DE102008014029A1 - Determining microbial contamination of water, comprises supplying sample in a reaction chamber, adding determined quantity of nutrient solution, which is a minimal medium, and intermixing the mixture of nutrient solution and water sample - Google Patents

Abstract

The method comprises supplying a sample (12) in a reaction chamber, adding a determined quantity of a nutrient solution (16), which is a minimal medium, intermixing the mixture of nutrient solution and water sample by circulating the sample/nutrient solution mixture in a circuit, measuring an optical characteristic of the mixture, transferring the measured value to a data processing unit, and repeating the intermixing, measuring and transferring steps for a determined time or until no significant change of the optical characteristic between two measurements is detected. The method comprises supplying a sample (12) in a reaction chamber, adding a determined quantity of a nutrient solution (16), which is a minimal medium, intermixing the mixture of nutrient solution and water sample by circulating the sample/nutrient solution mixture in a circuit, measuring an optical characteristic of the mixture, transferring the measured value to a data processing unit, repeating the intermixing, measuring and transferring steps for a determined time or until no significant change of the optical characteristic between two measurements is detected, and rinsing the reaction chamber with a cleaning solution (18). The water sample is obtained in a continuous flow by a bypass. The addition of the nutrient solution is carried out, so that a determined volume of the sample solution is replaced from the reaction chamber. The ratio of the sample to the nutrient solution in the reaction chamber is 1:1-1:100. The nutrient solution contains short-chain alcohols, short-chain organic acids and/or their salts as carbon source. The measured optical characteristic is the turbidity of the sample. The determined constant measured value is correlated at the end of the measurement and/or is correlated in the course of the measurement with the bacterial count of the sample. The change of the measured value is correlated in the course of the measurement with the bacterial count of the sample. The measurements are carried out with different nutrient solutions, which optionally contain special indicators. The process is automatically carried out in determined time intervals. An independent claim is included for a device for determining microbial contamination of water.

Description

Field of the invention

The The invention relates to a method for determining the germ load of fluids, in particular water, and a device for carrying out of the procedure.

State of the art

Untreated Water is never free of organisms, especially microorganisms. Therefore, the amount of these organisms in the water plays an important role Role in the assessment of water quality. Such is for example in Germany for perfect drinking water a total germ count of less than 10 colony forming units per milliliter (CFU / mL). This number indicates the number of colonies formed, which in incubation counted by one milliliter on a specific nutrient medium become. In view of the large number of different Microorganisms are not all possible in one sample prove existing microorganisms, since only certain Species can multiply under the chosen conditions. Therefore, in the determination of the total germ count always from the multiplication certain indicator microorganisms on the total bacterial count of the Sample closed.

in the Below is under germ count or total germ count always a more accurate Numerical value understood for example in cfu / mL while under germ load always an approximate value, such as high or low, is understood.

The However, germ contamination of water is not just for drinking water of great importance. Especially because of the increasing costs build more and more manufacturing processes on closed water circuits, for example for cooling. Because it is not possible is to be able to completely sterilize the entire system there are large amounts of microorganisms nesting there. Also in large water tanks, such as in example Steam power plants, microorganisms can become outstanding multiply. A monitoring of the burden of these systems is not only for reasons of safety at work, but also about the effectiveness of sterilization systems or measures to check for sterilization, there such measures often associated with high costs.

always then, if untreated water is to be used, must be sure that it is not too heavily loaded. So is especially in areas with difficult drinking water supply the use of untreated water, for example for irrigation of Garden or sports facilities advantageous, since thus the drinking water supplies can be spared. But equally important is the monitoring of public waters, such as streams, Rivers, lakes or their supply lines, such as sewage of sewage treatment plants, industrial wastewater, etc., as a strong Germ burden leads to major health problems. A fast method for determining the germ load of water is therefore necessary so as to be able to respond quickly to possible contaminations to be able to react.

