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and in some cases, subspecifi c characterization. Advances in technology have made
detection and identifi cation faster, more sensitive, more specifi c, and more convenient than traditional assays. These new methods include for the most part antibodyand DNA-based tests, and modifi cations of conventional tests made to speed up
analysis and reduce handling.
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In Pharmaceutical different grades of waters are used and they all must be tested firest before using it for manufacturing any products. Products sometimes get contaminated because of presence of endotoxins so they mus be checked by performing BET test
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In-House Microbial Isolates in Compendial Testing: Regulatory RequirementsRobert Westney
This presentation provides an overview of the current regulatory expectations for the use of in-house microbial isolates in compendial testing. It reviews regulatory, compendial and industry references on the topic. Importantly, it also provides a strategy for selection of these isolates.
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The detection and enumeration of microorganisms in food are an essential
part of any quality control or food safety plan. Traditional methods of detecting foodborne pathogenic bacteria are often time-consuming because of the need for growth
in culture media, followed by isolation, biochemical and/or serological identifi cation,
and in some cases, subspecifi c characterization. Advances in technology have made
detection and identifi cation faster, more sensitive, more specifi c, and more convenient than traditional assays. These new methods include for the most part antibodyand DNA-based tests, and modifi cations of conventional tests made to speed up
analysis and reduce handling.
Microbial analysis of water system and endotoxin estimationashapatel676
In Pharmaceutical different grades of waters are used and they all must be tested firest before using it for manufacturing any products. Products sometimes get contaminated because of presence of endotoxins so they mus be checked by performing BET test
Rapid sterility testing system is an automated solution for the rapid detection, response, and resolution of microbial contamination in filterable samples throughout the manufacturing process. Accurate, rapid sterility testing is not only critical for patient safety it also makes great sense for the compounders and other pharmaceutical manufacturers.
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* The time requirement of the redox-potential technique is significantly lower than that of the classical nutrient methods.
* While the classical methods use only 1 membrane in 1 Petri dish the redox-potential method makes possible to evaluate even 5 or more filters in one test cell. That means not only a 5 times lower detection limit of microbes but results in a remarkable cost reduction as well.
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* Very simple measurement technique.
* It does not require strict temperature control.
* Rapid method, especially in the case of high contamination.
* Applicable for every nutrient broth (impedimetric methods require special substrates with low conductance).
* Especially suitable for the evaluation of the membrane filter methods.
* Economic, effective and simple method for heat destruction measurements.
* Effective tool for the optimization of the nutrient media.
* The test costs are less than those of the classical methods, especially in the case of zero tolerance in quality control (coliforms, Enterococcus, Pseudomonas, etc.).
MPN is most commonly applied for quality testing of water, i.e., to ensure whether the water is safe or not in terms of the bacteria present in it. The presence of very few faecal coliform bacteria indicates that the water is likely free of disease-causing organisms.
video link: https://youtu.be/vOB9AQo54vU
A scientific presentation that describes the steps to perform antimicrobial sensitivity assay for water insoluble compounds and the type of data that can be obtained.
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Result and interpretation
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2. Introduction
MicroTester as a validated method is suitable for rapid
microbiological testing of mineral water, carbonated water, tank
and running drinking water and other types of water. The time
needed for a reliable detection of microorganisms is of key
importance: in water industry the real-time (or at least as fast as
possible) monitoring of the microbiological properties of the
production is indispensable; in public water supply the essential
basis of the epidemiological and public health measures is the
fast and reliable result of the microbiological inspection. Beside
the most important and most widely inspected microbiological
contaminants the most relevant disturbing flora was involved to
the validation process as well.
3. Theoretical base
The energy source of the growth is the biological
oxidation which results in a reduction in the
environment.
This is due to the oxygen depletion and the production of
reducing compounds in the nutrient medium.
A typical oxidation-reduction reaction in biological
systems:
[Oxidant] + [H+] + n e- [Reductant]
4. A typical redox curve of the
microbial growth
DC: Detection Criterion
TTD: Time to Detection
5. Microorganisms
The most frequently tested contaminant
microorganisms in mineral water productions
are:
Coliforms
Escherichia coli
Pseudomonas aeruginosa
Enterococcus faecalis
Total count (22 °C and 37 °C)
7. Selectivity 1.
Coliforms and Acinetobacter lwoffii in BBL.
