DE2657150A1 - METHOD AND DEVICE FOR DETECTION OF MICROORGANISMS - Google Patents

Description

Method and device for the detection of microorganisms

The invention relates to a method for the detection of microorganisms that are in or on any Sample are available to be tested for the presence of microorganisms. To this According to the invention, the purpose is in particular to place such a sample in a cell with one nutrient medium and two Introduced electrodes, one of which is protected from the action of microorganisms and the Change in potential between the measuring electrode exposed to the growing microorganisms and the reference electrode certainly.

At the moment some methods for the detection of microorganisms are known, which in different Samples may be present which include aqueous media such as blood, plasma, fermentation media, and the like.

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Dresdner Bank (Munich) Account 3939 BAi

Posischeck (Munich) Account 670-43-B04

_ £ _ B7835

These procedures are broadly divided into two categories of evidence, the first of which is one The "sieving test" is used to determine whether or not large numbers of microorganisms are present in a sample. If the result is positive, another test is carried out to determine the type and amount of organisms present performed. Most common are methods of determining the identity and amount of microorganisms on the basis of successive cultivation and observation steps. The growth rates in culture approaches are observed, obtained by multiple dilutions of the same sample. By determining the time in which If the diluted samples form observable populations, the microorganism concentration in the Original samples are determined.

The determination of the microorganism populations is usually carried out using one of three techniques: The first is a nutrient agar plate technique in which the microorganisms are grown on an agar substrate and the growth of the microorganisms is first observed visually and then microscopically. This method it is most widely used in the clinic.

The second technique involves several processes classified as chemical processes can. In such a process, microorganisms are

14 organisms in a growth medium with C-labeled

Glucose supplies. The microorganisms digest the radioactive glucose and develop COp, from which Samples are taken and counted. Although after this procedure positive results in a relatively short time Can be obtained, the method is complicated and laborious and also expensive and considering the time The need to handle radioactive samples

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dangerous. Another analytical method for determining the type and amount of those present in a sample Bacteria is based on the chemiluminescent reaction between luciferase and luciferin in the presence by ATP. For this, bacteria are grown in a culture and the cells then release the ones in it present ATP dissolved. The released ATP reacts with the luciferase-luciferin combination with emission of chemiluminescent light, which is detected with a photomultiplier and used to determine the amount of light present Organisms is used. The main disadvantage of this technique is that it uses expensive materials Need to become. Yet another chemical approach involves measurement based on Metabolic conversion of nitrite ion to nitrate ion. However, this method can only be used with some bacteria and yeasts.

The third type of techniques for detecting Microorganisms includes non-chemical methods. One method involves examining relatively clear ones Microorganism suspensions with modified particle counters. However, this procedure is not specific and does not allow a clear distinction between living microorganisms and dead organisms or even non-biological particulate matter. A second method involves the detection of microorganisms by impressing an alternating current between a pair of electrodes, which flows into a microorganism-containing Medium is brought and observation of the change in the resulting resistance as a function of growth of microorganisms. However, this technology is obviously not amenable to automation to the extent that as was expected for it.

A biochemical detector is disclosed in US Pat. No. 709827/1032

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3 403 081 (von Rohrback et al.) Known, which is used for the detection of trace elements and poisons in liquid and gaseous media. This detection device involves placing a measuring electrode such as an inert cylindrical wire mesh of nickel, platinum, stainless steel or the like impregnated with a colony of microorganisms or with an enzyme and a reference electrode such as a standard calomel electrode in an electrolyte. Leads from the individual electrodes are connected to a voltmeter. The microorganisms or enzymes, which are intended as impregnation or in close proximity to the measuring electrode, generate a current within the cell (caused by chemical reactions on the surface of the electrode or by promoting chemical reactions with the formation of materials, which in turn lead to depolarization reactions at the Lead electrode). In operation of the device, a trace element or poison is admitted into the device which deactivates the enzyme or kills or deactivates the microorganisms, thereby causing a change in the potential difference between the electrodes which is manifested in a change in the voltmeter reading. If one considers the fact that the enzymes or microorganisms promote or cause a considerable chemical reaction at the measuring electrode, which is detected by a standard voltmeter in the measuring circuit of the cell, it follows that the current generated within the cell must be substantial.

The electroanalytical device according to the invention, which is used to determine the type and amount of a
Microorganism is applied in a solution, on the other hand, does not require impregnation of the measuring electrode with an assessable colony of one

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Microorganism but rather it is operated to detect a microorganism in a solution, which gradually concentrates or enriches around the measuring electrode. That is, it is based on the previously unknown Phenomenon of this accumulation. Another difference between the method and the device according to the invention on the one hand and the method described on the other hand is that the circuit according to According to the invention, a high impedance potential measuring device (high impedance potentiometer) and not a conventional one Includes voltmeter, as used in the method described, since the present system is based on the Measurement of a quasi-electrostatic potential difference between the measuring electrode and the microorganisms in Solution based. With a standard voltmeter, the current flow through the voltmeter would be far too high, whereby the relatively delicate quasi-electrostatic potential difference between the measuring electrode and the microorganisms collected around the electrode surface would be disturbed or destroyed.

The invention therefore meets the existing demand for a method for rapid automatic and economic determination of types and amounts of different microorganisms by one of the Concept for simple and economical technology.

The present invention will be described in more detail with reference to the attached drawings; show it:

1 shows the potential profile during the growth of Pseudomonas in 10 ml of trypticase soy broth medium (TSB) as a function of time;

2 two curves for the potential profile as a function of the time from which the growth-inhibiting influence of bacteriophages on bacterial strains

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derived from E.coli;

3 shows an embodiment for an electroanalytical Cell according to the invention;

4 shows a further embodiment for an electroanalytical Cell according to the invention, wherein the cell is adapted to function as part of a syringe;

5 shows an embodiment of the invention Device "in which the measuring chamber has means for introducing a fluid sample containing microorganisms is equipped by suction of a vacuum;

6 shows an embodiment of the invention Device formed in part by a needle suitable for sampling body fluids;

7 shows a recording strip with the potential profile depending on the time for the culture of E. coli;

8 shows an analog recording strip for culture series of E.CoIi, in which the bacterial concentration of the initial inoculation in the individual samples was varied;

Fig. 9 comparison curves for the potential as a function of the time for a hydrogen-producing organism (E.coli) and a non-hydrogen-producing organism Organism (alkalescence);

Fig. 10 comparison curves for potential and radioactivity at the measuring electrode as a function of the cell concentration of radioactive alkalescence in TSB medium;

Fig. 11 two plots of the potential as a function from the time for a platinum wire / calomel electrode system and a nickel / nickel electrode system with the same TSB culture medium with alkalescence (the arrangement of the nickel electrodes corresponded to FIG. 3) i

12 shows the potential curve as a function of time for different approaches to the display of the growth-inhibiting influence of ampicillin (anti-

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biotikum) on S. epidermidis; and

Fig. 13 different potential curves showing the Growth-inhibiting influence of cephalothin on K. pneumoniae demonstrate.

