Download validation report ATP bac method final

MICROBI
MARIS
BIOTEC
Prof. Dr. Johannes F. Imhoff
(CEO
MicrobiMaris Biotech)
Report on the validation of a method for the determination of bacteria
in marine water samples based on the measurement of
cellular Adenosin-triphosphate (cATP)
Marine Microbiological Analysis of Ballast Water Samples
Content
Aim of the study ........................................................................................................................................................ 2
Material and Methods............................................................................................................................................... 2
Results ....................................................................................................................................................................... 3
Determination of the minimum cell number for determination of cATP in a water sample .................................... 3
Correlation of cATP concentration and organism density of marine bacteria .......................................................... 3
Validation of the cATP Method for marine water samples ....................................................................................... 3
Conclusion. ................................................................................................................................................................ 5
Appendix ................................................................................................................................................................... 6
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Aim of the study
A method for cATP-determination shall be used to evaluate the number of bacteria in marine
water samples. The method was validated regarding:
1. The concentration of cATP in filtered natural seawater in relation to the density of free
floating bacteria expressed as colony forming units-cfu/ 100 ml
2. The determination of the upper and lower sensitivity range of the method
3. The robustness of the method.
Material and Methods
Samples
An overnight Escherichia coli culture in TSB12 was prepared daily (28°C, 120 rpm) as standard
for the ATP method. Marine water samples were taken from the Kiel Channel and from the
Baltic with sterile Flasks submerged to 1 m below the water surface. Sampling took place at
different days (in September 2013). All samples were cooled and processed within 4 hours
after sampling. All samples were filtered over 1 – 2 µm glass fiber filters into new sterile
flasks in order to remove all larger (i.e. non-bacterial) organisms from the samples.
Dilutions
For measuring of cATP of over-night E. coli cultures, dilutions were made with 9‰ Saline in
sterile flasks under sterile conditions. Each of the dilutions was filtrated over 1 – 2 µm glass
fiber filters into new sterile flasks. To determine the lowest detectable concentration of
micro-organisms, dilutions from 10-1 to 10-5 were prepared of an E. coli culture.
Determination of cATP-content (“ATP-Method”)
The handling followed the test kit instructions for the Quench-Gone Aqueous Test Kit by
aqua-tools (Quench-GoneTM Aqueous - QGATM): For low-solids water-based samples, such
as drinking, cooling and process waters with less than 10% free oil and/or salinity). The
filtered water sample/E. coli culture (50ml, see table raw data, appendix) was submitted to
filtration through a filter attached to a syringe (QG-prep kit). Only living micro-organisms are
retained by the filter’s porosity. Then, lysis of microorganisms was performed directly on the
filter using UltraLyse 7 reagent and Intracellular ATP is released. The ATP solution was diluted
and the concentration of ATP was measured using Luminase reagent (Luciferine/Luciferase
complex). All measurements were done in duplicates or triplicates. An external standard
calibrated ATP solution, Ultracheck1, was used to calibrate the measurements and allowed
reliable quantitative results. The UltraCheck 1 (UC1) Calibration converts luminometer RLU
values into actual ATP concentrations. Final results were equated in pg ATP/ml. One
UltraCheck 1 calibration was used for each set of samples analyzed at the same time.
Determination of CFU/100 mL
To determine the CFU/100ml of the filtrated dilutions, appropriate volumes were incubated
on MacConkey agar plates at 37°C for about 20h. Alternatively, samples were filtered. 1, 5, 10
and 100 mL were filtered (membrane filter discs of nitrocellulose, Ø= 60 mm, Pore: 0,2 μm)
and the filters placed on McConkey agar (selective media for coliforms, colour of colonies
indicate coliform bacteria) and incubated overnight at 37°C. Colonies were counted, too low
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or to high number of colonies were ignored. The mean CFU/plate of three incubated plates
was used to determine the CFU/100ml.
Results
Determination of the minimum cell number for determination of cATP in a water sample
The lower sensitivity limit for the determination of cATP from bacteria in filtered, natural
seawater samples (based on the QGATM test with a volume of 50 mL) has been determined
to be:
a) 20 cfu/100 mL using an overnight culture of E. coli (cATP value of approximately 1 pg/mL)
b) 10 cfu/100 mL using marine water samples (cATP value of approximately 10 pg/mL).
An upper sensitivity range could not be determined by the experiments (samples of filtered,
natural seawater with a normal density of bacteria), as the values did not reach a maximum
level.
Correlation of cATP concentration and organism density of marine bacteria
1000 CFU of marine bacteria per 100 mL correlated to a value of approximately 700 pg
cATP/mL.
Validation of the cATP Method for marine water samples
The cATP method was used to measure different natural sea water samples of different
origin sampled at different days and processed by different executants. In parallel, RLU values
were meassured and the bacterial cell growth was examined by determination of colony
forming units on a complex medium.
