JPH05500311A - Method for electrochemically determining bacterial population - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Methods of Electrochemical Determination of Bacterial Population Prior Art and Its Limitations Over the decades, various electrochemical methods have been proposed for determining bacterial populations. It has been chanted. The basic method was developed by the inventor, H.P. Silverman (H.

P, Silverman) and Noei M. Blake (J, M, B r ak e), “Microbial population, enzyme activity and group size by electrochemical analysis” Method of Determining Micro vial Populations, Enzyme Activities, a nd 5ubstrate Concentrations by Elelc No. 3,506 entitled trochemical analysis) J. , No. 544. Patent No. 3.506.544 mentioned above is mentioned herein by reference.

The cited Nolberman/Blake patent includes a two-step reaction process, viz. First, a bacterial or enzyme-catalyzed oxidation/reduction reaction occurs, and second, the first Electrochemical acidification of reduced species (reduced forms, states or substances) produced in the process of It is described that the reaction was carried out to produce a measurable electrical current. More details Once the bacterial or enzyme sample is mixed with a special water-based reagent containing both oxidized species. This reagent/sample mixture was Different types of electrode systems are included, namely power electrodes, reference electrodes and current measuring electrodes. I put it in a container. In the control and measurement system: (a) with respect to the reference electrode; (b) by the action of bacteria or enzymes; The resulting reduced redox species is oxidized to its higher original oxidation state and (C ) The current obtained from this electrochemical reaction was measured.

Since the enzymatic activation reaction (first step) occurs throughout the entire fluid volume, the electrochemical reaction (second step) occurs only on the surface of the measurement electrode, but the reduced species produced by the enzyme The depletion due to electrochemical reactions is negligible compared to the increase in the concentration of reduced species. It was thought that Therefore, the concentration of reduced species increases continuously and electrochemically The current increased proportionally. It is the ``key'' to the Nruberman/'Blake method. ”, i.e., it is the determined rate of increase of the electrochemically generated current. It was. Except for significant changes and problems, the rate of increase in this current will depend on the increase in the concentration of reducing species. is proportional to the rate of the enzyme-catalyzed reaction, which in turn is proportional to the rate of the enzyme-catalyzed reaction; was proportional to the concentration of bacteria or enzyme in the original sample.

Therefore, the electrochemical methods presented in the prior art measure the electrical current associated with the sample. and the estimated relationship, i.e. (However, K is a calibration constant (or proportionality constant)). Bacterial concentration was determined by calculating the density of body numbers. prior art So, if 20K is constant, then 15. However, the applicant believes that in many cases it is I thought that it was not actually constant.

In addition to the major limitations of the prior art regarding non-constant calibration or proportionality constants, However, prior art systems had other significant drawbacks. These include:

That is, (1) Many bacterial species have the property of adhering themselves to surfaces and are susceptible to applied electric potential. uniformly on a surface (if on an electrode) with may be found in Therefore, traditional systems directly apply It was necessary to clean and sterilize the exposed electrodes after each test. this is the time It was a time-consuming and expensive job. Single-use disposable electrodes have been developed. However, these were very expensive, making the cost of testing prohibitive. even more troublesome The problem was the effect of bacteria on the measuring electrodes. Bacteria are sensitive to the properties of this electrode. and even significantly change its electrochemical activity during the test period. there were. In other words, the difficult part with prior art systems is the electrical activity in the fluid. Changes in the activity of measuring electrodes with respect to sexual substances and the effectiveness of such electrodes caused by a change in the product.

(2) There were significant problems with contaminants. In the context of this patent application, adulteration anything in the initial test sample (other than the bacteria themselves) that causes a current at the measuring electrode. It is defined as a substance. This accelerates the reaction in the "first step" mentioned above. may be due to contaminants (generally speaking, these contaminants may be caused by bacteria prior to the start of the test). (free enzyme). This also (or alternatively) indicates that the adulterant was used. This may occur when the substance is directly oxidized by the measuring electrode at the potential value. Ru. Both effects result in spurious currents and inaccurate bacterial counts. Contaminated in sample In addition to the basic problem of the presence of contaminants, there is also the problem of contaminant concentration for each sample. and hence the deviation of the current caused by the contaminant. There are many things. A strictly constant contaminant current per sample with a given system It was assumed that the calibration constants derived from independent plate counts of the sample were corrected. deer However, this is a rare case.

(3) A number of prior inventors working in the field of electrochemical measurements of bacterial populations have , precautions should be taken to prevent damage to bacteria from mechanical abuse. I didn't understand that I had to do it. Bacteria are subjected to sufficient mechanical stress. If the catalytic ability of the solution is catalyzed, those membranes can be destroyed and the Significant amounts of enzyme may be released, producing reduced species of spar (first step).

Even apparently mild disturbances can produce significant effects on bacteria. Book Applicant discloses the rapidly spinning and rotating methods commonly used in prior systems. electrodes, as well as other abrasive or destructive mechanical methods that have been used, It is believed that it may destroy the membrane of the

(4) Another feature of the prior art is that the current often varies over a wide range. there were. Such a fluctuating current may not explain the results or ensure the bacterial coefficient. made it difficult to

Due to various shortcomings of the prior art, electrochemical systems for determining bacterial populations The system has not proven to be of great practical effectiveness, nor is it of great commercial importance. It was something I couldn't hear. This is especially true for testing small numbers of bacteria. wait.

General purpose of the invention Among the (non-limiting) objects of the invention are the following. That is, (a) bacteria are the test method. turbulence that can destroy their cell membranes and thereby generate free enzymes. Not subject to harsh mechanical treatments and altering the ability of bacteria to catalyze chemical reactions Ensure that you are not subject to less severe disturbances that may occur. (b) Perform electrochemical measurements without any bacterial contaminants on the electrode surface. (C) Noku Tsu caused by background noise or by any contaminants present in the sample. determining or correcting that portion of the current scale that is (d) Contamination in the original sample Producing a "purified" sample before testing by first reducing the amount of Things. (e) Bacterial count density desired by many companies, scientists and experts. accurately, relatively quickly, and practically.

The above objectives and others are achieved in several ways. these are listed here do.

Summary of the invention The first new and improved method involves using a filter that collects bacteria from the sample to be counted. Use a tar cartridge. Transfer reagent fluids through cartridges like this electrochemical measurements of reduced species produced by bacteria in the filtrate from the cartridge. Do it with Next, we determined the bacterial count as a function of fluid flow rate, electrochemical signal, and time. to be determined. The method (according to one main aspect of the invention) provides an electrochemical signal characteristic of direct reactions against bacteria and at the same time neutralize contaminants. It is emphasized that

A second new and improved method involves transferring a fluid sample from a container through a filter. At the same time, the reduced species generated in the filtrate stream were electrochemically measured, followed by bacterial enumeration. as a function of varying bacterial concentrations, electrochemical signals and time. Including steps. An important aspect of this method is that the method uses by creating a second derivative of the chemical signal and at the same time nullifying the contaminant. be.

According to one main aspect of the invention, electrochemical measurements are performed (in the presence or absence of bacteria). Measurements are made in the filtrate as distinct from measurements made in chambers containing both fluid and bacteria.

Since the measurement is carried out in the filtrate, the bacteria must not be mechanically abused with the measuring electrode or otherwise. I can't find anything to use it. In addition, other devices beyond the measuring electrode or filter The possibility of bacterial contamination of the parts does not exist. In other words, the applicant It has been discovered that a significant improvement is achieved by incorporating the polar assembly into the filtrate. However, such a filtrate is the same as the filter previously used to collect bacteria from the original sample. - by Turquard Relan or at the outlet of the container containing the bacteria; produced by a filter located downstream from this.