The method and apparatus according to the invention are based on the determination that the presence certain population level of a given organism in a flowable medium in a measuring electrode and a cell containing a reference electrode because of a Difference between the living organism and the measuring electrode with respect to the electrostatic charge leads to changes in potential. The potential is determined with a high-ohmic potential measuring device, the is connected to the electrodes of the cell by electrical conductors. It was found that all microorganisms show a relatively negative electrostatic charge in solution compared to the electrode Measuring electrode is used, so that all microorganisms one Evidence according to the invention are accessible. It is believed that the microorganisms grow gradually migrate to the exposed surface of the measuring electrode, which is somewhat positive towards the microorganisms. So that a potential difference between the If the measuring and reference electrodes exist, it is essential that the reference electrode is only in contact with the growth medium and not with the microorganisms is or is insensitive to the charge of the microorganisms and that the microorganisms themselves around the Collect measuring electrode. For example, when a metal wire or the like is used as a reference electrode the electrode normally from the microorganisms through a liquid-permeable, but for the Organism shielded from impermeable substance. When using a device such as the calomel electrode as a reference electrode serves, it is usually carried out by the usual

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Glass envelope protected from microorganisms. The gathering of microorganisms around the measuring electrode changes the potential of the same relative to the reference electrode and therefore there is a change in potential between the electrodes, due to the charge / Charge interactions on the measuring electrode. The one caused by this electrostatic interaction Change in voltage is the means by which the presence of microorganisms in the solution is detected can be.

The cell that surrounds the growing microorganisms and that is provided with the electrodes is can be made from any suitable materials commonly used for the manufacture of electroanalytical Cells are used, such as glass, plastic and the like. Are made. Any material that is suitable for such use and does not interfere with the growth or viability of the organisms and does not affect the voltage generated inside the cell can be used.

The electroanalytical cell is provided with a culture medium for the growth or cultivation of the microorganisms that are brought into the cell. Any culture medium commonly used for culturing microorganisms can be used, and the type of culture medium used is therefore not critical. Suitable culture media include brain / heart infusions, trypticase soy broth (TSB), a phenol red / broth base + 1 % glucose, trypticase soy broth + CO ₂ , milk, beer, sodium glycolate and the like. The amount of growth medium provided in the measuring cell is of course not critical.

The measuring and reference electrodes 7 09827/1032 attached to the cell

can be made of the same conductive material or of different materials. Usually the measuring electrode is made of any suitable electrode material in any convenient form and attached to the cell such that a portion thereof is in contact with the liquid medium within the cell is. Suitable materials from which the measuring electrode can be made include precious metals such as silver, platinum, palladium, gold and the like, tungsten, molybdenum, nickel and the like. and different metal alloys such as stainless steel, winding chrome and silver palladium. The shape of the electrode is not critical and it can have any suitable shape and for example by a wire, a Strips, a coil or the like. Be formed.

The reference electrode can be any of the suitable electrode materials available for the measuring electrode be used, be manufactured. It is thus conceivable that the reference electrode could be made of the same material may like the measuring electrode, which is one of the most unusual electrode configurations for galvanic cells. Usually the electrodes consist of galvanic cells made of different materials so that the cell is operational. The reference electrode becomes the cell also added by any suitable means in some way so that a part (such as a End of a wire or tape) of the electrode is in contact with the liquid medium in the cell. The one with the part of the reference electrode in contact with the medium is usually prevented from contacting the Microorganisms shielded in the medium. If the measuring and reference electrodes are made of different metals or Alloys are, it is not necessary to shield the reference electrode as the microorganisms preferably the more positive of the two electrodes in

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the cell collect. If the measuring and reference electrodes are made of the same material, the reference electrode must be shielded. Any means by which the reference electrode can be shielded from the growing microorganisms, however at the same time allows contact with the liquid medium. For example, the exposed part of the electrode can in a gel such as agar gel, gelatin, dextran gel, carragheenanggel - acrylamide gels and the like. embedded will. Membranes, such as relatively large pore ultrafiltration membranes or even large-pore membranes, frits, ceramic layers or porous plastic films are generally suitable if they are impermeable to the microorganisms. When the reference electrode and the measuring electrode can be made of the same material as two stainless steel electrodes, such as it is the preferred embodiment of the invention, or two platinum electrodes or the like Sensing or measuring electrode cell resulting change in potential relatively free of thermocouple or element effects. This means that if the electrodes are the same, no voltage is generated, the (thermo) element effects would be attributable. If, on the other hand, the electrodes are made of different metals or alloys a (thermo) element effect can exist and the potential readings obtained should relate to the the part of the voltage attributable to the thermocouple effect can be corrected or "compensated". It is thus to note that a (thermo) element effect, which can occur with certain electrode combinations, is not a critical limitation and when working according to the invention it is only necessary take this factor into account.

In addition to the type described above, a standard reference electrode such as

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a standard calomel electrode or a mercury / mercury-Cli-sulfate-electrode, an Ag / AgCl-electrode or a quinhydrone electrode. When one of these standard types of reference electrodes is applied the part of the electrode within the liquid medium in the cell need not (extra) against the microorganisms shielded, as either the structure of these electrodes in a glass envelope is usually a Contact of the inner operating part of the electrode with the microorganisms in the solution excludes or the Electrodes have a potential that is not influenced by microorganisms.

For measuring the electroaralytic cell produced in the growing microorganisms Voltage it is necessary to use both electrodes of the cell with a high-ohmic potential meter or one Connect potentiometer circuit with high resistance. The type of measuring device used is not critical,

except that a high-resistance type is required. That

7 Potential meter must have an input resistance above

1 O A

to 10 ohms, preferably over 10 ohms. When a If a relatively low-ohmic potential measuring device is used, too much current flows through the measuring arrangement and is impaired so is the charge / charge interaction between the measuring electrode and the microorganisms. At a Collapses in the electrostatic potential at the electrode cannot obtain potential readings will. Of course, further Sj can be used with the high-resistance Potentiometer circuit compatible accessories, such as an amplifier and a recorder, into the Instrumentation to be included.