Next In terms of health, the germ burden also plays a role other areas an important role. So is the carryover of many thousands of microorganisms in the ballast water of ships a big ecological Problem. Therefore, in 2005 became a ballast water agreement closed, which determines when ballast water will be drained may. There are also limit values for the amount of organisms and the germ count in ballast water has been established as well as guidelines for ballast water management.

conventional Methods for determining total bacterial count, such as cast plate methods, Titre method and MPN method (most probable number) are since long standard procedure and known in the art. In the cast plate process An agar plate is incubated with the water sample and taken numbering colonies. For the other two procedures are dilution series of the sample with nutrient medium offset and from the dilution of still showing growth Sample determines the total germ count. These methods are not just complicated to carry out, since they work sterile and require a microbiological laboratory, but they need it also an incubation period of at least 2 days.

Next of the total bacterial count can also pathogenic germs, such as E. coli, by choice of nutrient medium or specific reagents be determined selectively.

The conventional methods require, as stated above, a high technical effort and are therefore not suitable, eg. to determine the germ load in the context of automated plants. The samples can not be timely for sampling be measured. Therefore, special measures need to be taken be taken against a change in the sample, like For example, sterile vessels and cooling the Sample.

Next the total germ count or germ count is for many applications also a reliable statement about the germ load sufficient, whether a certain number of germs is exceeded.

Out EP 0 304 406 A2 a device is known which can determine the germ load of liquids in a relatively short time. However, the device is complicated and requires different solutions and at least two measuring cells.

task

task The invention is to provide a method which in short time and as simple as possible the germ load or determine the total bacterial count of fluids. It is also the task of Invention to provide a device which the determination of Germ load or the total germ count allowed and thereby due operate in a fully automatic and self-sufficient manner thanks to its simple design can.

solution

These Task is by the inventions with the characteristics of the independent Claims solved. Advantageous developments The inventions are characterized in the subclaims. The wording of all claims hereby by reference to the content of this description. The invention also includes all meaningful and especially all mentioned Combinations of independent and / or dependent Claims.

in the Below individual process steps are described in more detail. The steps do not necessarily have to be in the specified Order to be performed, and the procedure to be described may also have other, unspecified steps.

At the The method according to the invention is a sample of to be tested fluid or liquid, in particular Water, fed to a reaction space. It may be appropriate be the reaction space several times with the liquid to be tested to rinse, to falsify the measurement by a contamination of the sample by previously present in the reaction space Minimize liquids.

After that is a certain amount of a nutrient solution placed in the reaction space. The nutrient solution is used as a growth medium for the microorganisms to be detected. The nutrient solution is not a complete medium, but a minimal medium. These are media that affect the microorganisms only a limited amount and type of nutrients available put. For example, the carbon source, Nitrogen source or certain essential amino acids a limiting factor. Just this property allows it, in the process according to the invention a high reproducibility to achieve. If there is too much growth of all microorganisms the danger that the measuring system gets too dirty and already low contamination of the sample to large measurement errors to lead. According to the invention, minimal media preferably, which only one or more carbon sources contain. These media are for conventional Method not suitable because it increases the growth of microorganisms To slow down too much compared to the full medium. It is important that the microorganisms preferred by these media are reliable Allow extrapolation to the total germ count of the sample. It will - like already executed - also in the conventional methods with full media always closed on the total germ count. It can the sample to be measured the nature of the most suitable medium determine.

at the appropriate choice of nutrient medium may be in reverse also to the presence of certain types of microorganisms, For example, certain pathogens are examined.

When Next, the liquid in the reaction space mixed, thus a homogeneous solution as possible of sample and nutrient solution arises. Of this Mix is immediately measured an optical property to the Starting value of this property, before the beginning of the multiplication of microorganisms to determine. It is important that the optical property is also changed by the growth of microorganisms. The optical property can be, for example, the turbidity the fluid or a change in absorption be of specific wavelengths. This change can also by the addition of certain substances, for example Color indicators, done to the nutrient medium, which, for example can indicate a change in the pH.

As already mentioned, changes the growth of microorganisms the measured optical property of the sample. Because these changes tion can be realized very sensitively, can already very much minor changes are registered and evaluated.

The Measurement will be either for a certain time or until no significant change in property between two Measurements more detected, repeated. A significant change is preferably a change from below the measurement accuracy or, for example, below 1% of the measured value. alternative Of course, only one more measurement after one specific incubation period.

All measured values are transmitted to a data processing unit and related to the measured starting value for evaluation.