The "electrochemical" methods of measuring reduced species referred to herein include Standards for electrochemical measurements, including measures of electrode potential at polar current and/or constant current and/or other voltmeters or ammeters to determine the concentration of electrochemically active substances. There is a method.

Measuring electrodes can be made of various chemically inert and electrically conductive materials, such as platinum, palladium, etc. aluminum, gold, carbon, graphite etc. and in numerous standard shapes, e.g. rotating, fixed , capillary, rod, wire, etc. In two electrode measurements , a reference electrode and a power electrode are combined to both detect potential and supply current. It can be any standard reversible electrode used. Three electrode system measures The four electrode system includes a measuring electrode, a reference electrode and a power electrode. Includes a power electrode and two reference electrodes.

There are various forms of embodiments of the invention in which electrochemical measurements are made in the filtrate, and each form has variations. There is a law. According to one method, bacteria are collected in a filter cartridge at the test site. After storage, the cartridge is transported to a laboratory where bacterial enumeration density is determined. The decision is , passing the reagent solution into and through the bacteria-containing filter cartridge. This ensures that the bacteria present in the cartridge produce substantial reduced species in the filtrate. It is done in such a way as to cause the formation of The reagent, along with its reduced species, is then flows into a chemical measuring device where it is absorbed by the electrochemical action produced by the reduced species. The resulting current is determined. This current is based on contaminants, background currents and Adjusted to the gas chemistry instrument scale to yield the bacterial count density of the original sample.

The average flow rate of the reagent solution through the filter cartridge is such that the bacteria have no significant reducing species. be made low enough to occur. Preferably, instead of using a constant flow rate, an intermittent Do things in such a way that there is a flow. This sets the flow rate to a predetermined time. After that, the electrochemical measurements are carried out by using a specific flow rate for a certain period of time, and the electrochemical measurements are carried out during the entire operation. It takes place during the production.

Discussing the above in detail and describing another aspect of the invention, the reagents are prepared in advance. A container containing a defined volume of bacteria from the original sample, i.e. a highly Alternatively, it can be introduced into a small volume filter cartridge. Reagent in cartridge Then, all the bacteria in it cause enough reducing species to be detected by the electrochemical measurement device. hold for a predetermined time long enough to produce a measurable current at the location.

The reagents are then passed from the cartridge to the electrochemical measuring device and the measurements are taken in the filtrate. conduct. Additionally, the same reagent, but not introduced into the cartridge, It is passed through an electrochemical measuring device separate from the cartridge. Take another measurement at this second port. Perform this on the reagents listed in . The difference between the measured values is multiplied by a calibration constant, but such a constant Numbers were determined by independent methods on a portion of the original sample. Next Then, repeat the operation on other samples and use the same process as described to obtain the predetermined Use fixed constants.

In another form of the method, reagents are transferred via bacteria-containing cartridges to various A low flow rate is passed and a measurement is made with an electrochemical measuring device for each flow rate. That's it This corrects for background effects.

Yet another method of determining bacterial enumeration density is typically the "cartridge" method described above. A much larger capacity will be used than in the case of the method. Original sample (preferably , the purified sample produced by pre-filtration) is passed through the filter to the room temperature outlet. put it in a container. A period of environmental conditioning for bacteria in the room during which reducing species are produced. After a certain period of time, the reduced species are extracted from the chamber and measured via an electrochemical measuring device. Let it pass. Measurement is 1. of two substantially different concentrations in (identical) fluids Performed on bacteria. These measurements are then used to determine the bacterial population. , there are no errors introduced due to the presence of any contaminants in the fluid. extremely preferable Alternatively, the two different concentrations can be obtained by filtering the liquid from the containers indicated above. and then electrochemical measurements are carried out in the filtrate.

Yet another form of bacterial count determination involves filtering the fluid and using an electrochemical measuring device. after the measurements have been carried out, leaving a portion of it behind. Determining the electrical signal produced by the m solution and transmitting it through an electrochemical device Compare with the first signal produced during the first pass. Second, there are contaminants Confirm whether or not this is the case, and make corrections regarding the measured bacterial count.

Drawing description FIG. 1 shows an apparatus for carrying out the method of the present invention, which can be referred to as the bus method. route map, and the center part of the filter housing is shown to show the filter. It is broken.

FIG. 2 shows an apparatus for carrying out the method that can be referred to as the flow-through method of the invention. This is a route map of the station.

Figure 3 shows the implementation of the method which can be referred to as the concentration change method and the stored sample method of the present invention. This is a route map of the equipment for this purpose.

Generation of purified samples Throughout this specification and claims, when the term "coarse filter" is used, Unless otherwise specified, the term "filter" means a microfilter. this is , there are a number of commercially available microfuges suitable for retaining bacteria in filtration. It is one of the filters. These filters are sterile and biologically neutral. Yes, and not cytotoxic, secant, or stratomycotic.

It is preferable to carry out an initial filtration step separately before starting the method described below. Ru. This is due to the significant amounts and types of contaminants present in many industrial systems. It's for a reason. For example, commercially available oxidants are typically used for bacterial and algal control. used. Chlorine, chlorine dioxide and acrolein are all electrochemically active and one that affects the electrochemical signal produced by the electrochemical measurement device. Boss. Numerous other biocides, herbicides and algaecides are used as well as various water conditioners. leads to errors.

Free enzyme is usually present even when chemical processing agents are not present in the original sample. There is. These enzymes are transferred from the bacteria themselves through their cell membranes. It is produced by the normal method of secretion. The presence of these enzymes increases the bacterial count density. High reading.

The purified sample is prepared by first passing the raw material sample through a coarse filter, and then Discard the coarse filter, and then reuse the filtrate filtered through the coarse filter. degree (by microfilter), and write a second like this Preferably, it is produced by collecting bacteria on a filter. like this Discard the raw filtrate from the procedure and begin the procedure described below.

It is emphasized that the bacteria remain viable and intact. Therefore, for example For example, they are not dried on filters, nor are they mechanically or cannot be abused in any other way. After the first filtration, the purified sample is 7, cleaning it, and reconstituting it with reagent fluid. is generated by.

In many cases, purified samples are purified as described in the previous paragraph by using the reagents themselves. 9, filter cartridges can be produced without a separate separation process. It can be placed at a suitable location in the industrial plant, for example on a test spigot.

A predetermined volume of fluid from the plant is transferred to the filter cart. Pass through the ridge. Next, fill the cartridge with reagent fluid so that the bacteria After making sure that the bacteria remain viable, plug both ends of the cartridge. cart Transport the ridge to the test room where the method is performed (or transport the method to that part of the plant). You can do it on the spot.

As a special example, when milk is collected in a milking parlor, a Spigo Soto with a coarse filter inside is used. and then gradually flowed through a filter cartridge. next After flowing the appropriate predetermined amount in this manner, remove the cartridge and place it in the bacteriometer. Collected in a laboratory for testing to determine number density.

Detailed explanation of bypass method As an aid to the explanation of what may be termed the bypass method, first, the route With reference to FIG. 1, an apparatus for use in carrying out the method is described. However However, the device of Figure 1 measures current, but not other electrochemical signals (e.g. potential). ) can also be measured. Preferably, but not necessarily, In the bypass method, a filter cartridge as shown in 10 is used. Become. This is a small volume airtight container, typically molded from synthetic resin. and a filter F that is coupled to prevent the passage of bacteria or allow the passage of liquid. It is stretched to seal it. The typical capacity of the cartridge is about lcc.

