Introduction to the application of redox-potential measurement in microbiological testing, including MPN. Calibration and validation characteristics are shown. Advantages of this method:
* 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.).
What is culture media
Bacteria culture
Importance of culturing.
Culturing and medium.
History of culture media.
How many types of growth media .
Basic components of culture media.
Classification
Consistancy
Nutritional components
Functional use
Aseptic condittion .
General steps for preparation of culture media .
Selective media .
Enrichment media.
Storage of culture media.
What is culture media
Bacteria culture
Importance of culturing.
Culturing and medium.
History of culture media.
How many types of growth media .
Basic components of culture media.
Classification
Consistancy
Nutritional components
Functional use
Aseptic condittion .
General steps for preparation of culture media .
Selective media .
Enrichment media.
Storage of culture media.
Electron microscope, principle and applicationKAUSHAL SAHU
Introduction
History
Resolution &Magnification of
Electron microscope
Types of electron microscope
1) Transmission electron microscope (TEM)
- Structural parts of TEM
- Principle & Working of TEM
- Sample preparation for TEM
- Advantages & disadvantages of TEM
Scanning electron microscope (SEM)
- Structural parts of SEM
- Principle & Working of SEM
- Sample preparation for SEM
- Advantages & disadvantages of SEM
3) Scanning transmission electron microscope (STEM)
Applications of electron microscope
Conclusion
References
This presentation include information about electron microscope & types of electron microscope i.e. SEM (Scanning electron microscope) & TEM (Transmission electron microscope).
An electron microscope is a microscope that uses a beam of scattered electrons as a source of illumination. It is used to get information about structure, topology, morphology & composition of materials. It has many advantages. Basically there are 4 types of electron microscope but here we will discuss only 2 types.
Transmission electron microscopy is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through it. Its resolution & magnification is about 10,000,000x. There are 5 types of transmission electron microscope i.e. BFTEM (Bright field transmision electron microscope), DFTEM (Dark field transmission electron microscope), HRTEM (High resolution transmission electron microscope), EFTEM (Energy filtered transmission electron microscope), ED (Electron diffraction). there are 4 techniques of TEM i.e. negative staining, shadow casting, Freeze fracture replication, freeze etching. It has many applications e.g, for the study of Cancer research, virology, chemical industry, electronic structure etc.
A scanning electron microscope is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. Types of signals produce by SEM include secondary electrons, back scattered electrons, X-rays, light rays. There are many advantages of SEM e.g, Btter resolution, fast imaging easy to operate, work with low voltage etc.
Why should you use Bottle Top Dispensers ?Microlit India
Bottle Top Dispensers can facilitate a broad range of applications and increase safety,speed and reliability of daily lab work.
Choosing a bottle top dispenser saves time,work and protects you and your samples.
This presentation by Microlit will help you to understand the need for using Bottle Top Dispensers.
Nutritional requirements of bacteria and nutrient media (2) copyvinaya warad
To understand nutritional requirements of bacteria
To study nutritional classification of bacteria
To study constituents of nutrient media
To understand types of nutrient media.
To understand uses of different nutrient media
Enumeration is counting of microorganisms present in a sample.
This is done to know the intense of presence of the spoilers in the spoiled food.
To detect which type of organism is responsible for the spoilage.
Mostly this is done two important methods.
Viable count
Total count
VIABLE COUNT:
A viable cell count allows one to identify the number of actively growing or dividing cells in a sample.
The plate count method or spread plate method relies on bacteria growing a colony on a nutrient medium.
Number of colonies can be counted.
Plate count agar is used for general count
MacConkey agar is used for Gram negative organisms.
TOTAL COUNT:
The initial analysis is done by mixing serial dilution of sample in liquid nutrient agar which is then poured into bottles.
The bottles are then sealed and laid on their sides to produce a slopping agar surface.
The colonies are then counted by eye.The total number of colonies are said as Total Viable Count. The initial analysis is done by mixing serial dilution of sample in liquid nutrient agar which is then poured into bottles.
The bottles are then sealed and laid on their sides to produce a slopping agar surface.
The colonies are then counted by eye.The total number of colonies are said as Total Viable Count.
Pour plate method:
The same procedure is done for this till serial dilution.
The serially diluted sample is then mixed with the molten nutrient agar.
Then poured onto the sterile petridish.
Incubated under appropriate temperature amd the colonies where counted.
ConclusionThe enumeration of these spoiled food samples are important to encounter the type of microbe is causing the spoilage.
And hence this is used to prevent the same type of spoilage.
This can be avoided by making the environmental changes which inhibits the organism which is responsible for the spoilage.