When carrying out a measurement according to the invention, a sample of microorganisms is in the Growth medium introduced inside the cell. at

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In one embodiment, a flowable sample of Microorganisms are injected into the cell through a self-sealing cap covering the cell from the atmosphere or a sample can be drawn into the cell by means of negative pressure. Microorganisms can also be introduced into the cell in the form of a gas sample, provided that the microorganisms get into the growth medium of the cell. A basic feature of the technique of the invention is in that a stable baseline can be established by the recorder (the one with the high-ohmic Potential meter is connected and plots the potential changes as a function of time) before the minimum detectable concentration (MNK) of the growing microorganisms has been reached. That is, the stable baseline corresponds to a value of zero for the function dE / dt. With the emergence of the Microorganisms, the population level reaches the minimum detectable concentration that is normal is about 5 x 10 to 5 x 10 organisms / ml for all but the slower growing microorganisms and the microorganisms can now be detected by the voltage that occurs. The readings in mV Voltage values always change in a negative direction, i.e. the measuring electrode becomes stronger negative. The reason for this is the negative charge of the microorganisms compared to the measuring electrode. For slowly growing microorganisms such as the tuberculosis bacillus however, the lower detectable concentration can be as low as 1 χ 1CK organisms / ml be. In cases where the method according to the invention for the detection of slowly growing microorganisms the cell may be agitated, for example, by shaking or any other suitable means or be stirred. Agitation of the solution containing microorganisms facilitates growth

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same in some cases and can thus reduce the delay time in the detection of the microorganisms. However, since according to the invention a significant accumulation of organisms on the measuring electrode is necessary so that the necessary electrical charge / charge interactions occur, too vigorous stirring could tear the microorganisms away and prevent the change in potential. The temperature range in the cultured microorganisms and optionally detected, ranging from 15 ⁰ C to 6O ⁰ C, preferably 32 to 37 ⁰ C. The pressure is not critical in the measurement, since growth and detection of micro-organisms under partial vacuum conditions as well how can take place under overprinting. Thus, the method according to the invention is accessible, for example, to the detection of microorganisms which grow under pressure (as in the production of beer).

The method according to the invention can be used for the successful detection of any types of microorganisms serve that can be cultivated in the nutrient medium that or provided in the electroanalytical cell this is added in a suitable concentration. It is also not absolutely necessary for the microorganisms to that growth or metabolism occurs, provided that they remain alive as it is their viability which causes its negative charge, which is lost when it dies. So is the method can be used for the detection of yeasts, fungi and bacteria. For specific examples of Bacteria that can be detected with the method according to the invention include those that do not produce hydrogen Bacteria Staphylococcus aureaus, Staphylococcus epidermidis, Streptococcus, Listeria monocytogenes, Pseudomonas aeruginosa, Moraxella, Shigella. alkalescens, Diplococcus pneumoniae, Bacillus subtilus

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and Hemophilus influenzae. Suitable examples of yeasts that can be detected include Candida species such as Candida albicans and Candida rugosa; Hansenula species such as Hansenula anomala, Pichia species such as Pichia membranaefaciens, Torulopsis species and Saccharomyces cerevisiae. Suitable examples of Fungi include the different types of Aspergillus such as Aspergillus auricularis, A.barbae, A.bouffardi, A. calvatus, A. concentricus, A. falvus, A. fumigatus, A. giganteus, A.glaucus, A.gliocladium, A.mucoroides, A. nidulans, A.niger, A.ochraceus, A.pictor and A.repens as well as the species of the large group of mushrooms known as Fungi imperfecti.

A number of bacteria make up a class of bacteria for their ability to give off hydrogen known during growth. These bacteria include Escherichia coli, Enterobacter aerogenes, Serratia marcescens, Proteus mirabilis, Citrobacter intermedium, Citrobacter freundii, Salmonella and Klebsieila. Since these bacteria migrate to the measuring electrode like all microorganisms, their hydrogen tends output to a gain around the measuring electrode.

The presence of hydrogen on the measuring electrode increases its properties very significantly, so that it becomes similar to the known hydrogen electrode. A completely different one effectively exists Type of measuring electrode for the measurement of hydrogen-producing organisms on the one hand and not hydrogen generating organisms on the other. The principle underlying the hydrogen electrode is that the following equilibrium on the metal surface, which usually consists of platinum or gold:

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2H ⁺ + 2e

That is, there is a balance between molecular Determine the hydrogen and hydrogen ions in the solution and the changes in this equilibrium the potential of the electrode. Once an equilibrium has been established on the electrode surface the electrode is referred to as "non-polarizable" or a reference electrode. The normal hydrogen electrode is produced by platinum (saturated with molecular hydrogen resp. rinsed with 1 at ^) at a defined temperature educated. A normal hydrogen electrode (NHE) has by definition a potential of 0.00 V and it forms the basis for the potential scale of all other electrode reactions as well as the basis for the voltage series of metals.

In a more specific aspect of the measurement of hydrogen producing bacteria, a calomel electrode is used used as a reference electrode in combination with a metal measuring electrode. The calomel electrode has a potential of about +0.23 V (based on the NHE) and since the growth medium is about neutral has a pH of 7, the hydrogen measuring electrode has a potential of about -0.42 V in relation to the NHE. the The measuring electrode for hydrogen-producing bacteria thus has a negative potential of about -0.65 V [J-O, 42) - (+ 0.23) = -0.65j compared to the calomel electrode. There in reality a pressure of 1 atm hydrogen at the measuring electrode due to atmospheric dilution effects by COp, nitrogen and the like. will never show the measured values obtained for hydrogen-producing bacteria have an adjustment potential of approximately -0.4 to -0.5 V. opposite the calomel electrode.

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In determining both hydrogen generating as well as microorganisms that do not produce hydrogen, a delay time is observed at the beginning, which extends from the time of inoculation of the analytical cell with the microorganisms to the time when the microorganisms reach a population level sufficient for detection. After that, the potential decreases in the negative direction continuously to until a maximum potential is reached, from where the potential remains at the same level. After that, the potential drifts back to more positive potential values as time progresses. For microorganisms that do not produce hydrogen, the total change in the readings is sufficient in mV from about 200 to 300 mV. In the case of hydrogen producing organisms, the initial ones are negative Increases in potential can only be ascribed to the presence of the organisms. After that, the potential decreases when the The effect of hydrogen build-up on the electrode becomes significant, sharply to considerably more negative potentials than can be achieved for non-hydrogen producing microorganisms. The negative increase in potential which is attributable to the presence of hydrogen on the measuring electrode amounts to about 500 mV.