The flow through the filter is typically slow, e.g. or less. for the filter cartridge 10 and for other components. The conduit that communicates with the filter preferably has a small inner diameter, and the Even a slow flow through the tube 10 will cause the filtrate to pass through the electrochemical measuring device. It should be understood that there is never too much time for

A reagent bag (or other container) 11 is connected to the pump 12 via a conduit. and communicates with a portion 13 of the bipolar two-way valve. In addition to the above valves component 14 is in communication with the first component 13, e.g. by solenoid means. , so that both components 13 and 14 operate simultaneously.

Valve component] If each of 3 and 14 is in a certain configuration, the sample from bag 11 The drug passes through pump 12 and component 13 to filter cartridge 10; From there it passes to the valve component 14 and then to the electrochemical measuring device 15. Fi router cartridge allows you to remove the cartridge from the system if desired. and allow new cartridges to be installed if desired. sequentially connected to valve components 13 and 14 by means of couplings 16 and 17 that You should understand that you are going through this.

A bypass 18 communicates between valve components 13 and 14 with a cartridge between them. ]Odor-introducing valve components 13 and 14 each have a different arrangement. If the reagent is used, the reagent is passed from the bag 11 and pump 12 to the measuring device via bypass 18. It flows to 15.

The illustrated electrochemical measuring device 15 has an insulating housing 19 in which a measuring device is placed. A constant electrode 20 is attached. The measurement electrode shown here is a cylindrical carbon electrode. pole, which is gradually rotated by the motor M within the housing 19 and They are fitted relatively closely together within the group. Prevent leakage with a suitable seal. Ru. The volume of the chamber inside the housing (external to the electrode) is small, preferably less than lcc. be.

The electrochemical measuring device further includes a power electrode 21 and a reference electrode 22, the latter being The measuring electrode 20 is substantially isolated from the surrounding chamber, such as by the conductive plug 23. It is. The measurement electrode, power electrode, and reference electrode may be any voltage source well known in the art. terminals and the current measuring device P, one example is the above-cited patent no. 506.544. Although the picoammeter is not shown, A device P is applied to measure the current flow at the measuring electrode.

The reference electrode 22 maintains a substantially constant potential and thereby the measuring electrode itself Measuring electrodes for which the body can maintain a substantially constant and selected electrical potential It should be understood that monitoring the potential of This can be done by suitable circuitry and This is done by applying an electric current from the force electrode 21 to the measuring electrode.

In the illustrated electrochemical measuring device 15, the fluid from the housing 19 is It passes through pole 21 and is discarded.

Proceeding to the method description, this is the population of bacteria to be determined. is collected (from the original sample of known volume) through a filter on the upstream surface. Including passing reagents. (The type of bacteria depends on experience with specific industrial systems, etc.) or by microscopic or other analysis. ) to the circuit before connecting the cartridge (as previously described) or through the filter. The first flow of reagent washes the bacteria and removes contaminants.

During at least the desired time after cleaning contaminants from bacteria, the pump 12 to ensure that the average flow rate of reagent through the filter cartridge 10 is sufficient. low enough that the bacteria in the cartridge produce enough reduced species that a measurable current is electrified. The measurement is performed by the physical measuring device 15.

To be more specific, one kettle of known volume of the original sample is stored in an industrial plant in a laboratory. is passed through a filter cartridge 10. This allows Kartlitz This results in the collection of bacteria on the upstream surface of the filter F. Then in the cartridge Fill with appropriate fluid and couple 1 with bacteria on the upstream surface of filter F. 6 and 17 into the circuit. Next, the pump 12 and valve components 13 and and 14 to transfer the reagent from the bag 11 to the filter cartridge 10 and 14. and bypass 18, alternately through the filter cartridge 18. The flow regime and flow rate used can be repeated (and repeated) for different samples. ).

Using an electrochemical measuring device 15, preferably measure the current for the entire time, Compare the amplitudes of. In other words, the current caused by the bypass is compared to the current caused by the cartridge. For example, if the condition is If the fluid in the chemical measurement device appears to be from a cartridge, the current is relatively high due to the effects caused by bacteria. On the other hand, electrochemical If the fluid in the target measuring device is from bypass 18, the current will The background current is relatively low, and the current is due to the action of bacteria in the cartridge. It is not typical.

The test ends when the amplitude of the current measured by the picoammeter repeats. can be done. In other words, the same current amplitude and the same (even lower) current amplitude occurs each time the fluid exits the bypass. The test may be terminated if this occurs every time. Furthermore, from the cartridge You need to get only one reading on the fluid and the one on the fluid from the bypass is , a given system has been thoroughly tested and the results can be repeated. If it is known that readings will be performed, alternating readings should be continued.

(As used herein, the term “amplitude” is used only if the same definition is used from test to test. can mean peak, integral, least mean square, etc., i.e. All variables derived in this way are constant parts shown below. ) The method is Furthermore, the electric current generated when the fluid in the electrochemical measurement device is from a bypass The current is reduced from the current that would occur if the fluid were from a filter cartridge. Including holding. Next, multiply this current difference by a constant or proportionality constant (K1), In this way, the colony forming units per milliliter (c Read the bacterial count density in f u's). This constant (K,) is Correlation of bacterial counts determined by this method when the operation is repeated and the counts were obtained by independent means on a portion of the original sample (e.g. (e.g., plate counting).

In each case when the fluid in the electrochemical measuring device is from the bypass If the current reading (by the picoammeter) of is very constant from reading to reading, then the fluid The picoammeter reading produced when coming from a cartridge is The picoammeter reading is slightly less than the reading if the fluid is from the bypass. It is significant even if it is only slightly larger. On the other hand, the reading of "bypass" is extremely low. ``Cartridge'' reading is more extreme than when referring to ``Bypass'' reading. It is desirable to conduct the test in such a way that the In the latter case, the The reading of ``ji'' changes the reading of ``bypass'' less often (both occur twice or three times). It is often desirable to

This bypass method with intermittent flow is one of the best embodiments considered by the inventor. Ru.

Constant or intermittent flow through the cartridge is controlled by valves 13 and 14. Whether the reagent from pump 12 is pumped on or off (of course) The entire time when the cartridge is in position to pass through the I can be there. Change the positions of valves 13 and 14 to ensure that the reagents are in the cartridge. If the cartridge is to pass through bypass 1-8 instead of through The flow through the pipe is zero.

The length of time for switching valves 13 and 14 to the bypass position is Since there is no flow through the The expected (or determined at an early stage of the study) details Related to bacterial counting. For example, if the bacterial count is low, the reagent will not flow and the cart The length of time left on ridge 10 is relatively long (on the other hand, a high bacterial count The length of time that the reagents are left in the cartridge 10 will be relatively short if . In both cases, the time is long enough for the bacteria to generate enough reduced species to be measured electrochemically. The objective is to allow the fixed device to make meaningful and meaningful measurements.

There are various constants that correlate current readings with bacterial count density. For example, cart One constant for zero flow for 3 minutes in the ridge and for 9 minutes in the cartridge. There is another constant for zero flow. Both of these constants are such as to yield corrected bacterial count density readings at both times of 3 minutes and 3 minutes.

Both constants are, as mentioned above, after they are repeated many times. Bacteria as determined by current reading and (for example) plate counting of identical volumes of sample It is determined by correlating with the count density.

Pump 12 receives flow from the bypass when the system is in the 1-bypass condition. reaches the electrochemical measuring device and purges it with the filtrate from the cartridge. It should be understood that it is preferable to stop immediately after This state is You will know it is present as soon as the current meter reads a minimum current. On the contrary, the system If the system is in the "cartridge" state, the pico ammeter will then show the maximum reading, so It was found that the filtrate was purged into the electrochemical measurement device with the reagents from the bypass. Ru.