The Incubation time depends on the conditions of the measurement, for example, the growth of microorganisms of temperature, pH, Amount of nutrient solution is affected. Prefers the conditions are chosen such that the measurement after a maximum of four hours, more preferably between one and three hours can be ended. However, in principle also measurements possible with a duration of only a few minutes.

To The measurement is the reaction chamber with a cleaning solution cleaned him up for the next measurement sterilized. As a cleaning solution can usual solutions suitable for sterilization, such as hypochlorite solutions, Hydrogen peroxide solutions or peracetic acid be used.

In An advantageous development of the invention is the fluid, preferably the water sample, taken from a continuous stream, for example, by a bypass.

The Addition of nutrient solution may vary Done way. When the reaction space gets bigger As the sample volume is, the nutrient solution can be added directly to the reaction space. It is preferred that the nutrient solution while displacing one certain volume, preferably the volume of added nutrient solution occurs. This has the advantage that the reaction space before the addition is complete filled with sample. As a result, the amount of sample is accurate certainly. Mistakes caused by mixing during The addition is caused by the always equal addition of nutrient solution taken into account, as they are also in control and standard measurements occur.

The Ratio of sample to nutrient solution the amount of nutrients that the microorganisms Available. The amount of nutrient solution is therefore a very important parameter. either no growth is observed or the microorganisms can to access other food sources in the sample and the measured values are not meaningful. In to high dilution of the sample decreases the sensitivity of the method, as the number of microorganisms in the diluted Sample is too small. Therefore, it is an advantage of a ratio of Sample to nutrient solution between 1: 1 and 1: 100, more preferably between 1:20 and 1: 100.

The Composition of the nutrient solution is also one very important parameter of the procedure. By their composition Not only growth, but also measured Determine the type of microorganisms. Advantageously contains the nutrient solution in proportions between 0.1 and 30%, preferably between 1% and 10%, more preferably between 5% and 8% of one or more carbon sources such as short-chain alcohols and / or short-chain organic Acids and / or their salts. With advantage are the one or more carbon sources selected from the group containing methanol, ethanol, propanol, isopropanol, butanol, formic acid, Acetic acid, propionic acid, acetates, formates, lactates and pyruvates. Preference is given to methanol, ethanol and / or acetates. Optionally, the pH of the nutrient solution be adjusted by the addition or preparation of a buffer.

In An advantageous development of the invention is considered to be changing optical property of the liquid the turbidity measured, preferably by measuring the attenuation of Transmission, for example by scattering. As measuring range can Wavelengths between 250 nm and 1500 nm are used preferably between 400 nm and 1100 nm.

In an advantageous embodiment of the invention, the after a determined time or in the course of the measurement determined constant value the optical property correlates with the total germ count of the sample. Preferably, a measurement after a maximum of four, preferably between two and three hours. The measured value can with an already measured standard curve, which is under the same Conditions measured were correlated with the total germ count of the sample.

In An advantageous development, the change the measured value of the optical property with the total germ count correlated to the sample.

The Temperature of the measurement determines the growth rate of the bacteria, Due to the high sensitivity of the process but at temperatures above or below the usual incubation temperatures be measured at 37 ° C. Preference is given to a temperature the measurement between 15 ° C to 37 ° C, especially preferably between 15 ° C and 22 ° C. This is under normal conditions no special control of the temperature the measurement necessary. However, if necessary, can be measured Liquid by appropriate measures on a Temperature in the specified ranges heated or cooled become.

In addition to the optical property of course, other parameters, such as pH, conductivity, oxygen content of the sample, etc. are measured. This information can also be used to determine sample properties vote.

There The process can be fully automated It is possible to self-sustain it at certain intervals perform. It also allows a direct, timely survey of fluid samples such as water samples within a short time. This is for example, when used on ships an estimate the required effort to degerminate the fluid, eg. of the water, possible.

Furthermore because of the simplicity of the process, either by parallelization of reaction spaces or by successive measurements, Measurements performed with different nutrient solutions which may contain special indicators, such as For example, dyes for pH detection to certain species of microorganisms in a sample.

The Procedure may also be approximate the germ load can be used. If only one statement about the suitability of the sample for a particular purpose, for example Germ load above or below a certain value, desired becomes. So it can be used as a quick test for the Microbial load of fluids such as water to be optimized.