The potential readings for the individual measurements give the sum of the due to the presence of the Microorganisms in the solution caused a change in potential and those changes in potential which can be ascribed to other solution and system factors that cause minor changes in potential. These Factors are characteristic of the particular liquid medium present in the cell as well as those used Electrodes and should be specifically designed by a potential reading at that of detectable levels microorganism-free medium to be tested. Based on the measurement of this background

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potentials one can therefore easily see the total change in potential determine by the absence of the Microorganisms. An interesting feature of the invention is that it is a Provides a method for the detection of a mixture of microorganisms in a sample to be checked. If a rehearsal, which contains a mixture of two or more types or kinds of microorganisms in the analytical Cell is cultivated, the microorganism is the one in which the minimum detectable limit is reached first, becomes, the microorganism detected by the system. With this in mind, it is therefore clear that the person skilled in the art knows the growth conditions such as temperature and type of culture medium to promote growth Modify one type of microorganism to the detriment of others and thus prefer one microorganism over can prove to others. For example, it can be used to detect a fecal coliform in a mixture of microorganisms the temperature of the growth medium for the preferential killing of the other microorganisms are raised so that only the coliform remains.

The volume of the liquid medium within the electroanalytical cell is not critical. Of course the liquid medium must be in ionic contact with both electrodes. The volume of the liquid growth medium can be made up by any reasonable amount which the skilled person can easily determine. Of course, as the volume of liquid increases, that is The microorganisms are diluted and the resulting response is reduced. It should be noted that the sealed electroanalytical cell may contain a growth medium before the microorganisms are introduced or the growth medium and the microorganism sample can be introduced into the cell at the same time.

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leads to be. The amount of microorganisms introduced into the cell is not critical and need not be within be any set limits.

A further understanding of the invention can be achieved with reference to Fig.1, the two simultaneous Potential / time curves for Pseudomonas aeroginosa with an initial inoculation with 10 cells / ml Frobe (cultivated in a trypticase soy broth medium at 37 C over a period of a few hours in two separate electroanalytical cells) shows. Both curves give the response of the potential to the microorganisms depending on the time again. For the first hour or two, the curve shows nothing significant Speak to. After that, the microorganisms begin to reach minimal detectable concentrations as out of the gradual increase in the potential reading in both Cells. The growth rate then begins after reaching its maximum value as a result of possible death of some of the cell population to gradually decline.

Heretofore, the invention has been made with reference to a direct measurement of the presence of living microorganisms described in an electrochemical cell. In another embodiment of the invention can Substances or events are detected which change or destroy the microbial viability, like contact of the microorganisms in the cell with an antibiotic, with serum antibodies, with chemical toxins, viruses, e.g. bacteriophages or with any other antimicrobial or anti-cellular phenomena. The effect of an antimicrobial agent or event can thus be determined by two methods be detected:

1) By adding an antimicrobial agent

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to standard dilutions of cultures at a certain concentration before reaching the MNC; and

2) by adding the antimicrobial agent to the MNK or above.

The inventive method is thus for testing the antimicrobial effectiveness in To study the activity of new antibiotics or to monitor developments in one Organism, such as the development of antibiotic resistance by the organism. In particular, the in vitro evidence of bacterial / antibiotic interactions predicting the in vivo effectiveness of a enable special drug by the clinician. The method of the invention is anti-micro-Meller when testing Sensitivity useful when the organism in question does not respond predictably to antibiotics, as shown below in Table 1 and also ^ if the organism is invariably susceptible to is a special drug, as shown in the table below.

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TABLE 1

Microorganisms with an unpredictable response

Antibiotics

Organisms

Means to which the organisms can respond

Staphylococcus aureus Eschen'chia coli Klebsiel la pn euiuoniae Pseudoinonas aeruginosa Proteus nn'rabilis Proteus vulgaris Enterobacter aerogenes Salmonella

Shi gel Ia

Listeria nionocytogenes

Penicillin G., methicillin, cephalothin, vancomycin

Ampicillin, cephalothin, tetracyclines, kanamycin

Cephalothin, tetracycline, Kanamyci η, C hl orampheni col

Gentamyci η, Tobramyci η, Carbenicillin, polymixin B

Penicillin (moderately resistant) ampicillin, kanamycin

Tetracyclines, kanamycin, Chloramphenicol

Tetracyclines, chloramphenicol Kanamycin

Ampicillin, tetracyclines, Cephalothin, kanamycin

as before ( Salmonella )

Penicillin G, erythromycin, Chloramphenicol

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TABLE 2

Microorganisms with a predictable response

Antibiotics

Organisms

Means to which organisms respond equally

Meisseria cjonorrhoeae Dipl ococciis p ne umoniae

Streptococc us pyog enes Clostridium perfringe ns

Haeinophiiu s in fluenzae '

Haeinophii iis pertussis Brucelia

Corynebacter i um diphtheria Neisseria men ingi tidis

Penicillin G, erythromycin

All penicillins, Q.'phalothin Erythroinyciη, Vanconiyciη

like Diplococcus

Λ1 lePenicillins, Cephalothin, Tetracycline

Penicillin G, ampicillin, Tetracyclines, chloramphenicol

how

I. influenzae

Tetracyclines

Erythromycin, penicillin G, penicillin G, ampicillin

FIG. 2 shows another example of a means with which the growth of a microorganism is interrupted. Curves A "and B" show the potential profile as a function of time for the growth of an E. coli strain in a growth medium to which different concentrations of bacteriophages JZ-, u.zw. 10 phages / ml or 1Cr phages / ml were added to the growing microorganism. The time until the maximum potential is reached after adding the virus is inversely proportional to the logarithm of the virus concentration at the time of addition, corresponding to:

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log C (virus)

According to this technique, the virus concentration can be analyzed can be reached in less than an hour.

The following drawings show different designs of the electroanalytical cell according to the invention:

3 shows an embodiment of an electroanalytical Cell in which the chamber 1 is closed with a self-sealing resilient cap 2. the Measuring electrode 3 is passed through an opening (not shown) in the chamber wall and fixed. Of the Part 4 of the electrode 3 extends through the wall so that its surface is the culture medium 5 in the chamber is exposed. A reference electrode 6 is passed through an opening (not shown) at the bottom of the chamber. The part 7 of the reference electrode inside the chamber is protected by a shielding means 8 against all microorganisms, that are present in the culture medium, shielded. The shield 8, measuring electrode 3 and reference electrode 6 can be formed from the materials mentioned above. When applying the above described device is a suitable amount of a microorganism-containing flowable sample injected into the chamber through the cap. The external connections of the measuring and reference electrodes are then connected to the terminals of a high-ohmic potential measuring device via appropriate lines and the arrangement monitored during microorganism growth.