The particular electrochemical method described above provides an output for determining bacterial population. Although current measurement is used as the signal, the method is not limited thereto. Determination of reduced species be replaced by any other method suitable for the measurement, e.g. using a potentiometer. be able to.

In a number of applications, data processors are used, as described under the subheadings below. It is preferable to

Detailed explanation of flow-through method The flow-through method involves the collection of bacteria, i.e., a predetermined amount of bacteria from the original sample. Transfer reagents through the filter cartridge (or other container) containing the sample. and measuring reduced species in the filtrate stream. Two sets of data, that is. A bacteriometer can be used in a data processor using flow rate versus time and current versus time indicating reduced species. Determine the number density.

The flow-through method is another best embodiment considered by the inventor.

Referring to Figure 2, one form of apparatus for use in carrying out a flow-through method state is shown. The electrochemical measuring device 15 is the device 15 described with respect to FIG. is the same as As in FIG. 1, the filter cartridge 10 is placed in the device 15. in communication, the cartridge is removable due to the presence of couplings 16 and 17. Ru. In FIG. 2, the reagent pump is illustrated as a syringe 25, whose piston 26 is driven by a motor 27. The motor 27 is a motor and/or or piston position, and the flow data is processed as illustrated at 28. in combination with detection means or other means for communicating with the device and the bacterial reader.

Device 28 also provides current data from the measurement electrodes. Flow to device 28 The transmission of this data is indicated by arrow 29, whereas the transmission of current data is indicated by arrow 29. Indicated by arrow 30. The application equipment shown with respect to Figure 1! and the current measuring device P is shown in Fig. It also exists linearly or equally in the two devices. For example, in Figure 2, “Electrochemical measurement The box labeled ``Standard Device'' is a combination of the ``Force and Current Measurement'' box in Figure 1. It can also be absorbed. Alternatively, for example, a picoammeter may be connected to the data processing block 28. May be given.

If there is a constant flow of reagent through filter F in cartridge 10, all That is, when the motor 27 is operated at a constant speed, the bacterial count density is The average current detected by the measuring device 15 and transmitted to the data processing device 28 Equal to the constant to adjust. Such a constant flow rate may occur if the concentration of bacteria is extremely high. , or run the pump very slowly (than in the examples described below under this subheading). It can also be used when operating to produce a flow (which is clearly slow).

In a preferred form of the flow-through method, bacterial counts are determined through the cartridge. This is achieved by making measurements at two different flow rates.

Whether the flow is constant, intermittent, or variable can vary. repeated for each test on samples of Furthermore, all conditions are kept the same from test to test. be done. Then, for each type of exam, i.e. For each computer program (where determined by A constant (F2) that correlates with the actual bacterial count density is known. This constant is For each type of test, (e.g.) derived by obtaining a plate count .

Then, for the latter test (of all embodiments of the invention), the same constants are repeatedly used. Ru.

The flow rate through the filter in the cartridge is for a predetermined time. If zero, reduced species will accumulate in the cartridge 10. Next, flow If it is started at the end of a predetermined time, the flow is thus The accumulated reduced species are transferred to and into the electrochemical measurement device 15.

The integral of the current generated in device 15 is the result from the transfer of specific accumulation to device 15. The area is based on the current pulse (current vs. time curve) generated by the current pulse. flow program If the ram can be accurately repeated and repeated for each sample (e.g. There is a constant ratio between the amplitude and area of the pulse based on the curve (generated in processor 28). exists. Therefore, the bacterial enumeration density is determined by the current received from the cartridge. If the amplitude of the pulse obtained with device 15 is due to the presence of accumulation, the current is adjusted. Equal to the clause constant.

The fundamental difference between this flow-through method and the prior art is that the flow-through method This means that we do not measure the rate of change of current to determine . Instead, This flow-through method measures the average current flowing through the measurement electrode at a constant average flow rate. Ru.

According to one embodiment of the preferred form of the flow-through method, especially when bacterial densities are low, , the reagent is passed through the filter in the cartridge 10 to ensure that very few reduced species are produced. The object is passed at a very high speed, almost as if the object is moving. From filters to electrochemical measuring devices The result is that most of the liquid that passes through is only reagent and not reduced species.

The resulting current delivered to the data processor is appropriately considered a background current. Accounting factors including any residual contaminants that may be present in the bacteria after washing and cleaning This is the background current considered as

To give an example of the flow-through method, the dimensions of the filter are (capacity is lcc, The first pulse of reagent (with a flow rate of e.g. lees/min) is applied to the filter cartridge. It is assumed that the dimensions are such that most of the residual contaminants are washed away from the pipe. Next A second pulse, also at ICC 11 cc/min, produces a background current. Jiru. This again allows the bacterial enumeration density in the cartridge to be reached at lcc/min. It is assumed that this is low enough to result in fewer reduced species. Next, this After the first two pulses, the pump is stopped and a delay of e.g. 15 minutes is applied. bring about Then, after that delay, start the pump again and run it through the filter Flow is induced at a rate of lcc/min. This is a pulse, slap or volume of filtrate. The quantity is advanced to the electrochemical measuring device 15. Next, use a pico-ammeter or data processor. The current exhibited by the chemical 28 indicates that such pulses containing substantially reduced species are present in the device. 15, i.e., the subsequent signal indicates the concentration of the reducing species. related to. If such a slap passes through the measuring device, the signal will be , return to essentially the same background current as previously measured (this is the base line). At that time, the bacterial count will be performed using a slap bank with less than ground current. is equal to a constant (F2) that adjusts the average amplitude of the current produced by holding.

The bacterial count density is determined by a data processor, e.g. per milliliter of the original sample. It can be read in Coney forming units (cfu's/ml).

The method includes making current measurements at two different fluid flow rates. 2 like this Different types of flow rates electrically measure the concentration of two different types of bacteria in exactly the same fluid effectively providing a means to ensure that the effective bacterial concentration in the filter cartridge is , which is inversely proportional to its flow rate. The relationship is 2. influence of contaminants This is a relationship that eliminates it from the calculated reading of bacterial counts.

To paraphrase the above, (any electrical current other than that produced as a result of bacterial action) The background current (obtained from the source) filters the reagent fluid into the cartridge. are excluded from the test results if they are alternately moved at two different flow rates through the

Two types of current measured by an electrochemical measuring device are used together with the measured flow rate. and calculate the bacterial count.

The applicant has determined that the component of the current (rlJ) generated by the action of bacteria has two types of flow rates. (Fl and F2), but the background current components from other sources are They found that the amount was virtually constant. Therefore, two unknowns with only two Give the equation and solve for B (bacteria).

In general, ■ (measured value) = 1 (bacteria) + ■ (background) [However, ■ (background) ground) is constant].

However, the current value (L, 12) and flow rate (Fl, F2) are all measured. F2 is the amount determined by an independent method of counting bacteria in a portion of the original sample (e.g. is the proportionality constant derived using plate counting).

For example, if the average flow rate of lcc/min produces a 12.1 nanoamp reading, then 1/ A 10 cc/min average flow rate produces a 16.8 nanoampere current, F, = 0. I L =16. 8 Detailed explanation of concentration variation method A concentration variation method is described with respect to FIG. 3, thereby providing an explanation for implementing such a method. 1 shows one form of apparatus that can be used. Electrochemical measuring device 15 in FIG. is shown as identical to that of FIG. 1 and the same reference numerals are used in connection therewith.

