0 5.0 ml
Sample 3 1.0% 990.0 10.0 ml
Sample 4 2% 980.0 20.0 ml
Sample 5 5% 950.0 50.0 ml
Sample 6 10% 900.0 100.0 ml
Sample 7 20% 800.0 200.0 ml
Sample 8 50% 500.0 500.0 ml
Sample 9 100% 0 1000.0 ml
Total Volumes 8114.0 1886 ml
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Results of 2nd Dilution Series
The lab commissioned to produce the dilution series accidentally used sterile water instead of autoclaved sample to
produce the dilution series. Nevertheless, the results have been evaluated.
ColiMinder Results of the second dilution series show a
good correlation with the dilution factor except the
sample which should contain only 0.1% of raw sample.
This dilution seems to have been produced incorrectly.
Comparing the only the 3 lab results of undiluted sample the results range between 1.5 Mill and 18 Mill CFU while
each result is produced twice with relatively low variation. The results of the three independent laboratories are not
satisfactory.
approximately correct ranking of the samples and even
in this laboratory the factors of dilution were not nearly
reproduced.
Taking all lab results together the box plot below shows
at least a ranking of the samples while the individual
results differ at factors up to several thousand from
each other.
As the results have not been satisfactory and as the dilution series has been produced using sterile water instead of
autoclaved sample, the dilution series experiment has been repeated.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Results of 3nd Dilution Series
Again, the ColiMinder Results of the third dilution
series show a good correlation with the dilution
factor except the sample which should contain
only 0.1% of raw sample. This dilution seems to
have been produced incorrectly. As this is
occurring for the second time it is most likely that
the lab which prepared the series mad a
systematic error in preparation.
Mill CFU which is much better than in the second
Dilution Series. None of the trey labs has been
able to produce a correct ranking of the samples.
Again, the single results of the sample doublets
seem amazingly close.
shows no ranking of the samples. The individual
results differ at factors up to several thousand
from each other. Only the 50% results match
across the different labs.
Conclusion After three dilution series measured with the ColiMinder and 130 parallel lab tests it appears obvious that the
ColiMinder is perfectly able to reproduce the gradation of contamination produced by diluting the sample. The lab
tests in contrary have been unexpectedly unreliable in many respects.
The main question arising is why the lab results are not able to reproduce the actual contamination levels produced
by diluting the sample. As all the labs recognized the blank sample as sterile, there is no doubt that the raw sample
has been diluted with sterile sample/water.
The results produced by the ColiMinder also suggest that the dilution series have been produced correctly, except
some minor deviations.
As none of the labs has been able to deliver reasonable results it has to be assumed that there is an underlying
mechanism which prevents the lab results to reproduce the concentrations produced by diluting in their results.
In the following a hypothesis will be presented which might explain the observations.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Hypothesis Why the Lab Results do not Reproduce the Dilution Factors
Background 3 Dilution Series have been performed.
1st Dilution Series prepared with autoclaved sample, has been measured by the ColiMinder (in triplets) and by one
Lab (single samples).
As the Lab results did not reproduce the dilution factor. The dilution series has been repeated.
2nd Dilution Series prepared with sterile water, has been measured by the ColiMinder (in triplets) and by 3
independent labs in doublets.
The Lab results still do not reproduce the dilution factors but at least a trend visible in the lab results. The dilution
series has been repeated.
3rd Dilution Series prepared with autoclaved sample has been measured by the ColiMinder (in triplets) and by 3
independent labs in doublets.
During 3 dilution series the lab results have not been able to reproduce the dilution factors while the ColiMinder®
(enzymatic activity) results reproduced the dilutions perfectly well across the range. At the same time all Labs
correctly identified the blank sample – the autoclaved or sterile water which has been used for preparing the
dilutions.
The question is why did the lab results not match the dilution factors and why are the results of the dilutions using
sterile water better than the dilutions prepared using autoclaved sample?
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Why did the 130 Lab results not reproduce the dilution factors? – A Hypothesis.
The exemplary below shows three jars,
• first with original sample
• second with autoclaved sample still containing particles but no bacteria
• third sterile water containing no bacteria.
According to the hypothesis the bacteria in the sample tend to stick to particles.
An agglomeration like this would still produce only one CFU.
The Traditional Method would see 12 individual bacteria as 12 CFU but 12 bacteria agglomerated on one particle
would appear as 1 CFU. The Enzymatic Method in contrary does not see a difference between 12 bacteria that are
agglomerated and 12 bacteria which are separated.