Another embodiment of the electroanalytical Cell according to the invention is shown in Fig.4. With this arrangement the cell is essentially through a syringe with the chamber 11 and the plunger 12 is formed. The bottom of the chamber is covered with a suitable one

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hollow conduit 13 is provided, which is passed through a hollow needle, a rubber hose or the like. Can be formed with one end 14 through the chamber wall and a shield 15 protrudes against microorganisms in the mass of the culture medium 16 within the chamber. The device is equipped with a measuring electrode 17 which is attached to the chamber through an opening (not shown) is. The part 18 of the electrode is in contact with the culture medium. A reference electrode 19 is on the ground the chamber passed through an opening (not shown) and set. The part 20 of the electrode inside the chamber ends inside the shield

The device according to Figure 4 can be used for an immediate Removal of fluids from a living body into the culture medium or an immediate Be used to expel liquids and immediately connected to a potential measuring device, so that the growth of any microorganisms present in the body fluid can be monitored can.

Yet another embodiment of a cell according to the invention is shown in FIG. Inlet and vacuum line (31 _f 32) protrude through a resilient sealing cap 33 inwardly, but it should be noted that both lines may be added by the walls of the chamber 30 as desired. When a fluid sample is drawn into the device by vacuum suction, it falls onto the culture medium 34 and is mixed with it. A measuring electrode 35 is attached to the device through an opening (not shown) such that the measuring end 36

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the electrode protrudes into the culture medium. A reference electrode 37 is similar to the device attached to the bottom of the chamber so that the end 38 protrudes into the chamber in a shield 39, which protects the end of the electrode from microorganisms in the culture medium. The device can be used in the same Way used for sampling by vacuum suction or by injection through the sealing cap will.

Yet another embodiment of the device according to the invention is shown in FIG Functional parts are housed within a needle. Thus the device is essentially made up of a line 40, which can be formed by a hollow needle, manufactured in which an electrical insulation 41 is concentric is arranged. A conduit 42 is connected to the outer surface of the needle in such a way that it can be used as a Measuring electrode acts. A wire 43 is disposed within the inner hollow core 44 of the insulation and fixed at the bottom of the insulating core 45 by a shield 46. The needle apparatus will then connected to an evacuated enclosed chamber (not shown) containing a growth medium can. When used, the needle is driven into a body and the microorganisms to be examined containing liquid drawn up. The lines attached to the needle can then be connected to a high-ohmic Potential measuring device to determine the microorganisms contained in the liquid are connected.

According to a further (not shown) embodiment of the device according to the invention, the Arrangement according to FIG. 3 for generating a vacuum within the device closed under negative pressure will. The bottom of the chamber is then covered with a

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sealed hollow needle provided, the connecting end through the bottom of the chamber and the shield protrudes into the culture medium. This device. can be used for growing microorganisms containing Liquid into the chamber by simply sticking the free end of the needle into the body, for example of a living being, the puncture and / or the fluid pressure sealing the The needle is destroyed, so that liquid enters the device due to the negative pressure prevailing in the chamber is absorbed.

The invention can be used in many ways, since it can always be used when the detection of the The presence and amount of microorganisms in a sample is desired. So the invention can be used as evidence the presence of bacteria in a biological or non-biological fluid medium will. The method according to the invention can be used as a "sorting technique" for antibiotics to determine the effectiveness of an antibiotic agent. For example, a microorganism in an inventive Cell cultivated and treated with an antibiotic. At the same time the potential curve Monitored in the medium to determine the influence of the addition of antibiotics. Biological applications include the detection of microorganisms in such body fluids urine, blood, cerebro / spinal fluids, sputum, amniotic fluid, synovial fluid, Plasma and dialysates from artificial kidneys. The present technique is also used for investigation of throat tissue cultures, vaginal and cervical tissue cultures as well as tissue biopsies are suitable. The invention is also useful for the detection of microorganisms in Alcohol-producing media such as wood, grain, molasses, sulphite and in "sewage" from the formation of wine and

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Beer and the formation of glycerine are useful. Yet another area of application is in fermentation processes for the production of organic acids such as lactic acid, citric acid, fumaric acid and acetic acid. Furthermore, the food processing industry offers a general field of application, in particular the packaging and production of beverages and canned food. Still other uses include rural, urban, and regional water supplies, water holding tanks, and septic tanks.

The invention is explained in more detail below with the aid of examples.

example 1

For the specific experimental procedure described below, the following were established Hydrogen producing bacteria obtained from the American Type Culture Collection (Rockville, Maryland) U.S.A.:

E. coli (12014), E. aerogenes (13046), S. marcescens (13880), C.intermedium (6750), C. freundii (8090) and P. mirabilis (12453).

The microorganisms were stored at 5 ° C. in the form of trypticase soy agar slants (TSA, BBL) and broadcast monthly.

Vaccine preparation, active counts and media

Inoculation preparations for hydrogen measurements were made by ten-fold dilutions of a 24-hour trypticase soy broth culture (BBL) with sterile 0.05% peptone broth and adding 3 ml of suitable dilutions to 27 ml of phenol red broth base with 1.0 % glycose (Difco) after preheating 35 ° C. Representatives were given for a limited number of tests

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of the Coliform group in lauryl tryptose broth (Difco). Active counts (viability) were done by spreading appropriate dilutions of the ten-fold series on TSA and colony count after 35 h incubation at 35 ⁰ C. active counts were performed also in the individual organisms at the time of hydrogen evolution, and after 24 hours of incubation.

Hydrogen measurements

The test facility for measuring the evolution of hydrogen through the test organisms consisted of a test tube (25 x 90 mm) with 2 electrodes and broth and organisms placed in a 35 ° C water bath. The leads to the electrodes were connected to a DC buffer amplifier (Type 122, Neff, Inc., Duarte, Calif, ”), which in turn is connected to a Schreiber (Model 194, Honeywell Industrial Div ", Fort Washington, Pa.) Was attached. The buffer amplifier was used to adapt the high-ohmic resistance of the electrode test system to the (strip cards) -Scriber. The evolution of hydrogen was observed as an increase in voltage in negative (cathodic) Direction measured and recorded by the recorder.

A standard calomel electrode (SCE Beckman Instruments Inc., Fullerton, Calif.) Was used as a reference electrode, which was cemented into a plastic cap, and a platinum electrode in the form of a platinum strip adapted to the circumference of the test tube served as a measuring electrode. A section of the platinum was placed outside the test tube for connection to the amplifier line. During the During operation, the platinum electrode and the test tube were steam-sterilized using conventional autoclave techniques.

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The reference electrode (SKE) attached to the plastic cap was exposed to two UV lamps (15T8, General Electric Heights, Ohio) sterilized in a clear plastic box. A number Tests showed that this technique enables effective sterilization of the reference electrodes.

The registration of the mV response curve for 1.9 × 10 E. coli cells per ml is shown in FIG. In the manner characteristic of hydrogen-producing organisms, the registration curve shows a lag time in which the microorganisms grow but are present in populations that are insufficient to achieve a signal or value. Once the microorganism population has reached a sufficient level, a measured value attributable to the microorganisms is obtained. When the amount of hydrogen evolved becomes sufficient, the electrode response characteristics change rapidly as shown by the sudden increase in potential. The drop after reaching the maximum potential (400 to 500 mV) takes place over a period of 3 to k hours.