Electron microscope, principle and applicationKAUSHAL SAHU
Introduction
History
Resolution &Magnification of
Electron microscope
Types of electron microscope
1) Transmission electron microscope (TEM)
- Structural parts of TEM
- Principle & Working of TEM
- Sample preparation for TEM
- Advantages & disadvantages of TEM
Scanning electron microscope (SEM)
- Structural parts of SEM
- Principle & Working of SEM
- Sample preparation for SEM
- Advantages & disadvantages of SEM
3) Scanning transmission electron microscope (STEM)
Applications of electron microscope
Conclusion
References
This presentation include information about electron microscope & types of electron microscope i.e. SEM (Scanning electron microscope) & TEM (Transmission electron microscope).
An electron microscope is a microscope that uses a beam of scattered electrons as a source of illumination. It is used to get information about structure, topology, morphology & composition of materials. It has many advantages. Basically there are 4 types of electron microscope but here we will discuss only 2 types.
Transmission electron microscopy is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through it. Its resolution & magnification is about 10,000,000x. There are 5 types of transmission electron microscope i.e. BFTEM (Bright field transmision electron microscope), DFTEM (Dark field transmission electron microscope), HRTEM (High resolution transmission electron microscope), EFTEM (Energy filtered transmission electron microscope), ED (Electron diffraction). there are 4 techniques of TEM i.e. negative staining, shadow casting, Freeze fracture replication, freeze etching. It has many applications e.g, for the study of Cancer research, virology, chemical industry, electronic structure etc.
A scanning electron microscope is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. Types of signals produce by SEM include secondary electrons, back scattered electrons, X-rays, light rays. There are many advantages of SEM e.g, Btter resolution, fast imaging easy to operate, work with low voltage etc.
Why should you use Bottle Top Dispensers ?Microlit India
Bottle Top Dispensers can facilitate a broad range of applications and increase safety,speed and reliability of daily lab work.
Choosing a bottle top dispenser saves time,work and protects you and your samples.
This presentation by Microlit will help you to understand the need for using Bottle Top Dispensers.
Nutritional requirements of bacteria and nutrient media (2) copyvinaya warad
To understand nutritional requirements of bacteria
To study nutritional classification of bacteria
To study constituents of nutrient media
To understand types of nutrient media.
To understand uses of different nutrient media
Enumeration is counting of microorganisms present in a sample.
This is done to know the intense of presence of the spoilers in the spoiled food.
To detect which type of organism is responsible for the spoilage.
Mostly this is done two important methods.
Viable count
Total count
VIABLE COUNT:
A viable cell count allows one to identify the number of actively growing or dividing cells in a sample.
The plate count method or spread plate method relies on bacteria growing a colony on a nutrient medium.
Number of colonies can be counted.
Plate count agar is used for general count
MacConkey agar is used for Gram negative organisms.
TOTAL COUNT:
The initial analysis is done by mixing serial dilution of sample in liquid nutrient agar which is then poured into bottles.
The bottles are then sealed and laid on their sides to produce a slopping agar surface.
The colonies are then counted by eye.The total number of colonies are said as Total Viable Count. The initial analysis is done by mixing serial dilution of sample in liquid nutrient agar which is then poured into bottles.
The bottles are then sealed and laid on their sides to produce a slopping agar surface.
The colonies are then counted by eye.The total number of colonies are said as Total Viable Count.
Pour plate method:
The same procedure is done for this till serial dilution.
The serially diluted sample is then mixed with the molten nutrient agar.
Then poured onto the sterile petridish.
Incubated under appropriate temperature amd the colonies where counted.
ConclusionThe enumeration of these spoiled food samples are important to encounter the type of microbe is causing the spoilage.
And hence this is used to prevent the same type of spoilage.
This can be avoided by making the environmental changes which inhibits the organism which is responsible for the spoilage.
experiment of
1. isolation of microbes from soil
2. enumeration of microorganism
3. identification of microorganism
(must includes background, topic, objective, research question , ways to solve it)
This presentation gives an overview of : Validation of microbiological methods , Considering some of the limitations and
Key criteria that may be applicable for assessment.
Application of MicroTester for detection of low microbial contaminationOlivér Reichart
Advantades of the redox method in the evaluation of membrane filtration:
* 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.
A portable biosensor system for bacterial concentration measurement in liquid...Marco Grossi
A portable biosensor system for bacterial concentration measure using the impedance technique is here presented. The system is intended for in situ measurements in industrial environments.