Assumed a Lab achieved a result of 10CFU/100ml for the sample and zero for both sterile samples and the
ColiMinder would deliver 10 mMFU/100ml and zero for the sterile samples.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
A 50% dilution using autoclaved sample:
In case the original sample is diluted by autoclaved sample. The autoclaved sample contains the same concentration
of particles as the original but no bacteria.
The Hypothesis assumes that a part of the bacteria occupies the additional particles, reducing the bacterial density
on the particles.
In the culture-based evaluation method each agglomeration of bacteria is appearing as a single colony after
incubation.
In the example the original sample gets diluted with autoclaved sample, after mixing the bacterial concentration
shown in the culture based lab evaluation appears to be the same as in the original sample, as bacteria have
occupied most of the additional particles which have been contained in the autoclaved sample.
Therefore, the culture-based lab tests of the diluted samples show a similar number of CFU as the original sample.
The enzymatic approach in contrary, is not affected by agglomerations of bacteria as the enzymatic activity is
proportional to the number of bacteria independent of the arrangement of the individuals within the sample
volume.
This explains why the enzymatic results correctly reflect the dilution factor while the lab results do not.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Remaining question is why the dilution series produced with sterile water is at least showing some tendency of
reduced contamination in dilutions while the series produced with autoclaved sample does not.
According to the Hypothesis the reason is the following.
As the sterile water does not contain any particles the total number of particles is not increased by the dilution.
The Bacteria therefore tend to stay with “their” particle instead of separating from it. This is seen as the reason why
this dilution series shows a reduction of contamination with increased dilution.
ColiMinder Measurement Parameter
The technology is based on the direct measurement of living indicator bacteria present in the sample.
It is based on the fact that all living bacteria/organisms need to have a metabolism in order to live.
This metabolism is catalysed by enzymes. By measuring the activity of enzymes which are specific to the target
bacteria/group, the contamination level is measured through a parameter which is directly linked to the activity of
the living organisms itself.
The measurements need no incubation time; therefore, the measurement can be done in 15 minutes.
The measurement approach is fully compatible with the most important concept of indicator organisms, which has
been the basic concept of microbiological evaluation for more than 100 years. The enzymatic approach increases the
value of this concept, as in the enzymatic measurement all living indicator organisms produce a signal, while in the
culture-based methods only culturable bacteria representing a (small) subset produce a result.
E. coli, indicator of faecal contamination. Result: β-Glucuronidase activity/Volume
UNITS: [mMFU/100ml] – Thousandth Modified Fishman Units per 100 ml of sample.
Unit Definition - MFU (Modified Fishman Unit): One MFU will liberate 1.0 μg of phenolphthalein from
phenolphthalein glucuronide per hour at pH 6.8 at 37°C.
Besides the activity measurements the ColiMinder also produces a transmission measurement timeline. The
“transmission” signal represents the amount of light transmitting the sample in comparison to the transmitted
intensity when measuring the rinsing water (without reagents).
Contents
INTRODUCTION 1
Introduction 1 1st Dilution Series 2
ColiMinder® Results 2 Lab Results 3 Correlation between Lab Results and ColiMinder Readings 3
Preparation of the 2nd Dilution Series 4 Preparation of Samples 4
Results of 2nd Dilution Series 5 Results of 3nd Dilution Series 6 Conclusion 6
HYPOTHESIS WHY THE LAB RESULTS DO NOT REPRODUCE THE DILUTION FACTORS 7
Background 7 A 50% dilution using autoclaved sample: 9
ColiMinder Measurement Parameter 10
Hong Kong October 2019
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
ColiMinder® - Rapid Microbiological Measurements
Figure 1: ColiMinder “Finn” at Stone Cutters Island Sewage Treatment Works.
Introduction
A ColiMinder® is a fully automated measurement device used to measure the microbiological contamination of
water.
A ColiMinder® (with device name Finn) has been purchased by the DSD and installed at Stone Cutters Island Sewage
Treatment Works (SCISTW) on 4th December 2017. Since this time the ColiMinder is monitoring the sample stream
producing 24 measurements per day.
In preparation of the IWA ASPIRE Conference a dilution experiment has been planned in order to evaluate the
performance of the technology across the full measurement range. Thankfully, DSD has provided its ColiMinder to
perform these experiments.
As the accuracy and reproducibility of the ColiMinder as well as its measurement range and especially the
correlation between lab results and ColiMinder results is of great interest, a dilution series has been produced and
measured 3 times by the ColiMinder.
The same samples have been sent to a lab in order to compare the ColiMinder results to the classic culture-based
lab results.