The relationship between the amount of vaccination and the length of the delay time for different E.coli vaccine preparations is shown in Fig.8. These delay times range from 1 hour for 10 cells / ml up to 7 hours for 10 cells / ml, from which it can be seen that (1) every tenfold increase in cells leads to a decrease in Delay time leads to 60 to 70 minutes and that (2) the mean cell concentration at the time of rapid formation of hydrogen was 1 χ 10 cells / ml. Since there were no differences with regard to the response to washed or un-

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> If *

washed cells were found, they were done

Investigations with unwashed cells. In addition, no differences in the response curves or delay times were found for E. coli, E. aerogenes, and C.intermedium when tested in lauryl tryptose broth or phenol red broth with glucose. A gas chromatographic analysis of the gas space above the cultures showed that the Hp level for 2k hours of cultures in the present case was between 4 and 10% by volume. The only exception was S.marcescen3, in which Hp was only determined in traces (έ 1%). Limited research indicated that the pH did not change appreciably before or during the time of the Hp detection.

Example 2

Fig. 9 shows a comparison of the potential characteristics a 'hydrogen producing bacterium (E.coli) and a non-hydrogen producing bacterium (Alkalescens). Both types of bacteria were inoculated in a lauryl tryptose glucose broth cultured from 1 ml samples with 100 cells / ml. The response characteristics were measured with a platinum-calomel electrode combination measured. The registration curves showed a far greater response in the system with Hydrogen producing bacteria compared to the non-hydrogen producing bacteria. To notice is that the same lag time is observed when the initial inoculation and growth rates are the same.

Example 3

The data from FIG. 10 enable an assignment; the change in the potential of a culture medium is shown or a culture of alkalescence depending on the cell concentration. A 1 ml sample of radioactive Alkalescens (initial dose: 100 cells / ml)

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was added to a 10 ml trypticase soy broth medium Cultivated at 37 C. A sample was taken from the electrode together with the reading of the potential and checked for radioactivity. The plotted curves clearly show the corresponding increases of potential and radioactivity depending on the cell concentration. The values show that the cells increasingly collect around the surface of the electrode and that the potential reading through this Cells build up around the electrode.

Example 4

A 1 ml sample of alkalescence was cultured in 10 ml trypticase soy broth medium for 7 hours. The growth of the bacterium was monitored simultaneously with two different electrode systems with a platinum wire / calomel system and a nickel / nickel system in which a nickel electrode is replaced by a Glass frit was shielded from the organisms. How out 11, the two curves obtained are essentially the same, only the nickel / nickel electrode system appears not being quite as sensitive as the platinum / calomel system.

Example 5

A 1 ml sample of Pseudomonas aerogenosa was in trypticase soy broth in a manner described with reference to FIG Cell cultivated, with both the measuring device and the reference electrode made of stainless steel (type 3 or 4) were. In this example, peptone agar with 10 mg / ml NaCl was used as the shielding material. When the cell population reached a value of 9 × 10 cells / ml, the change in potential was detectable, as in Fig.1 is shown.

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Example 6

The procedure of Example 5 was repeated, however, with E. coli as the culture organism in a medium of trypticase soy broth. A potential reading was observed at a population level of 1.2 × 10 cells / ml.

Example 7

The procedure of Examples 5 and 6 was repeated, but with sodium glycolate medium as Growth medium and Shigella alkalescens as an organism. A potential reading was taken at a population level of 8.6 10 cells / ml was observed.

Example 8

The procedure of Examples 5 to 7 was repeated, but with sodium _{glycolate + O 9} 5 ml of human blood as the growth medium and Hemophilus influenzae as the microorganism. A potential reading was observed at a population level of 2.4 × 10 cells / ml.

Example 9

The procedure of Examples 5 to 8 was repeated, except that Bacillus subtilus in 1% nutrient broth with 0.5 % NaCl at 30 ° C. was used as the organism. A potential reading was observed at a population level of 2.3 x 10 6 cells / ml.

Example 10

The procedure of Examples 5 to 9 was repeated, except that 10% ± ge nutrient broth with 30% dextrose was used as a Candida organism Tropicanas and as a growth medium at 30 ⁰ C. Evidence was provided at

3.1 x 10 ⁵ cells / ml.

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Example 11

The cell of Figure 4 was used for culturing of the fungus "Aspergillus niger" in trypticase soy broth used at 25 C. A potential reading was taken at a population level of 10 cells / ml observed.

Example 12

A culture of Pseudomonas aeroginosa was grown in a non-nutritive buffer (phosphate buffer solution or PBS) and "starved" for 48 hours at 15 ° C. The cells obtained under this condition show neither division nor metabolism, but they are technically alive and therefore still have a distinct negative charge, these cells were placed in a cell according to Figure 4 with stainless steel electrodes, however, the nutrient broth was replaced by PBS. With a population of 2 10 viable cells / ml a signal was observed.

Example 13

Two cow milk samples were added to measuring cells of the type described with reference to FIG. One of the samples was "pasteurized" while the other was made from "raw" milk. The milk served both as growth medium and for inoculation in both cases. The electrodes were stainless steel and the membrane was saline agar. The experiment was carried out at a temperature of 37 ° C. Both samples provided a signal at around 10 cells / ml, but the raw milk showed this signal after 2.5 hours, while the pasteurized milk only reached Λ0 cells and provided a signal after 4.5 hours.

Example 14

Testing of the antimicrobial effectiveness

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Reagents and Media **

Identical amounts of media and antimicrobial agents were used in the efficacies performed. Sensitivity tests used. Served as media Trypticase Soy Agar (TSA) and TSD from Baltimore Biological Laboratories (BBL), Cockeysville, Maryland. The 12 antimicrobial used in these experiments Agents selected based on their clinical usefulness were laboratory reference standard agents and included the following antibiotics: Penicillin G Potassium, Cephalochin Sodium, Tobramycin, vancomycin hydrochloride and streptomycin sulfate from Eli Lilly and Co., tetracyclines and carbenicillin from Pfizer Laboratories; Ampicillin and sodium nafcillin from Wyeth Laboratories; Kanamycin and methicillin from Bristol Laboratories; Gentamicin from Schering.