The same applies to the application and current measuring device P.

Has a capacity many times that of the filter cartridge 10 in most applications A container 32 is provided with a vented cap 33. Reagent container 34, e.g. The bag communicates with container 32 via valve 35. The filter 38 is the container 3 2, and the top surface of the filter is in direct communication with the interior of such a container. It is preferable to be there. The filter 38 is placed in a filter housing 39 and This communicates with the pump 37, which communicates with the housing 19 of the measuring electrode 20. Ru.

Gas injector 40 removes oxygen from the fluid in container 32 and agitates it. In order to do this, an inert and oxygen-free gas is introduced into such a fluid. other The element is combined with a container, e.g. for introducing a mechanical agitator and a cleaning fluid. It is a means of The gas is supplied via a gas injector 40 and preferably to an acid Passed into a fluid in a container containing nitrogen-free gas. Sample inlet not shown is provided on the container cap or cover 33 to ensure that such To eliminate the need to remove the cover.

The liquid passing through the electrochemical measurement device will be discarded, i.e. its This is not recycled to container 32. Therefore, valve 41 is applied to the waste line. and remains open during the concentration variation method.

Preferably, the concentration variation method includes a preliminary step of introducing a known volume of sample into the container 32. stomach. While valve 35 remains closed, pump 37 pumps all liquid into the container. and processed to pass to waste. Therefore, the fine particles in the container Bacteria are present on the top surface of the filter 38.

Next, pump 37 is stopped, valve 35 is opened, and reagent is poured from bag 34. 32 to fill it to the desired level. Next, close the valve 35. Jiru. Using gas injection means 40 alone or in combination with a mechanical stirrer, Purge the reagents with oxygen and (preferably) homogenize the reagent-bacteria mixture. Hold. The device is now ready to begin testing for bacterial counts.

In general, the basic theory of the concentration variation method is to determine the concentration of bacteria in one and the same liquid (filter). (by removing fluid from vessel 32 via a filter) and reducing species. The rate of increase in concentration is measured (by means of a suitable electrochemical measuring device).

Therefore, two sets of data are obtained, one is the concentration of reducing species vs. time, and the other is the concentration of reducing species versus time. One is the concentration of bacteria versus time. Using these data, count bacteria per unit volume. determine and negate the effects of any contaminants that may remain after cleaning. many dark Any one of the degree programs (operations) may be used, each with its own data It has a data processing algorithm that allows it to detect two functions that are characteristic of bacteria. Use the set of data.

A preferred method of bacterial concentration uses filtration as shown in FIG. pump 32 Upon activation, the volume of bacteria-containing reagent in container 32 is adjusted to an initial volume (which may be referred to as Vt). volume to a substantially lower volume (which may be referred to as V2). in advance At a given time, for example, the volume of liquid in container 32 is vI; When the amount is V2, the electrochemical signal at each concentration is measured using the electrochemical measuring device 15. Read Gunar. Preferably, the electrical signal is derived from an electrochemical measurement device. a volume signal from pump 37 or associated with vessel 32. The information is transmitted from the detector to the processor.

The preferred form of concentration variation method uses a data processor to determine volume versus time and signal. from an electrochemical measurement device and from a pump or supplies /gunar from a container. A pump 37 pumps the reagent into the container 32. Operation is started immediately after introduction from the pump 34, and the pump is operated using an electrochemical measuring device. Continuously measure the current during this period.

Next, the data processor determines the second derivative of the knobal present at the measurement electrode 20. Set. Such a second derivative is proportional to the bacterial count (B). signal is current , the general formula is , where B is the bacterial count: 3 is the bacterial count by using plate counting etc. is the proportionality constant derived (in the manner described above): and I is the measuring voltage This is the polar current.

Any desired flow program can be used, but the program is Must be repeated for each charge. As before, keep all conditions the same from test to test. Must have.

Converting the reading produced by the measuring electrode in the concentration variation method to bacterial count density The second measure is as follows. Start the pump 37 and apply three types of current. Measured by a gas-chemical measuring device, the current is measured at regular intervals and in time.

Specifically, the current 11 is measured with the capacitance V1. Next, further fluid is passed through the container 32. and allow it to pass through so that capacity ■2 exists. Next, the current I2 is measured. I, and I2, and the capacity remains v2. During this period, measure current (3).

The bacterial count (B) is then determined by the equation below.

B=Kn (I4+I2 2I3) As before, the constant 4 can be calculated by an independent counting method, e.g. plate counting, into the equation determined to interpret the results to actual bacterial count density in cfu/ml. It is something that was given.

The concentration variation method described above eliminates any residual contaminants. Concentration of bacteria in reagent rises as reagents are filtered from the bottom of the container and pumped to waste. However, it is noted that there is no corresponding increase in the concentration of contaminants.

The contaminant concentration remains the same while the bacterial concentration increases. therefore , Mathematically, contaminants can be excluded from the equation, so they cannot be produced by bacteria. Only the same effects are used to generate the bacterial count density.

Detailed explanation of storage sample method Referring to Figure 3, the equipment used in the pooled sample method is similar to that previously described with respect to the concentration change method. It can be said that it is substantially the same as the one posted. However, valve 42 has the reference number 43, which extends into a container (e.g., a bag).

The pooled sample method removes any residual contaminants in the bacteria (remaining after the preparation of the purified sample described above). Significantly reduce or eliminate the effects of foreign substances in the following ways:

The first part of the method is the same as described above for the concentration variation method. But long electrochemical during the test (when readings are taken by the electrochemical measuring device). The liquid that passes through the measuring device does not pass into the waste, but instead into the bag. 43, valve 42 is opened and valve 41 is closed. Furthermore, the filtrate and reagents from the filter housing 39 to the pump 37 and the electrochemical measuring device. During the passage to valve 42 through the be exposed.

Therefore, substantially later, liquid stored samples can be measured via an electrochemical measuring device. and pass it again. For example, the conduit combined with the valve 42 may be drained or Separate the bag 43 and transfer the stored sample from the bag into the pump 37 and into the electrochemical This may also be done by introducing it into a physical measuring device. Although the introduction is not shown, , by means other than the container 32. Next, make a second current measurement on the stored sample. implement.

In this way, it can be determined whether contaminants were present when the first reading was taken. Set. Contamination by a second passing current signal equal to the first measured current It is proven that the object did not exist. On the other hand, increasing with respect to fluid storage samples The current generated indicates the presence of contaminants. In the latter case, the amount of increase in any such current is used as a correction factor. In other words, the electric charge obtained from the second pass of the liquid Calculate the difference between the current and the current obtained from the first pass of the liquid. This difference, the liquid Subtracted from the current measured on the first pass through the electrochemical measuring device.

Next, the bacterial count density is determined using the current corrected in this way. (the result is used in conjunction with the constant determined by plate counting to obtain the density. ) test , repeat the same method again for each sample.

Concrete example Example 1 Enzyme test showing the possibility of bypassing fermentation from tryptic soy broth The combination of certain oxidants and substrates, such as methylene blue and gelcose, Known to catalyze reactions. Using this feature, we can use reduced species (in this case reduced species) Bypass method by determining the current in response to an increase in methylene blue) We tested the possibility of

Reagents and trypsin soybean buoy in a small plastic container with a capacity of approximately 1/2 ml. 50150 mixture was added. Enter and release the connection to the cartridge, Can be used as a substitute for filter cartridges in bypass devices. (Figure 1). Place a small container into the test loop and wait 15 minutes. selected to produce a constant concentration of reduced methylene blue through enzyme catalysis. I set it. The reagent had the following composition.