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Microbiological inspection of mineral water by redox-potential measurement Olivér Reichart
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STERILITY INDICATORS : Physical Indicators, Chemical Indicators
Biological Indicators
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2. Problems in microbiological
quality control
Classical methods
Long incubation time (1-4 days)
The applicability, reliability and test price of the
methods are concentration-depending:
High concentration: dilution and colony
counting in the range of
30-300 cfu/ml.
Low concentration: MPN method
Membrane filtering
6. In biological systems
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]
7. The electric effect of the changing could be expressed
by the Nernst equation:
RT [oxidant] [H+]
Eh = E0 + ------ ln ----------------
nF [reductant]
RT [reductant]
Eh = E0 - ------ ln ----------------
nF [oxidant] [H+]
Where Eh is the redox-potential referring to the normal
hydrogen electrode (V)
E0 is the normal redox-potential of the system (V)
R is the Gas-constant R = 8.314 J/mol K
F is the Faraday constant F = 9.648˙104 C/mol (J/V mol)
n is the number of electrons in the redox system (n=1)
9. Typical redox-curve of the
microbial growth
E. coli 37 °C, TSB
-400
-300
-200
-100
0
100
200
300
400
500
0 1 2 3 4 5 6 7 8 9
t (h)
Eh(mV)
3
4
5
6
7
8
9
lgN
Eh lg N
|dE/dt|>DC
lg Nc
lg N0
TTD
10. The detection time (TTD) is that moment when
the absolute value of the rate of redox potential
change in the measuring-cell overcomes a value
which is significantly differing from the random
changes (e.g. |dE/dt| 0.5 mV/min).
This value is the detection criterion. As the
critical rate of the redox potential decrease
needs a determined cell count the detection time
depends on the initial microbial count.
11. Redox-curves of several bacteria
-400
-300
-200
-100
0
100
200
300
400
500
0 5 10 15 20
t (h)
Eh(mV)
Campylobacter B. subtilis L. monocytogenes
Ent. faecalis Ps. aeruginosa E. coli
12. Effect of the initial Cell-
concentration on the redox-curves
E. coli in TSB
-400
-300
-200
-100
0
100
200
300
400
0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960
t (min)
Eh(mV)
Steril steril lgN=0,09 lgN=2,38
lgN=3,39 lgN=4,25 lgN=4,80
TTD for the redox-potential measurement is: |DE/D t|>1mV/min
13. Effect of the initial cell
concentration on TTD
E. coli in TSB
0
1
2
3
4
5
6
2 3 4 5 6
lgNo (cfu/inoculum)
TTD(h)
14. Determination of calibration
curves
1. External calibration curve
Known microflora
The equation of the calibration curve is
calculated by linear regression from the log
N (determined by classical cultivation) and
the TTD (is determined instrumentally)
15. Determination of calibration
curves
2. Internal calibration curve
Unknown microflora
This method is applied when the composition of the
microflora is not known and previously constructed
calibration curve cannot be taken. In this case, the
redox potential measurement is combined with the
MPN method. Based on the last dilution levels still
showing multiplication, the initial viable count is
calculated using the MPN-table. Based on the
obtained microbe count and TTD values, the internal
calibration curve can be constructed.
21. Test microorganisms and culture
media of the tests 2.
Microorganisms Redox
potential
Plate
counting
Pseudomonas
aeruginosa
Cetrimide,
TSB
TSA,
Cetrimide
Pseudomonas
fluorescens
Cetrimide,
TSB
TSA,
Cetrimide
Enterococcus
faecalis
Azide, TSB TSA, Slanetz-
Bartley
Total count TSB TSA
22. Validation characteristics of the
method 1.
Selectivity
it depended on the media used for
identification.
Linearity
from 1 to 107cfu/test flask.
23. Validation characteristics of the
method 2.
Sensitivity
Detection limit
1 cell/test flask.
Quantitation limit
The theoretical quantitation limit is 10 cell/inoculum
(1 log unit), which is in agreement with the
obtained calibration curves.
min13060
Nlg
TTD
24. Validation characteristics of the
method 3.
Range
On the base of the calibration curves the range
lasted from 1 to 7 log unit. Below 10 cells the
Poisson-distribution causes problems, over 107
cells the TTD is too short comparing to the
transient processes (temperature-, redox-
equilibrum, lag-period of the growth).
Repeatability
Calculated from the calibration curves:
SDlgN = 0.092
SDN = 100.092 = 1.24 = 24%
25. Validation characteristics of the
method 4.
Robustness
The most important parameter is the
temperature, which has a double effect on the
results – the growth rate of the microorganisms
and the measured redox-potential are
temperature depending. Performing the
measurements at the temperature optimum of
microorganisms, the growth rate in a ±0.5 °C
interval does not change. The effect of the
temperature on the measured redox-potential
was determined experimentally. The results
showed that the effect of the temperature
variation is negligible.