The effect of an antibacterial drug on a susceptible bacterium is either bacteriostatic or bactericidal. A bacteriostatic agent merely inhibits bacterial growth, the "reversibly" resumed upon removal of the antibacterial agent. Bactericidal agents result an irreversible killing effect on the microorganisms susceptible to the drug. Of those mentioned 12 antibiotic agents are all bactericidal with the exception of tetracyclines, which have a bacteriostatic effect. Penicillin G, ampicillin, nafcillin, methicillin, and carbenicillin all belong to the penicillin family and thus, their mode of action includes a disruption of the synthesis of bacterial cell walls. This also applies to Cephalothin (a cephalosporin) and vancomycin (a glycopeptide) too. The aminoglycoside antibiotics kanamycin, Streptomycin and gentamycin cause specific reading errors in the genetic code in the ribo-

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somal area, as a result of which the protein synthesis of the bacterium is disturbed. This also applies when using Tetracycline, which specifically prevents the attachment of amino acid-activated transfer RNA to the ribosomes.

Cultures

The following cultures were from Hoffmann-La Roche Inc. and were from the American Type Culture Collection (ATCC) Organisms as indicated below: Escherichia coli, "Seattle Strain" ATCC 25922, Klebsiella pneumoniae ATCC 27736; Pseudomonas aeruginosa, ATCC 9721. These bacteria are gram negative. The following Bacteria are gram positive: Staphylococcus aureus, "Seattle Strain" ATCC 25923; Staphylococcus epidermidis ATCC 14990; and Streptococcus pyogenes ATCC 10389. In addition, clinical specimens of the same six Organisms from Sensitivity Isolates from the University of Virginia, Hospital Department of Clinical Pathology, Bacteriology Selection obtained. These organisms, both pathogenic and non-pathogenic (Staphylococcus epidermidis) were selected because they are some of the most frequently enumerated organisms of clinical Are isolates or preparations.

Vaccine preparation and active counts

Inoculants were made by diluting a hundredfold χ 2 of one overnight TSB culture of the microorganisms to be tested were generated in sterile TSB preheated to 35 ° C. This means that the total dilution of the overnight batch was 10 ". 0.3 ml of the 10 series were placed in a test tube (Falcon, Abgabe ^ t2057) with 2.7 ml TSB for growth control or 2.6 ml TSB +0.1 ml of a specific concentration of suitable antibiotic to be tested Funds transferred. The antibiotic was on a

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Analytical balance weighed in accordance with the respective activity standard and diluted with sterile distilled water in a volumetric flask to obtain a stock solution of 1000 μg / ml, which was then frozen in small aliquots. Further dilutions as required for effectiveness or sensitivity tests were also obtained by adding sterile distilled water to this stock concentration. Active counts were performed by plate culture of appropriate dilutions of the 10 series on TSA and colony count after 24 hours incubation at 35 ⁰ C. active counts were performed also in the individual micro-organisms when reaching the MNK and again at the end of the 24 hour test time for confirming the minimalinhibierenden concentration (MIC) .

The experimental device used in this investigation to determine the growth inhibition by the addition of antibiotic agents consisted of up to and including 8 test tubes of 17 x 100 mm in size, each one sterilized (by boiling treatment) Pt / SKE electrode combination (Sargent-Welch, S-3O1O1-15) as well as TSB, microorganisms and antibiotics (where appropriate) and placed in a heating block at 35 ° C were arranged. Connection lines led from the electrodes to a high-ohmic ( > 10 Ohrn) potential measuring device or a voltmeter, which in turn with a Schreiber (Hewlett-Packard Type 680M) were connected. The microbial growth was about an increase in tension measured in the negative direction as a function of the time recorded by the recorder.

Fig.12 shows the registration of the antimicrobial curves Response from a 4 channel experiment with a clinical sample of the non-hydrogen generating gram-positive bacteria Staphylococcus epi-

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dermidis when treated with 0.012; 0.5 and 1.0 / Jg / ml ampicillin (curves B, C and D). Curve A shows the response curve of a control sample of S. epidermidis that had not been treated with ampicillin. 13 shows a registration curve for the antimicrobial response in a 6-channel experiment with an ATCC culture sample of the pathogenic hydrogen-producing gram-negative bacteria Klebsiella pneumoniae, treated with 0.3; 0.4; C, 5 as well as 1,0 and 2, O _x UgZmI cephalothin (curves B ^!, C, D ¹ , E ¹ and F '). Curve A ¹ shows the response behavior of a control sample of Kapneumoniae that had not been treated with cephalothin. Both figures show that the growth of the respective bacteria is increasingly inhibited by the specially used antibiotic material. The minimum inhibitory concentration (MIC) is defined as that amount of an antimicrobial agent which completely inhibits the growth of the microorganisms tested. Tables 3 and 4 below show the MIC values for various antibiotics for a number of microorganisms as determined according to Invention were determined.

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Table 3

Electroanalytical determination of the MIC values of gram-negative bacteria

-4 O CD OO

JSJ

Antibiotics; concentration in iTiCQ / ml Carb Ceph letra Ka na Genta Strep Tobra Organisms 4.0 8.0 1.0 3.0 0.5 9.0 - E. coli (ATCC) A: r.p 5.0 B ₅ O 4.0 4 _v 0 1.0 9.0 - E. coli (clinical) 4.0 > 128 1.0 5 _.; 0-6 _? 0 3.0 0.5
i 5..0-6, O - K. pneiimoniae (ATCC) 6.0 > 128 - 5.0-6.0 4.0 ύ, 5 6.0 - K. pneu ~, cniae (clinical) 32 54 > 360 64 32-54 2.0 - 4.0 P. aeruginosa (ATCC): - 64 > 360 64 64 4.0 - 8.0 P. aeruginosa (clinical) - -128 • 128

cn

Table 4

Electroanalytically determined MIC values of gram-positive bacteria

CO OO ro

O Wl

Orgäni smaa
S. aureus (ATCC) "-ntibiotics < Concentration in mca / ml Arno 0.12 - Naf [• 'eth Ceoh Tetra Vanco Ka na Strep j S. aureua (elinical) Pen G 0.05b, 12-0.6 1.0 - 0.5 0.7 0.15-0 _; 2 2 _; 0 1.0 0.2 ε. ο S. epidermidis (ATCC) 0.05 0.1 0.8-1.0 0.5 0.7 0.8-1.6 2.0 2.0 0.5-1.0 ε, ο S. epidermidis (clinical) 0.08-0.1 - 0.5-1 _; 0 0.5-1.0 0.1 1.0-2.0 1.5 0.4 4.0 S. pyogenes (ATCC) - D.5-1.0 0.5-1.0 Q 5-1.0 2.0 2.0-3.0 0.6 4.0-6.0 S. nvoaenes (clinical) - ■ - - - 0.1 -0.5 O .1 - - - I 0.5 0.1

CD

Claims (25)