Introduction
In order to proof the accuracy, reproducibility and the linearity of the ColiMinder® results across the measurement
range, a dilution series has been produced using a sample from a wastewater treatment plan effluent stream.
This sample has been diluted in 8 steps from 100% sample down to 0.1%.
Therefore, the results of the measurement of the diluted sample should be a fraction - corresponding to the dilution
percentage - of the result for the undiluted sample. From the result of the undiluted (100%) sample, a theoretical
value was calculated for each dilution.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
1st Dilution Series A lab has been commissioned to produce a dilution series using autoclaved
sample to dilute the original sample.
10 samples have been prepared: 100%, 50%, 20%, 10%, 5%, 2%, 1%, 0.5%,
0.1%, 0.0%.
In order to validate the correlation with conventional laboratory results, the
diluted samples were split into two parts each. Of the 500 ml prepared from
each of the 10 samples, 450 ml samples were sent to an independent
laboratory and the remaining 50 ml were measured by ColiMinder. The
ColiMinder performed 3 measurements of each sample.
ColiMinder® Results The ColiMinder® measurement have been produced fully automated using the 10-fold multisampling module.
Linearity
FIGURE 2 LEFT: TABLE OF THEORETICAL VALUE (CALCULATED FROM THE 100% RESULTS DOWN) AND MEASURED VALUE (AVERAGE FROM 3
MEASUREMENTS) RIGHT: CORRELATION BETWEEN THEORETICAL VALUE AND MEASURED VALUE
The measurement results of the dilution series proof that the measurement system is performing extremely linear
across the full measurement range.
Reproducibility
Each of the samples has been measured 3 times in order to evaluate the reproducibility of the results.
FIGURE 3 RESULTS TABLE FROM 3 MEASUREMENTS PER DILUTION
The results show that the variation of results is far below 5% for the most part of the series. Only at very low
contaminations the variation is higher. This is most likely to be explained by the fact that these low contaminated
samples can be easily influenced by a minimal carry over from highly contaminated samples, when measured in the
same apparatus.
FIGURE 4 SINGLE MEASUREMENT RESULTS OF 3 MEASUREMENT SERIES LEFT: LINEAR SCALE RIGHT: LOG SCALE
Dilution Series 1 Series 2 Series 3 Average Standard Deviation Percent
0.0% 7.38 0.59 3.98 3.40 85.2
0.1% 6.12 4.24 5.62 5.33 0.79 14.9
0.5% 20.07 19.04 17.64 18.92 1.00 5.3
1.0% 42.43 40.16 38.87 40.49 1.47 3.6
2.0% 88.32 84.03 84.70 85.68 1.88 2.2
5.0% 224.81 215.41 215.39 218.53 4.44 2.0
10% 436.51 425.20 414.71 425.47 8.90 2.1
20% 901.48 880.82 858.01 880.10 17.76 2.0
50% 2289.15 2254.99 2269.08 2271.07 14.02 0.6
100% 4484.67 4503.12 4430.95 4472.91 30.61 0.7
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Lab Results The respective theoretical value for each dilution has been calculated from the lab result of the 100% sample
applying the respective dilution factor.
FIGURE 5 LEFT: TABLE OF THEORETICAL VALUE (CALCULATED FROM THE 100% RESULTS DOWN) AND LAB RESULTS; RIGHT: CORRELATION BETWEEN
THEORETICAL VALUE AND MEASURED VALUE
The lab results do not show any useful accuracy, the blank control showed zero CFU and from the 10% dilution
upward the tendency is correct but the correlation with the actual ratio of bacterial content, produced by the
dilution, is not reproduced.
This test should be reproduced not in single tests per sample but in duplets or triplets.
Correlation between Lab Results and ColiMinder Readings
Since the actual dilution, respectively the reduction of contamination, produced by the diluting the original sample,
has not been reproduced by the lab results, it seems not meaningful to correlate both results.
Based on highes Value
0.0% 0 0
0.1% 930 110000
0.5% 4650 330000
1.0% 9300 580000
2.0% 18600 490000
5.0% 46500 560000
10% 93000 490000
20% 186000 690000
50% 465000 870000
100% 930000 930000
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Preparation of the 2nd Dilution Series
As in the first dilution series lab evaluation of the samples did not deliver any useful results a second dilution series
has been produced. This time the diluted samples have been sent in doublets to three independent labs for
evaluation.
Again, a lab has been commissioned to produce a dilution series from 100% sample down to 0.1% using autoclaved
sample (WWTP effluent) as dilution medium.
Preparation of Samples
In order to produce the dilution series 15 liters of sample have been collected from the WWTP discharge stream.