The The invention also relates to a device for determination the germ load and / or the total bacterial count of fluids, in particular water.

The apparatus comprises means for supplying a fluid sample, for example a water sample, 10 , This is preferably a pump 12 which conveys the sample into a reaction space. The sample can be taken via a separate sampler, a bypass or manual sampling. If necessary, the sample can be previously cleaned by filtration of larger turbidity. If desired, accurate metering of the amount of sample is possible.

According to the invention, reaction space is understood to mean the entire space in which the measured microorganisms multiply during the measurement. It therefore contains at least one measuring cell 14 , The volume of the reaction space can be selected depending on the sample to be measured. For example, it is between 1 mL and 1000 mL, preferably between 1 mL and 100 mL. Advantageously, the largest part of the volume of the reaction space is part of the measuring cell. Since the total volume can be chosen small, the amount of required solutions and the amount of wastewater is low.

In addition, the apparatus includes means for supplying a nutrient solution to the reaction space 16 , The means must be selected so that the volume supplied can be dosed as precisely as possible, so that the ratio of nutrient solution to sample can be set exactly.

For cleaning the reaction space after a measurement, the device also contains means for supplying a cleaning solution to the reaction space 18 ,

Around to be able to mix the liquid well in the reaction space, the reaction space still contains means for mixing.

In addition to the means just mentioned, the reaction space must also have means for regulating the amount of liquid 20 contain. Advantageously, this is an overflow 23 , via which the maximum amount of liquid in the reaction space is automatically limited and determined. Alternatively, or in addition, still a valve 22 can be used, which is optionally infinitely controllable. Advantageously, it is appropriate that the measuring cell is completely emptied when the valve is open. This makes it possible to rinse the reaction space faster before a measurement.

To supply microorganisms in the liquid in the reaction space with oxygen, the reaction space is still provided with means for supplying oxygen, preferably air 24 , Optionally, this supply still contains an air filter to protect against contamination of the reaction space.

In order to ensure a supply of nutrient and rinsing solution in an automatic operation, the device still contains reservoir for both liquids 26 . 28 ,

To determine the growth of the microorganisms, the device also contains means for measuring an optical property of the liquid in the measuring cell 30 . 32 , The optical property can be, for example, the turbidity, absorption or transmission of the liquid.

The measured values are sent to a data processing unit 34 transmitted, which evaluates the data and, if necessary, forwards. There, the measured optical property of the liquid is correlated with the germ count of the sample. Whether a certain germ count or the total germ count is determined depends on the nutrient solution used.

In an advantageous embodiment of the invention, the reaction space is a cycle 36 , which by a pump 38 is mixed. This has egg few advantages. On the one hand eliminates the impairment of the measurement in the measuring cell 14 through the mixing. Almost the entire volume of the measuring cell and thus also a large part of the volume of the reaction space can be used for the measurement. By circulating the circuit, the reaction space can be thoroughly rinsed. The addition of the nutrient solution can also be carried out simply by displacing a certain volume of the sample. As a result, in principle only the nutrient solution must be metered precisely, since the amount of sample is predetermined by the completely filled reaction space. Advantageously, the pump is for adding the nutrient solution 40 a peristaltic pump, as this type of pump ensures a particularly uniform pumping capacity and low contamination of the sample. In addition, this construction allows a low dead volume of the feeds and high modularity, since all feeds can optionally be connected directly to the reaction space.

Will also pump for feeding the nutrient and cleaning solution 40 . 42 , preferably used peristaltic pumps, so the entire device is optionally only with a valve to be switched, namely the purge valve 22 , out. This makes it possible to minimize the dead volume of the device and the susceptibility to errors.

Advantageously, the optical property of the liquid is measured by the transmitted light method with a light source 30 and a detector 32 executed. For this purpose, almost the entire volume of the measuring cell can be used as the measuring volume. This allows a very sensitive measurement.

In an advantageous development, the device with a GSM module and / or a TCP / IP connection for remote maintenance and control. This makes it possible autonomously operating devices to construct, which must be visited only for maintenance.