Claims \ ij method for detecting the presence of a microorganism in a flowable sample, characterized by the cultivation of the microorganism in a liquid growth medium which is in contact with a measuring electrode and a reference electrode and detection of the change in potential between the electrodes with a high-resistance potential meter. 2. The method according to claim 1, characterized by the cultivation of a hydrogen generating Bacterium in such a way that the developed hydrogen collects around the measuring electrode, which is modified in this way and achieves the peculiarity of a hydrogen electrode; Establishing a stable response baseline the potential meter values before reaching the minimum detectable concentration of the bacterium and Monitoring of the potential between the modified electrode and the reference electrode. 3. The method according to claim 1 or 2, characterized in that the high-ohmic potential measuring device 7 10 has an input resistance of 10 to 10 ohms. 4. The method according to claim 1, characterized in that that the part of the reference electrode exposed to the growth medium is protected from the microorganisms by a for the Growth medium permeable / but impermeable to organisms Shielding is shielded, which is preferably formed by a gel, in particular by agar gel will. 709827/1032 ORIGINAL INSPECTED 5. The method according to claim 4, characterized in that the measuring electrode and the reference electrode from the are the same metal and are made in particular of nickel, gold, platinum, silver or stainless steel. 6. The method according to claim 4, characterized in that the electrodes are made of different metals are. 7. The method according to claim 1, characterized in that that the measuring electrode is a metal electrode and the reference electrode by one of the standard calomel electrodes or the mercury-mercury (I) sulfate electrode is formed. 8. The method according to claim 1 or 2, characterized in that the growth medium from the brain / heart infusions., Trypticase soy broth, phenol red broth base + 1 % glucose, trypticase soy broth + CO ^, milk, beer, sodium glycolate formed Group is selected. 9. The method according to claim 1, characterized in that the cultivated microorganism by bacteria, Fungi or yeast is formed. 10. The method according to claim 9, characterized in that the bacteria do not generate hydrogen Organisms from the group Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus, Listeria monocytogenes, Pseudomonas aeruginosa, Moraxella, Shigella alkalescens, Diplococcus pneumoniae, Bacillus subtilus, and Hemophilus influenzae are. 11. The method according to claim 9, characterized in that that the yeast is caused by a type of Candida, 709827/1032 - ί / [ _ B 7835 . 3 · Hansenula, Pichia, Torulopsis or Saccharomyces formed will. 12. The method according to claim 1, characterized in that the microorganisms are cultivated ^{at a temperature of 15 to 6C 0 C.} 13. The method according to claim 1, characterized in that that the microorganisms are formed by a mixture of at least 2 types of microorganisms will. 14. The method according to claim 2, characterized in that the bacteria generate hydrogen Organisms from the Escherichia coli, Enterobacter aerogenes, Serratia marcescens, Proteus mirabilis, Citrobacter intermedium, Citrobacter freundii, Salmonella and Klebsiella are formed group. 15. Method for determining the influence of an antimicrobial agent or event on growing Microorganisms, characterized by the cultivation of the microorganisms in a liquid growth medium, that is in contact with a measuring and a reference electrode, which is connected to a high-ohmic potential measuring device with an input resistance of 10 to 10 ohms are conductively connected, with the growing bacteria gradually accumulating around the measuring electrode with the formation of a charge / charge interaction between the measuring electrode and the accumulated Microorganisms; Establish a stable response baseline for potential meter information Creation of the minimum detectable concentration of microorganisms; Bringing about the action of a antimicrobial agent or event on the 7 0 9 8 2 7/1032 B 7835 Microorganisms; and monitoring the change in potential between the electrodes under the influence of the antimicrobial agent or event on the microorganisms. 16. The method according to claim 15, characterized in that the microorganisms are the antimicrobial Exposure to agent or event prior to reaching the minimum detectable concentration in the growth medium will. 17. The method according to claim 15, characterized in that the microorganisms are the antimicrobial Exposure to agent or event after reaching the minimum detectable concentration in the growth medium will. 18. The method according to claim 15, characterized in that the microorganisms are caused by bacteria, Fungi or yeasts are formed. 19. The method according to claim 15, characterized in that that the antimicrobial agent is an antibiotic, serum antibody, chemical toxin or Virus is. 20. Device for the electrochemical detection of living microorganisms in a flowable medium, characterized by an orifice chamber (1) with a suitable growth medium (5) for the cultivation of microorganisms; Means (2) for tightly closing the chamber opening, which according to Piercing through a means for delivering a sample containing microorganisms into the chamber in a self-sealing manner works; one of the chamber externally attached and fixed measuring electrode (3), one end of which (4) 709827/1 032 B 7835 s- enters the interior of the chamber and is in contact with the growth medium based on charge / charge interaction responds to microorganisms enriched in its environment; one of the chamber (1) of externally attached and fixed reference electrode (6), one end (7) of which protrudes into the interior of the chamber and through a permeable for the growth medium Shielding (8) is covered, which prevents contact with the microorganisms; and a potential measuring • 7 ΛΓ) device with high input resistance of 10 'to 10 ohms, that is conductively connected to the measuring and reference electrode. 21. The device according to claim 20, characterized in that the measuring electrode (4) and the reference electrode (7) are made of the same metal, preferably stainless steel. 22. The device according to claim 20, characterized in that that the shielding is formed by a gel, preferably agar gel. 23. Apparatus according to claim 20, characterized by inlet means (13 or 31) for the introduction a fluid sample with microorganisms in the cell and means (12 or 32) for generating or applying a vacuum in the cell, the inlet and vacuum means preferably of the chamber itself or the Closing means (33) are attached. 24. The device according to claim 20, characterized by the bottom of the chamber (11) and the shield (15) of the reference electrode (2C) penetrating line (13) whose open end (14) into the culture medium (16) protrudes for the introduction of the material to be examined into the chamber and a piston (12) 709827/1032 - ^{B 7835} O. in the open end of the chamber (11), which is freely movable and containing the absorption of a microorganism Sample into the chamber or expulsion from the chamber. 25. Device for electrochemical detection living microorganisms in a flowable medium, characterized by: a conduit (40) having a hollow core and a fluid sample receiving end; an electrical lead (42) to said line (40), which acts as a measuring electrode and on Charge / charge interaction is triggered by the accumulation of microorganisms around the electrode; a second line (43) which extends over the length of the first line (40) and is arranged in this is; an insulation (41) with a hollow core, which is arranged concentrically within the bore of the first line, through which the second conduit (43) passes; Means (46) for connecting the second line (43) to the insulation (41) at the fluid sampling end of the first line (40), which connecting means for the microorganisms in the fluid sample so impermeable are that the sample does not come into contact with the second line (43); and a potential meter with a high input 7 10 resistance of 10 to 10 ohms, the one with the measuring electrode (40) and the second line (43) is electrically connected. 7098 2 7/10 ??