After 30 min of settling, the upper 11 liters (without sediments) have been used as raw sample.
9 liters of raw sample have been autoclaved to be used as diluent. The remaining 2 liters of raw sample have been
kept at 4°C until the dilutions where produced.
In order to also validate the correlation with traditional lab results the diluted samples have been split into parts.
• 1000ml of sample has been produced of each dilution.
• This 1000 ml have been split in 6 times 150ml to be sent to Labs.
• The remaining 100ml have been used for ColiMinder Measurements
60 bottles of sample have been used to supply three independent labs with 20 samples each -two samples of each
dilution.
Number of samples per Lab 2 10 1000 ml
Number of Labs 3
Volume Per Sample 150 ml Bottles to be sent to the Lab´s
ColiMinder Samples Volume 100 ml Nr Bottles of Volume
Requried Volume Per Sample 1000 ml 60 150 ml
Number of Samples 9 50 ml conical tubes ColiMinder
Control - Blank 1 10 50 ml
10 50 ml
Autoclaved Sample 8114 9000 9 litres 11%
Sample 1886 2000 2 litres 6%
Total Volume Sample 10000 11000 11 litres 10%
Sample Series Prepare 1000 ml per dilution
Dilution Percentage Autoclaved Sample Volume Sample ml
Control - Sample 0 0.0% 1000.0 0.0 ml
Sample 1 0.1% 999.0 1.0 ml
Sample 2 0.5% 995.0 5.0 ml
Sample 3 1.0% 990.0 10.0 ml
Sample 4 2% 980.0 20.0 ml
Sample 5 5% 950.0 50.0 ml
Sample 6 10% 900.0 100.0 ml
Sample 7 20% 800.0 200.0 ml
Sample 8 50% 500.0 500.0 ml
Sample 9 100% 0 1000.0 ml
Total Volumes 8114.0 1886 ml
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Results of 2nd Dilution Series
The lab commissioned to produce the dilution series accidentally used sterile water instead of autoclaved sample to
produce the dilution series. Nevertheless, the results have been evaluated.
ColiMinder Results of the second dilution series show a
good correlation with the dilution factor except the
sample which should contain only 0.1% of raw sample.
This dilution seems to have been produced incorrectly.
Comparing the only the 3 lab results of undiluted sample the results range between 1.5 Mill and 18 Mill CFU while
each result is produced twice with relatively low variation. The results of the three independent laboratories are not
satisfactory.
approximately correct ranking of the samples and even
in this laboratory the factors of dilution were not nearly
reproduced.
Taking all lab results together the box plot below shows
at least a ranking of the samples while the individual
results differ at factors up to several thousand from
each other.
As the results have not been satisfactory and as the dilution series has been produced using sterile water instead of
autoclaved sample, the dilution series experiment has been repeated.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Results of 3nd Dilution Series
Again, the ColiMinder Results of the third dilution
series show a good correlation with the dilution
factor except the sample which should contain
only 0.1% of raw sample. This dilution seems to
have been produced incorrectly. As this is
occurring for the second time it is most likely that
the lab which prepared the series mad a
systematic error in preparation.
Mill CFU which is much better than in the second
Dilution Series. None of the trey labs has been
able to produce a correct ranking of the samples.
Again, the single results of the sample doublets
seem amazingly close.
shows no ranking of the samples. The individual
results differ at factors up to several thousand
from each other. Only the 50% results match
across the different labs.
Conclusion After three dilution series measured with the ColiMinder and 130 parallel lab tests it appears obvious that the
ColiMinder is perfectly able to reproduce the gradation of contamination produced by diluting the sample. The lab
tests in contrary have been unexpectedly unreliable in many respects.
The main question arising is why the lab results are not able to reproduce the actual contamination levels produced
by diluting the sample. As all the labs recognized the blank sample as sterile, there is no doubt that the raw sample
has been diluted with sterile sample/water.
The results produced by the ColiMinder also suggest that the dilution series have been produced correctly, except
some minor deviations.
As none of the labs has been able to deliver reasonable results it has to be assumed that there is an underlying
mechanism which prevents the lab results to reproduce the concentrations produced by diluting in their results.
In the following a hypothesis will be presented which might explain the observations.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Hypothesis Why the Lab Results do not Reproduce the Dilution Factors
Background 3 Dilution Series have been performed.
1st Dilution Series prepared with autoclaved sample, has been measured by the ColiMinder (in triplets) and by one
Lab (single samples).
As the Lab results did not reproduce the dilution factor. The dilution series has been repeated.
2nd Dilution Series prepared with sterile water, has been measured by the ColiMinder (in triplets) and by 3
independent labs in doublets.