In an advantageous development contains the device a plurality of means for supplying nutrient solution to the reaction space, as well as a plurality of storage containers for nutrient solutions. Optionally required this also several different washing solutions. Thereby are measurements with different nutrient solutions possible to examine the different aspects of the allow to be tested liquid. The simple construction allows the construction of such embodiments.

The Device can in addition to the means for measuring an optical property the liquid in the measuring cell still means of measurement other properties, such as pH, conductivity or contain oxygen content.

The Device according to the invention is characterized all through the simplicity of construction and the concomitant Robustness. So the device comes with a minimum of solutions and moving or contaminable parts. This allows for example also the construction of compact portable devices for the mobile application. Because the results of the measurement already in a short time Time available, such devices can also used as automatic monitoring stations become.

Further Details and features will become apparent from the following description of preferred embodiments in connection with the dependent claims. Here, the respective features alone or in combination with each other be realized. The possibilities to solve the task are not limited to the embodiments. For example, ranges always include all - not - intermediate values and all conceivable subintervals.

The Embodiments are shown schematically in the figures. The same reference numerals in the individual figures indicate same or functionally identical or with regard to their functions corresponding elements. In detail shows:

1 Schematic representation of an embodiment of the invention.

1 shows a preferred embodiment of the invention. The sample 10 is doing by the pump 12 into the measuring cell 14 pumped. To the measuring cell are via different connections, the supply of the nutrient solution 16 , the supply of cleaning solution 18 , the circuit for mixing 36 , the drain 20 with flush valve 22 , an overflow 23 and the oxygen supply 24 connected. The connections do not have to be made directly on the measuring cell, but can also be realized via a common connection. Thus, for example, the sample feed and the cleaning solution can be supplied via a common connection. This ensures that the cleaning solution has no influence on the measurements.

The reaction space of the device contains the measuring cell in this embodiment 14 , the circuit for mixing 36 and the pump for mixing 38 , The diffusion from the tributaries into the reaction space need not be considered separately, since it is the same for all measurements.

The Construction allows the dead volume of the tributaries, i. H. the ways of the pumps to the reaction room as possible short to choose.

To measure the optical property of the liquid at the measuring cell, a light source 30 and a photodetector 32 appropriate. In the embodiment, the turbidity of the sample is measured. Since the mixing is realized outside the measuring cell, a particularly large volume is available for the measurement. This is advantageous because such a high sensitivity can be achieved since the measurement can be made through the entire width of the measuring cell, or a measuring cell with maximum width can be used. For this purpose, at least two opposite sides of the measuring cell are made of transparent material for the wavelength, for example sapphire.

To prepare a measurement, the reaction space is rinsed with the fluid to be tested, such as, for example, water by supplying sample for a longer period of time while the mixing pump 38 is turned on. The excess liquid in the reaction chamber passes over the overflow 23 from. As a result, the entire reaction space can be rinsed and filled with a certain volume of sample. If necessary, in order to accelerate the filling, the measuring cell must first be emptied via the flushing valve. The sample supply is then stopped and a certain amount of nutrient solution pumped into the reaction space. This displaces the same volume of sample. This sets a specific ratio of sample to nutrient solution. By circulation in the reaction space sample and nutrient solution are mixed. At the same time the turbidity at the beginning of the measurement is determined in a first measurement. For the later evaluation of the data this point represents the zero point of the turbidity measurement. For the germ load the increase of the turbidity by the growth of the microorganisms is always important, a constant turbidity by possible turbidity can be considered in this way. The mixing of the sample is then continued, optionally with repeated measurement of the turbidity. After two to four hours, preferably after two to three hours, the measurement of turbidity is terminated. The now measured value is compared to a data stored in the data processing unit data from reference samples. As a result, the total germ count of the sample can be determined. The accuracy is about 20%, preferably 5-10%. Depending on the dilution of the sample with the nutrient solution, the measuring range of the device ranges from 0 to approx. 10 ⁶ cfu / mL.

example 1

from Expiration of a sewage treatment plant, a sample was taken and measured by the method according to the invention. The measuring time was 2 hours, 4 mL nutrient solution at a total volume of the reaction space of 50 mL. One received 154566 cfu / mL. The same sample became a standard method sample measure (nutrient plate cultivated, 72 hours at 32 ° C). A value of about 160000 cfu / mL was obtained.