H2O75ml -Basic phosphate 0.4g K2HPO40.8g Glucose 0914g Methylene blue 0.005g Potassium chloride 0.35g It had the composition of tritripone soybean broth.

H2O100ml Casein pancreatic juice digest 1.7g Papain digested product of soybean meal 0.3g NaC] 0.5g K2HPO40.25g Dextrose 0.25g During the last minute of the waiting period, the reagents are pumped through the electrochemical to the measuring device and zeroed the current (no background current was flowing). (Even if it was, it was zero) At the end of the waiting period, bypass the reagent stream. Transfer from the path and through the container containing the reagent/soy broth mixture. It flowed. A stack of stagnant liquid held in a plastic container is electrochemically 120 nanoamps (zeroed back) when passed through the measuring device. Record the peak current (above ground or baseline) and then return it to This brought the value close to zero when the stack passed through. With this test, If the reduced species is produced by biological action, the concentration of the reduced species is It can be easily measured by the peak current generated by a physical measuring device. is confirmed.

Example 2: Bacterial quantitative test using bypass method In this exam, Nalge Company (o Bacteria were retained using syringe filters from Star, NY).

The cellulose acetate sterile filter membrane has a diameter of 25 mm and a porosity of 0.　 It was 45 microns. Remove the filter top and add a new top to complete the filter. The volume of space on the router surface was increased to approximately 1/2 ml. Escherichia coli (E, col) The pure culture of i) (approximately 1 ml) was filtered through a filter membrane. Next, Add the methylene blue/reagent used in Example (1) above to the filter cartridge. Filled with fluid, the cartridge was placed into an apparatus loop as shown in FIG. of reagent A hold time of 15 minutes was used after introduction.

As in Example (1), near the end of the retention time (by bypassing the reagents) and an electrochemical measuring device, where the current was set to zero.

The reagent stream was then moved and passed through a filter cartridge. Retention 4.5 nanoamps when the stack of fluid reaches the electrochemical measuring device. A peak current of .

By moving the accumulated stack from the cartridge, new reagents are added to the filter. It ended up being in a Luther cartridge. Hold this again for 15 minutes and change the current. The determination was repeated as above. , recorded a peak current of 4.6 nanoamps. and thereby for a specific waiting time (measured by an electrochemical measuring device). The concentration of the resulting reduced species was found to be essentially constant.

The new reagent solution occupying the filter chamber is now held again for 30 minutes. I did it in between. Determining the peak current for this retention time, for a retention time of 15 minutes The current was found to be 9.4 nanoamperes, almost twice the current found in the previous study.

This test shows that the amount of reduced species produced is proportional to the time held in the filter chamber. It turns out that 9. The peak current exceeds the expected value of 2 (2X4.6) A slight increase in flow was determined by performing the test three times on the same bacterial sample in the cartridge. This is likely the result of an increase in total bacterial numbers that occurred during the time required to

Example 3; Qualitative tests on various microorganisms and various reagent compositions Regarding the presence of bacteria and reagents with various oxidants and substrates If peak currents occur due to the pulse mode of the bypass method, two additional The decision was made through experimentation.

Conditions Example (1) Example (2) Microorganisms, yeast B. globigii (B, Globigi i) Oxidant: Phenazine methosulfate Methylene blue Substrate: D-glucose Sodium succinate buffer/Phosphate Phosphate In each case test, the peak current is easily measurable after a holding time of 15 minutes. It has been shown.

The present invention has devised the ability to accurately and sensitively measure the concentration of reduced species; This makes electrochemical measurements practical and useful. In a preferred method, the resulting As proposed for prior art methods of measuring the rate of change of current, electrochemical Directly measure the magnitude of the current generated by the measuring device. Furthermore, in the present invention, reagents, contaminants Current measurements are completely freed from background contributions from physical and electronic noise. to correct. This transfers reagents, including oxidants (oxidized species) and substrates, through the bacteria stagnant in the filter cartridge and, in turn, the magnitude of the current. This is accomplished by moving the sample through an electrochemical measurement device that measures the temperature.

The same reagents can then be transferred to the filter cartridge via bypass. gives a measure of the background current, which can be applied to the filter cartridge subtracted from the total current measurement in the edge channel to determine the true current product caused by bacterial action alone. give a minute. This current component is sequentially pre-established to be proportional to the bacterial population. It was found that the concentration of the reduced species was proportional to the concentration of the reduced species. Therefore, for a novel method , bacterial individuals determined by an independent method of injection plate counting (plate counting) The numbers should show sufficient correlation with the novel electrochemical method of the present invention. is expected.

Additional disclosures The Silverman/Blake patent cited above describes the applied voltage, pH1 temperature, oxygen Since factors such as desorption of (other than briefly referenced above) are not described herein. identification Other factors described in the cited patents, such as those relating to However, since it is not already described in the patent, it is only partially mentioned in this specification. . .

The portions of the cited patents specifically mentioned herein relate to the determination of bacterial populations. It is something to do. However, the present method of the applicant mainly depends on the number of viable bacteria. Emphasis is placed on the decision.

Said washing of soluble contaminants from bacteria is preferably carried out with distilled water.

The picoammeter used herein by applicant was manufactured by Kais, Cleveland, Ohio. Lee Instruments, Inc. (Keithley Instruments) 485 manufactured by Ruments, Inc.). The applicant is The filter cartridge used here was manufactured by Narji, Rochinister, New York. Catalog number 190-2045, manufactured by E. Co., Ltd.

The electrochemical measuring device used here by the applicant is made of stainless steel as the power electrode. It includes a tube 21. The reference electrode used and preferred herein is available from a number of sources. A commercially available silver/silver chloride reference electrode. Other reference electrodes, e.g. The calomel electrodes mentioned in the patents mentioned above can also be used.

A preferred measurement electrode 20 is manufactured by Phantom Elements, Fullerton, California. Quantum Electro Development.

pment). This measuring electrode is made from polycarbonate. a rotating solid rod of graphite housed in a cylindrical chamber 19 constructed of The gap between the chamber walls is only 0.005 inches. The length of the rod is 1 to 7/16 inches. inch and has a diameter of approximately 172 inches. The holding capacity of the assembly is approximately 1/4 CC. Collect the 316 stainless steel stirring rod by passing the rod through its center. and communicates with the drive shaft by means of a sealing means. drive motor housing Attached to one end of the chamber. The electrode assembly runs smoothly and with minimal sonic noise and noise. and electrical noise. The entire electrode surface is rotated at exactly the same speed throughout the fluid. However, this speed is relatively slow compared to the prior art. The preferred rotation speed of the electrode is 40 0 times per minute. (The graphite was produced at StackBot in St. Mezies, Pennsylvania. Stackpole Carbon Compa Obtained from ny). Specification values are density 1.85 and grade 1336. ) Numerous installations including rotating peddles, wire loops, dripping liquid metal, capillaries, flat plates, rods, etc. It is emphasized that there are measuring electrodes for the meter. Disposable devices (as described above) were also used. .

The present invention uses a process that prevents bacteria from coming into contact with measurement electrodes, so It is believed that much less electrode change occurs than in the prior art.

However, the applicant prefers that the measuring electrodes be tested hourly. ing. The measuring electrodes are therefore protected from bacterial contamination and frequent sterilization methods. However, it is necessary to ensure that the measuring electrode activity is adequate and constant.