(K.o.: Klebsiella oxytoca, Ent.: Enterobacter aerogenes, Citro.: Citrobacter freundii,
E.c.: Escherichia coli, Acin.: Acinetobacter lwoffii)
Coliforms and Acinetobacter in BBL
-400
-200
0
200
400
600
0 5 10 15 20
t (h)
Eh(mV)
K.o.lgN=3,55 Citro lgN=3 Ent lgN=3,48
E.c. lgN=3,67 Acin. lgN=3,65
8. Selectivity 2.
Micrococcus and Enterococcus in Azid broth
Enterococcus and Micrococcus
0
50
100
150
200
250
300
350
400
0 5 10 15 20
t (h)
Eh(mV)
Enterococcus Micrococcus
9. Selectivity 3.
Growth in Cetrimide broth
0
100
200
300
400
500
0 5 10 15 20 25
t (min)
Eh(mV)
Ps. aeruginosa Ps. fluoresc. E. coli Enterococcus
Pseudomonas aeruginosa, Pseudomonas fluorescens,
E. coli and Enterococcus faecalis in Cetrimid broth.
10. Linearity
The linear relationship between the logarithm of the
cell concentration and TTD values is demonstrated by
the calibration curves. From the concentrated
suspensions of the test microorganisms tenfold dilution
series were prepared in physiological salt solution. From
the members of the dilution series the redox-potential
test flasks were inoculated with 1.0 ml suspension and
the TTD values were determined.
11. Linearity
Calibration curves of Coliforms
Coliforms in BBL
y = -1.699x + 17.004
R2
= 0.9958
y = -1.471x + 14.26
R2
= 0.9714
y = -1.3506x + 12.896
R2
= 0.9941
y = -1.1775x + 10.184
R2
= 0.9907
0
5
10
15
20
0 1 2 3 4 5 6 7
lgN (cfu/cell)
TTD(h)
Citrobacter Klebsiella oxytoca Enterobacter E. coli (37 °C)
12. Linearity
Calibration curve of E. coli
Escherichia coli
y = -0.8393x + 7.1607
R2
= 0.9988
0
2
4
6
8
0 1 2 3 4 5 6 7
logN/100 ml
TTD(h)
17. Detection limit
The detection limit is 1 cell/test flask, so the
system is suitable for the absence/presence tests,
so considerable costs and time could be saved
with more membrane filters joined together.
On the base of the calibration curves the range
lasted from 1 to 7 log unit.
18. Repeatability
The repeatability calculated from the calibration
curves:
SDlgN = 0.092
SDN = 100.092 = 1.24 = 24%
which complies with the requirements of
microbiological methods.
19. Quality control tests
72 bottles tested for Coliform
Testing method of Laboratory
Membrane filtering of 3x250 ml mineral water with 1
filter. Cultivation Tergitol agar at 37 °C for 48 h. One
Petri dish represents 3 bottles of mineral water.
Redox-potential measurement method
Membrane filtering of 3x250 ml mineral water with 1
filter, placing 4 membranes into 1 test flask containing
BBL broth. Temperature: 37 °C. One test flask
represents 12 bottles of mineral water.
Positive control: 1 ml of Citrobacter freundii suspension
(lgN = 3.66)
21. Quality control test
Bottles 1.-12. 13.-24. 25.-36. 37.-48. 49.-60. 61.-72.
Laboratory negative negative negative negative negative negative
Redox negative negative negative negative negative negative
Results of 72 bottles test
22. 66 bottles tested for Coliforms
Testing method of Laboratory
Membrane filtering of 3x250 ml mineral water with 1 filter.
Cultivation Tergitol agar at 37 °C for 48 h. One Petri dish
represents 3 bottles of mineral water.
Redox-potential measurement method
Membrane filtering of 3x250 ml mineral water with 1 filter,
placing 3 membranes into 1 test flask containing BBL broth.
Temperature: 37 °C. One test flask represents 9 bottles of
mineral water.
Besides the mineral water two technological water samples were
tested for Coliforms
Positive control: 1 ml of Escherichia coli suspension (lgN = 6.7)
23. Quality control test
Results of redox-potential measurement of 66 bottles
66 bottles
-400
-300
-200
-100
0
100
200
300
400
500
0 2 4 6 8 10 12 14 16 18 20 22 24
t (h)
Eh(mV)
1.-9. 10.-18. 19.-27. 28.-36. 37.-45. 46.-54.
55.-63. 64.-66. E.coli (+) Negativ
24. Quality control test
Samples 1.-66. Bottles Water sample 1. Water sample 2.
Laboratory results negative negative negative
Redox method negative negative negative
Results of 66 bottles test
25. Detection time of one cell
Microbe One cell detection time (h)
Escherichia coli 11
Citrobacter freundii 23
Pseudomonas aeruginosa 24
Enterococcus faecalis 15
26. Results of industrial tests
Microbe
All
measurements
(piece)
Match the
standard
test (%)
False
positive
results
(%)
False
negative
results
(%)
Escherichia
coli
942 99,89 0,11 0,00
Coliform 4674 99,87 0,00 0,13
Enterococcus 3000 99,93 0,00 0,07
Pseudomonas
aeruginosa
3372 99,82 0,06 0,12
27. Advantages of the redox-potential
measurement
Very simple measurement technique.
Rapid method, especially in the case of high
contamination.
Applicable for every nutrient broth
Especially suitable for the evaluation of the membrane
filter methods.
The test costs are less than those of the classical
methods, especially in the case of zero tolerance
(Coliforms, Enterococcus, Pseudomonas, etc.).