The Lab results still do not reproduce the dilution factors but at least a trend visible in the lab results. The dilution
series has been repeated.
3rd Dilution Series prepared with autoclaved sample has been measured by the ColiMinder (in triplets) and by 3
independent labs in doublets.
During 3 dilution series the lab results have not been able to reproduce the dilution factors while the ColiMinder®
(enzymatic activity) results reproduced the dilutions perfectly well across the range. At the same time all Labs
correctly identified the blank sample – the autoclaved or sterile water which has been used for preparing the
dilutions.
The question is why did the lab results not match the dilution factors and why are the results of the dilutions using
sterile water better than the dilutions prepared using autoclaved sample?
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Why did the 130 Lab results not reproduce the dilution factors? – A Hypothesis.
The exemplary below shows three jars,
• first with original sample
• second with autoclaved sample still containing particles but no bacteria
• third sterile water containing no bacteria.
According to the hypothesis the bacteria in the sample tend to stick to particles.
An agglomeration like this would still produce only one CFU.
The Traditional Method would see 12 individual bacteria as 12 CFU but 12 bacteria agglomerated on one particle
would appear as 1 CFU. The Enzymatic Method in contrary does not see a difference between 12 bacteria that are
agglomerated and 12 bacteria which are separated.
Assumed a Lab achieved a result of 10CFU/100ml for the sample and zero for both sterile samples and the
ColiMinder would deliver 10 mMFU/100ml and zero for the sterile samples.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
A 50% dilution using autoclaved sample:
In case the original sample is diluted by autoclaved sample. The autoclaved sample contains the same concentration
of particles as the original but no bacteria.
The Hypothesis assumes that a part of the bacteria occupies the additional particles, reducing the bacterial density
on the particles.
In the culture-based evaluation method each agglomeration of bacteria is appearing as a single colony after
incubation.
In the example the original sample gets diluted with autoclaved sample, after mixing the bacterial concentration
shown in the culture based lab evaluation appears to be the same as in the original sample, as bacteria have
occupied most of the additional particles which have been contained in the autoclaved sample.
Therefore, the culture-based lab tests of the diluted samples show a similar number of CFU as the original sample.
The enzymatic approach in contrary, is not affected by agglomerations of bacteria as the enzymatic activity is
proportional to the number of bacteria independent of the arrangement of the individuals within the sample
volume.
This explains why the enzymatic results correctly reflect the dilution factor while the lab results do not.
VWMs GmbH - Vienna Water Monitoring Solutions, Favoritenstrasse 4-6 / 13, 1040 Wien Lab: Dorfstrasse 17, A-2295 Zwerndorf, AUSTRIA Tel: +43 2284 20188-0, Mail: [email protected], www.v-w-m.at
Remaining question is why the dilution series produced with sterile water is at least showing some tendency of
reduced contamination in dilutions while the series produced with autoclaved sample does not.
According to the Hypothesis the reason is the following.
As the sterile water does not contain any particles the total number of particles is not increased by the dilution.
The Bacteria therefore tend to stay with “their” particle instead of separating from it. This is seen as the reason why
this dilution series shows a reduction of contamination with increased dilution.
ColiMinder Measurement Parameter
The technology is based on the direct measurement of living indicator bacteria present in the sample.
It is based on the fact that all living bacteria/organisms need to have a metabolism in order to live.
This metabolism is catalysed by enzymes. By measuring the activity of enzymes which are specific to the target
bacteria/group, the contamination level is measured through a parameter which is directly linked to the activity of
the living organisms itself.
The measurements need no incubation time; therefore, the measurement can be done in 15 minutes.
The measurement approach is fully compatible with the most important concept of indicator organisms, which has
been the basic concept of microbiological evaluation for more than 100 years. The enzymatic approach increases the
value of this concept, as in the enzymatic measurement all living indicator organisms produce a signal, while in the
culture-based methods only culturable bacteria representing a (small) subset produce a result.
E. coli, indicator of faecal contamination. Result: β-Glucuronidase activity/Volume
UNITS: [mMFU/100ml] – Thousandth Modified Fishman Units per 100 ml of sample.
Unit Definition - MFU (Modified Fishman Unit): One MFU will liberate 1.0 μg of phenolphthalein from
phenolphthalein glucuronide per hour at pH 6.8 at 37°C.
Besides the activity measurements the ColiMinder also produces a transmission measurement timeline. The
“transmission” signal represents the amount of light transmitting the sample in comparison to the transmitted
intensity when measuring the rinsing water (without reagents).