A preferred and accurate method of providing such assurance is to calibrate the electrode activity from time to time; This calibration is then used in the final calculation of the bacterial count. Therefore, Honde The applicant shall be permitted to take the test before or after conducting the test, or if it is considered expedient to do so. Whenever possible, use a means of measuring the sensitivity of an electrode. Calibration of electrochemical measuring equipment , an electrochemical reaction produced by a known amount of a specific reduced species measured during bacterial enumeration. All this can be done by determining the actual current measured by the measuring device. can. A “standard solution” is used for this test and may be referred to as the electrochemical calibration fluid. can. Thereby, the variable area, variable hydrodynamics and surface activity of a single (in units of current per mole of reduced redox species) to be combined with a calibration constant. An electrochemical measurement device moves a fluid through it, and the fluid has a known concentration. calibrate by measuring the resulting current. The emphasis is on being corrected.

Furthermore, redox (oxidation/reduction) pairs exist in two oxidation states in equilibrium. can be changed from one state to another by a chemical or electrochemical reaction. organic compounds that can change into states, such as methylene blue, It is emphasized that this is a pair of Lembres. The oxidizing species of the selected redox couple is the reagent Present in fluids. Its function is normally controlled by bacteria, as any oxidant Anything used in its natural environment has a role to play (02. 504=, N0s- or C03=, etc.). This new oxidant (or medium ) reacts with the substrate (further provided by the reagent) to create a new enzyme for the bacteria. This results in an energy cycle.

A given new oxidant can be (a) used in the energy cycle by bacteria; and (b) produce reduced species that are readily electrochemically reoxidized. Can be done. Many substances can be used as oxidants or two or more cocktail mixture. Suitable oxidants are known dye media. such as methylene blue, 2,6-dichloroindophenol, indigo diphenol, It can be selected from sulfate, phenosafranine, phenazine methosulfate, and the like.

Other substances, such as benzoquinone and ferrocyanide, have also been found to be useful. It was.

Substrates are redox partners in reactions catalyzed by bacteria (or their enzymes). A substance that combines with selected oxidized species to produce the desired reduced species. There are many It has been found that organic materials are useful in a variety of applications for this purpose. to these is a monosaccharide such as glucose, lactose, dextrose, fructose, etc. and other intermediates in the oxidation cycle, e.g. pyruvate, succinate, lysate. These include malate, citrate, -ketoglutarate, acetate, and lactate. Oxidized species Cocktail mixtures of two or more substrates may be advantageous, as in .

Reagents provide components needed by bacteria for bacterially catalyzed reactions. A specific solution that supplies reduced species to produce reduced species. The reagent is the oxidation state of the organic redox couple. (For example, methylene blue in the redox couple methylene blue/lucomethylene blue) blue), the group W required for the catalytic reaction (e.g. glucose) and the desired Contains appropriate buffer components to maintain pH. Reagents are oxygen-free and sterile. Ru.

The foregoing detailed description is only as clearly presented by the drawings and examples. It is to be understood that the spirit and scope of the invention is determined by the following claims. limited.

Submission of translation of written amendment (Article 184-8 of the Patent Law) May 1992 72I Ai 1

Claims (43)