10

sample flow

12

pump for feeding the sample

14

cell

16

medium for supplying the cleaning solution

18

medium for feeding the nutrient solution

20

procedure

22

flush valve

23

overflow

24

air supply

26

reservoir for nutrient solution

28

reservoir for cleaning solution

30

light source

32

photodetector

34

Data processing unit

36

circulation for mixing

38

pump for mixing

40

pump for the supply of nutrient solution

42

pump for supplying cleaning solution

List of quoted literature:

• EP 0 304 406 A2

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• EP 0304406 A2 [0011]

Claims (18)

Method for determining the germ load of Fluids, in particular water, characterized by the following steps: (A) Feeding a sample into a reaction space; (b) Addition a certain amount of a nutrient solution, wherein (B1) the nutrient solution is a minimal medium; (C) Mixing the mixture of nutrient solution and fluid sample, in particular water sample; (d) Measurement of an optical property the mixture of nutrient solution and sample; (e) Transmission the measured value to a data processing unit. (f) repetition of steps (c) to (e) for a certain time or until no significant change in the optical property between two measurements are detected; (g) rinsing the reaction space with a cleaning solution; Method according to claim 1, characterized in that that the fluid sample, in particular the water sample, through a bypass is taken from a continuous stream. Method according to one of claims 1 or 2, characterized in that the addition of nutrient solution such that a certain volume of sample solution is displaced from the reaction space. Method according to one of the preceding claims, characterized in that the ratio of sample to Nutrient solution in the reaction space between 1: 1 and 1: 100 lies. Method according to one of the preceding claims, characterized in that the nutrient solution as Carbon source short-chain alcohols and / or short-chain organic Contains acids and / or salts thereof. Method according to one of the preceding claims, characterized in that the one or more in the nutrient solution existing carbon source are selected from the group containing methanol, ethanol, propanol, isopropanol, butanol, formic acid, Acetic acid, propionic acid, acetates, formates, lactates and pyruvates. Method according to one of the preceding claims, characterized in that the mixing of the sample by circulating of the sample / nutrient solution mixture in a cycle he follows. Method according to one of the preceding claims, characterized in that the measured optical property the turbidity of the sample is. Method according to one of the preceding claims, characterized in that either the at the end of the measurement and / or the constant measured value with the total germ count determined in the course of the measurement the sample is correlated. Method according to one of the preceding claims, characterized in that the change of the measured Value is correlated during the measurement with the total germ count of the sample. Method according to one of the preceding claims, characterized in that several measurements each with different Nutrient solutions is performed, which may also contain specific indicators. Method according to one of the preceding claims, characterized in that the method automatically in certain Time intervals is performed. Device for determining the microbial load of fluids, especially water, characterized in that it comprises: (a) means for supplying a fluid sample, in particular water sample ( 10 ); (b) a reaction space, wherein (b1) the reaction space is a measuring cell ( 14 ) contains; (c) means for supplying a certain volume of nutrient solution to the reaction space ( 16 ); (d) Means for supplying cleaning solution to the reaction space ( 18 ); (e) means for mixing the liquid in the reaction space; (f) means for regulating the amount of liquid in the reaction space ( 20 ); (g) storage container for the nutrient solution ( 26 ); (h) storage tank for the cleaning solution ( 28 ); (i) means for supplying oxygen, preferably air to the reaction space ( 24 ); (j) means for measuring an optical property of the liquid in the measuring cell ( 30 . 32 ); (k) a data processing unit which processes the measured values ( 34 ); Apparatus according to claim 13, characterized in that the reaction space is a circuit ( 36 ), which by a pump ( 38 ) is mixed. Device according to one of claims 13 or 14, characterized in that the means for regulating the amount of liquid in the reaction space an overflow ( 23 ). Device according to one of claims 13 to 15, characterized in that the means for measuring an optical property of a light source ( 30 ) and a photodetector ( 32 ). Device according to one of claims 13 to 16, characterized in that the device is a GSM module and / or has a TCP / IP connection for remote maintenance and control. Device according to one of claims 13 to 17, characterized in that the device has a plurality to means for supplying nutrient solution and a plurality of storage containers for Nutrient solutions.