26. Advantages of the redox-
potential measurement 1.
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.
27. Advantages of the redox-
potential measurement 2.
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.).
28. Application of the redox method
1. Quality control
Foods
Water
Surfaces
2. Heat destruction of bacteria
3. Activity of bacteria
4. Media optimization
5. Efficiency of disinfectants
29. Quality control 1.
Foods
Enterobacter and total count in raw milk
Nyerstej, 1/2 TSB (T=30 °C)
-400
-300
-200
-100
0
100
200
300
400
500
0 5 10 15 20 25
t (h)
Eh(mV)
0. hig. 1. hig. 2. hig. 3. hig. 4. hig
5. hig 6. hig 7. hig.
30. Quality control 1.
Foods
Enterobacter and total count in raw milk
Nyerstej belső kalibrációs görbe
(1/2 TSB, T=30 °C)
y = 2,6486x + 1,34
R
2
= 0,9895
0
5
10
15
20
0 1 2 3 4 5 6 7
hígítás
TTD(h)
Összcsira Enterobacter
MPNEnterob.=2,3x102
/ml
MPNÖsszcsíra=2,3x106
/ml
37. Quality control 3.
– The microflora present on the swab is directly
measurable without washing. There is no statistically
significant difference between the microbial counts
obtained with redox-potential measurements and the
plating method.
– By help of internal calibration curve, the viable count
of surfaces with unknown microflora may also be
determined. In further studies of surfaces with
identical microflora, the already established
calibration curve may be applied as an external
calibration curve. Observing the shape of the redox-
curves both the total count and Enterobacterial count
can be determined simultaneously, applying non
selective nutrient broth (TSB) in a single, common
measurement system.
38. Quality control 3.
– Comparing the time requirement of the methods, the
traditional plating method demands 3 days for the
determination of total count while by the redox
method, using internal calibration and depending on
the level of surface contamination, the viable count
can be determined within 15-20 hours or using
external calibration curve (depending on the level of
the surface contamination) it may be determined
within 4-8 hours.
– Applying external calibration curve, when washing of
swabs and the preparation of dilution series are not
necessary, the duration of the examination, the
material, tool and labor requirements can significantly
be reduced.
41. Calibration diagrams
Campylobacter in different selective broths y = -176,56x + 2026,1
R
2
= 0,9738
0
200
400
600
800
1000
1200
1400
1600
1800
2 3 4 5 6 7 8
42. Heat destruction experiments
3 different models:
Classical isotherm model
Redox isotherm model
Redox anisotherm model
43. Thermal death curve –
Classical isotherm method
Classical isotherm
thermal death curve y = -0,086x + 5,3621
R2
= 0,9987
-0,5
0
0,5
1
1,5
48 53 58 63
T (°C)
lgD
Z=11.62°C
44. Thermal death curve –
Redox isotherm method
Thermal death curve y = -0,1012x + 6,2336
R2
= 0,954
-0,5
0
0,5
1
1,5
50 52 54 56 58 60 62 64 66
T (°C)
lgD
Z=9.88°C
45. Thermal death curve –
combined isotherm results
Combined thermal death curve y = -0,092x + 5,7014
R2
= 0,971
-0,5
0
0,5
1
1,5
48 53 58 63
T (°C)
lgD
Z=10.86°C
46. Simplified determination of z-
value
Calibration curve: lgN=a-b·TTD
Decimal reduction time:
D=-Δt/ΔlgN= Δt/(b· ΔTTD)
lgD=lgΔt-lgb-lg(ΔTTD)T
From the thermal death curve:
z
1
T
Dlg
47. Simplified determination of z-
value
z
1
T
TTDlg
T
blg
T
tlg
T
Dlg
D
D
lgΔTTD is a linear function of temperature,
from the slope the z-value can be calculated
T
z
1
ATTDlg D
48. Determination of z-value from
anisotherm heat treatment
On the base of calibration curve: z=9.37 °C
Thermal death curve
y = -0,1067x + 5,5218
R2
= 0,9779
-0,8
-0,7
-0,6
-0,5
-0,4
-0,3
-0,2
-0,1
0
54 55 56 57 58 59
T (°C)
lgD
49. Determination of z-value from
anisotherm heat treatment
On the base of TTDs: z=9.37 °C
Anisotherm heat treatment y = 0,1067x - 3,5787
R2
= 0,9779
1,5
1,8
2,1
2,4
2,7
3
54 55 56 57 58 59
Ti(°C)
lgΔTTD