Note: The QGA kit measures only Intracellular ATP (cATP) and allows direct quantification of
all living microorganisms. Intracellular ATP is the ATP content in living cells. This is the true
indicator of total viable flora. Measuring living biomass through the quantification of cATP
parameters includes quantification of cultivable, difficult to culture and non cultivable
microorganisms. cATP values can be equated into Equivalent Microorganisms/ml (1pg ATP =
1000 Equivalent microorganisms). Equivalent microorganisms include cultivable
microorganisms and non cultivable microorganisms and this level is not directly comparable to
CFU results from culture methods. However, as a rough estimation, cfu on complex media may
be used as an indicator for bacterial content of sea water from areas under anthropogenic
impacts.
As a consequence, the cATP concentration in a natural sea water sample with a natural
occuring density of marine bacteria is much higher compared to pure cultures of Escherichia
coli with the same estimate of cfu/mL, as many non-cultivable microorganisms are in the
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sample. These organisms will be detected by the cATP-methods but will not grow on the agar
plates used for the colony counting for the cfu determination.
Figure 1 shows five different experiments in comparison. The curves of the five samples
range in the same order of magnitude. However, deviations occurred with the variation of all
parameters: sample origin, day of sampling and executants
900
800
cATP pg/mL
700
600
Sample 1
500
Sample 2
Sample 3
400
Sample 4
300
Sample 5
200
100
0
1,00E+00
1,00E+01
1,00E+02
1,00E+03
cfu/100 mL
Fig. 1 :Correlation of cATP content and density of marine bacteria in filtered, natural seawater
Note: The use of a glas fibre filter (1-2µm) as pre-filtration step reduced the cell number of a
sample of cultured Escherichia coli culture by the factor of 100. It has to be assumed that this
reduction by pre-filtration also happens when analyzing natural seawater samples. A reason
for this phenomenon may be the aggregation of bacteria to larger pellets in a solution (i.e. the
culture of E. coli) or the attachement of bacteria to particles. Both, particles and pellets would
be removed by a filtration step.
An upper sensitivity limit could not be determined by the experiments, as the values for
samples of natural seawater with a normal density of marine bacteria did not reach a
maximum level. A minimum volume of 50 mL filtered, natural seawater is recommended for
the method. Larger sample volumes lead to a higher sensitivity of the method.
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Conclusion.
1. Correlation of the cATP content to the density of bacteria expressed as cfu/100ml
1000 cfu Bacteria/100 mL correlated to cATP of approximately 700 pg cATP/mL. The cATP
concentration in a sea water sample with a normal density of marine bacteria is much higher
compared to a sample of cultured Escherichia coli with the same cfu/mL.
2. Upper and lower sensitivity range
The lower sensitivity limit of method is 10 cfu/100 mL (50 mL of filtered, natural seawater).
An upper sensitivity limit could not be determined by the experiments, as the values did not
reach a maximum level (samples of filtered, natural seawater with a normal density of
marine.
3. Robustness of the method
The method reacts very sensitive with deviations between samples, day of sampling and
executants The ATP method gives very fast answers concerning the presence or absence of
bacteria in relation to a given maximum number of bacteria acceptable in the water sample.
4. Time requirements
Including all preparatory steps, such as dilution, filtration, etc. the method requires 13-15
minutes from sample to result.
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Appendix
List of consumables needed for 400 analyses
Item
Luminase
Luminometer tube
Calibration*
per 1
analysis
per 400
analysis
100µl
100µl
40000µl
14 bottles of Luminase, AQ
1pc
1
Necessary supplies
(AQ : available through Aqua Tools, France)
1pc
400pcs
5 bags 100 each, AQ
GF filter 2 µm
1pc
400pcs
4 boxes 100 each
Syringe, 60 mL
2pc
400pcs
7 boxes 60 each, AQ
QuenchGone filter
1pc
400pcs
5 bags 100 each, AQ
UltraLyse 7
1ml
400ml
2 bottles, AQ
UltraLute
9ml
3600ml
7 bottles, AQ
Extraction tube
1pc
400pcs
11 boxes 25 each, AQ
*1: it is highly recommended to execute several calibrations, during the course of the entire analysis
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Preetz, Germany, December 21st, 2013
Prof.Dr. Johannes F. Imhoff
(CEO Microbi Maris Biotec
MICROBI
MARIS
BIOTEC
Headquarters:
Möwenstieg 6
24221 Preetz, Germany
Tel: 01520-9494251
microbimaris@email.de
Tax-Id.No. : DE255688982
Account details : Commerzbank Kiel, BLZ 21040010, Konto 710437500
Laboratory:
Am Kiel-Kanal 44
24106 Kiel, Germany
Tel: +49 431 600-4450/4433
jimhoff@geomar.de