[Claims] 1. A method for electrochemically determining bacterial population, the bacterial population being measured. providing a sample of a fluid containing bacteria and filtering the sample to obtain a filtrate; carried out to prevent bacteria from being present in the nuclear filtrate; Electrochemical measurements on the filtrate are carried out using electrode means that do not come into contact with the bacteria. Therefore, do it, and using the results of the electrochemical measurement to determine the population of bacteria in the sample. The method described above. 2. 2. The method of claim 1, wherein said bacteria are live bacteria that have been substantially unexploited. . 3. The method includes the electrochemical measurement including a redox couple and a measuring electrode. It is carried out by using an electrochemical measuring device, and the measuring electrode of the device is 3. The method of claim 2, further comprising exposing to the filtrate but not to the bacteria. 4. The bacteria are live bacteria, and the method includes: (1) washing the bacteria; Contaminants are removed therefrom, and (2) the washed bacteria are oxidized to redox couples. (3) causing said bacteria to progressively convert said oxidized species to reduced species; (4) filtering said bacteria from said reagent, and (5) said step (4). using an electrochemical measuring device that is in contact with the filtrate obtained from the bacteria but not with the bacteria. 2. The method of claim 1, further comprising: performing said electrochemical measurements. 5. The method described above causes the bacteria in contact with the reagent to undergo sufficient conversion of the oxidized species to reduced species. long enough that a significant reading can be taken by the electrochemical measurement device. 5. The method of claim 4, further comprising maintaining the temperature for a long time. 6. The above method can be carried out by means of a small capacity filter cartridge containing a filter. 2. The method of claim 1, further comprising performing the filtration step. 7. The method described above is performed at the outlet of a relatively large volume container containing the bacteria in the reagent. by providing a filter on the side and filtering the reagent through the filter. 2. The method of claim 1, further comprising performing the filtration step. 8. The above-mentioned method combines the above-mentioned steps in the same way but with different numbers of individuals. repeated on different samples containing bacteria, thereby further comprising determining a bacterial population, and further comprising: determining a bacterial population; using a constant that equates the chemical measurements, said constant being applied to a portion of said sample. of claims 1 to 7, determined by carrying out a non-electrochemical counting of related bacteria. The method described in any one of the above. 9. A method of determining bacterial population comprising: from there, through a filter provided downstream of said container, and then through a filter provided downstream of said container. From this, bacteria from the sample to be measured are transferred to the filter via the electrochemical measuring device. present in said initial volume upstream of the ter and not present on said device side. a second volume of the fluid separately from the container and the filter. There is substantial fluid in and to the device that has passed through the filter. introduced at some point in time, said second amount is free of bacteria. , using said device, when at least a portion of said first amount is present in said device. at least once and at least a portion of said second amount is present in said device At least one electrochemical measurement is performed at each time and the bacterial population is determined on the filter. the average signal measured by the device when the fluid from the tank is present in the device; the device if the fluid that is not from the filter is present in the device. The method described above is calculated from the difference between the average signal and the average signal measured by the position. 10. The method provides a common source of the fluid and provides the bacteria in the container. , then, during a period of time, directing the fluid from the feedstock through the container and the filter. and then, during another time, pass the fluid from the feedstock through the device. and passing the device separately from the container and filter through a 10. The method of claim 9, comprising: 11. Said method includes said passage of said first amount of fluid through said filter. Claim further comprising: having the bacteria present on an upstream surface of the filter prior to 9. 11. The method described above can be accurately repeated for the various bacterial samples to be measured. 10. The method of claim 9, further comprising repeating. 12. The above method uses a small capacity cartridge containing the filter as the container. 10. The method of claim 9, further comprising using. 13. The method further comprises using a container having a substantial capacity as the container. 10. The method of claim 9, comprising: 14. The method described above uses a reagent containing an oxidized species of a redox couple as the fluid. and such that the oxidized species is progressively catalyzed by the bacterium to reduced species. adapted so that the electrochemical measuring device produces a current that is related to the amount of the reduced species. , the method is adapted to pass through the container and the filter into the apparatus. The device is implemented to produce a significant electrical current when the fluid is present; the filter when there is fluid in it that has not passed through the filter. generates a background current and converts the difference in current into a non-electrical By correlating the above difference to bacterial counts previously obtained by of claims 9 to 13, wherein the bacterial count is correlated by using a constant obtained by The method described in any one of the above. 15. The method comprises discharging at least a portion of the first amount of fluid in the container. further comprising maintaining the portion of the fluid for a qualitative period of time, the period of time being such that the portion of the fluid remains in the filter. after passing the fluid from said second quantity to said device. a signal that is significantly greater than the signal produced by the device if present 10. The method of claim 9, wherein the method is long enough to cause . 16. A method for determining bacterial population, the method comprising: providing a bacterial sample; Bacteria from the sample are collected upstream of the filter in a filter cartridge and added to the sample. providing medicinal fluids; moving the reagent fluid through the filter cartridge at a measured average flow rate; , The filtrate obtained from the last mentioned step is electrochemically measured to determine the reagent flow. Determine the results of the bacterial action on the body and the results obtained from the last-mentioned steps above. Using the measured electrochemical measurements and the average flow rate measured above, The aforementioned method comprising calculating the bacterial population. 17. The reagent fluid is an ice-based fluid containing an oxidizing species and a substrate material of an organic redox couple. and the substrate substance induces a catalytic chemical reaction between the oxidizing species and the substrate. claim 1, wherein said bacterium produces a reduced species of said redox couple. 6. The method described in 6. 18. The method includes continuous flow of the reagent fluid through the filter cartridge. 17. The method of claim 16, further comprising moving the device. 19. The method includes intermittent flow of the reagent fluid through the filter cartridge. 17. The method of claim 16, further comprising moving the device. 20. The method further comprises performing the measurement using a measurement electrode, and the method is based on the integral signal generated at the measuring electrode during the measuring time. 17. The method of claim 16, further comprising: 21. The method includes various flows of the reagent fluid through the filter cartridge. further comprising moving at an amount, said measurement being measured during a period of relatively high flow rate. 17. The method according to claim 16, which is carried out by measuring the signal amplitude with an electrode for Law. 22. A method for determining bacterial population, the method comprising: providing a sample of bacteria; providing a reagent fluid; producing a second sample by mixing the sample of bacteria with the reagent fluid; by passing a sample of the bacteria through a filter that retains the bacteria. providing a bacteria-free filtrate from a second sample; electrochemically measuring the results of the bacterial action of Using the above measurements obtained by the last-mentioned step, the first-mentioned step said method comprising calculating the number of bacteria in the sample obtained. 23. The first sample listed above was prepared by precipitating the bacteria onto the filter in commercial equipment. 23. The invention according to claim 22, wherein the invention is produced by: 24. The method described above involves storing the filtrate and measuring it electrochemically once again at a later time. electrochemical measurements resulting from the presence of contaminants in said second sample. 23. The method of claim 22, further comprising determining an effect on a determination. 25. A method for determining bacterial population, the method comprising: providing a sample of bacteria; A reagent containing an oxidizing species of an organic redox couple and a substrate substance that can be utilized by the bacteria. and induce a catalytic chemical reaction between the oxidizing species and the substrate to reduce the redox couple. Generate the original seed, provide electrochemical measurement means; The reagent and the bacteria are allowed to interact for a sufficient period of time such that a sufficient amount of the reduced species is produced. the reduced species and the electrochemical measurement means are in contact with each other such that the reduced species and the electrochemical measurement means are in contact with each other; produce a significant signal in the electrochemical measurement means when the reagent or passing the bacteria and reduced species through a filter to produce a filtrate free of the bacteria; The filtrate is brought into contact with the electrochemical measuring means, and the filtrate is brought into contact with the electrochemical measuring means. and read the signals generated as a result of said contact between them and the filtrate. stomach, Any contaminants that may be present in the filtrate then contain significant additional amounts of reduced species. The filtrate is stored for a sufficient period of time to produce a contacting the electrochemical measuring means and reading the resulting signal; Correlate the first listed signal with the second listed signal. ,and using the results of said correlation to determine the population of bacteria in said sample. Method. 26. A method for determining bacterial population, the method comprising: providing a bacterial sample; providing a reagent fluid; mixing the sample and the fluid with each other to provide a first mixture of bacteria and fluid; , which has a first concentration of said bacteria in said fluid, and said concentration of said bacteria in said fluid. The same fluid can increase a second concentration of bacteria in the fluid by changing the the bacterial action in the fluid at the first concentration and the second concentration. determined chemically, and calculating the population of bacteria in the sample using the results of the electrochemical measurements. The method described above. 27. The reagent is a water-based fluid containing an oxidizing species and a substrate substance of an organic redox couple. the substrate material is available to the bacteria and the contact between the oxidized species and the substrate material is inducing a mediatic chemical reaction to produce a reduced species of said redox couple and said concentration change; 27. The method according to claim 26, wherein said is carried out by filtration of said bacteria. 28. The electrochemical measurement value is determined when the bacteria are in contact with the filtrate obtained as a result of the filtration. 28. The method according to claim 27, obtained by non-contact electrochemical measuring means. 29. A flow-through method for determining bacterial populations in which liquid is passed through a filter. a pump adapted to pump the filter, and an electrical connection downstream of the filter; provide a chemical measurement device, provide a sample of bacteria in a reagent, the reagent comprising an organic redox; A fluid containing oxidizing species of bacteria and substrate material available for use by the bacteria; inducing a catalytic chemical reaction between a species and the substrate to produce a reduced species of the redox couple; pumping the reagents through the filter and into the electrochemical measurement device using a pump. during at least a portion of said pumping time; Significant reducing species are generated by the catalytic action resulting from the presence of the bacteria. carried out in a manner that yielding a curve of signal versus time, the signal detecting a portion of the electrochemical measurement device. the number of bacteria in the sample that is present at the measuring electrode and using said curve. Flow-through method as described above comprising determining body count. 30. The method includes generating the curve in a data processor and determining the area under the curve. 30. The method of claim 29, further comprising determining a bacterial population in said step. Method. 31. After the method collects bacteria from the sample upstream of the filter, further comprising contacting bacteria with the reagent, and further comprising removing contaminants from the bacteria. 30. The method of claim 29, comprising contacting the bacteria with the reagent after washing. 32. The above method collects the bacteria on the upstream surface of the filter and then exposes the bacteria to the reagent. further comprising contacting the reagent with an initial amount of the reagent through the filter. cleaning contaminants from the bacteria, and further , all measurements obtained with the filtrate resulting from said washing were performed on said bacteria. 30. The method of claim 29, comprising ignoring in determining population size. 33. The method includes introducing an additional amount of the reagent through the filter and into the electrochemical Pump into the measurement device to eliminate background signals and any residual contaminants Determine the background or baseline that shows the results of the the background or baseline from the next measurement by the electrochemical measurement device. further comprising subtracting from the background signal so that the result is 33. The signal of claim 32 is substantially independent of signals generated by or contaminants. How to put it on. 34. The method includes introducing an additional amount of the reagent through the filter and into the electrochemical Pump into the measuring device to eliminate background currents and any residual contaminants. Determine the background or baseline that shows the subtract the round or baseline from the next measurement by the electrochemical measurement device. further comprising detecting a background signal or 30. The signal of claim 29 is substantially independent of signals generated by contaminants. Method. 35. The method includes intermittently pumping the reagent through the filter. and the reagent is applied to the measuring electrode of the electrochemical measuring device at a relatively high signal. contact with the bacteria for one or more periods of time long enough to cause the bacteria to be present therein. 30. The method according to claim 29, comprising leaving. 36. The above method calculates the area under the signal-time curve from the above determined bacterial population. 36. The method of claim 35, further comprising: 37. The method uses the amplitude of the pulses of the signal to determine the population of the bacteria. further comprising: the pulsing causes the reagent to generate a significant reducing species. after being in contact with the bacteria for a substantial period of time sufficient to 36. The method of claim 35, wherein the result is a reagent passed through the electrochemical measurement device. 38. A method for determining the population of bacteria in a predetermined volume of a bacterial sample, comprising: , provide electrochemical measurement equipment; The oxidizing species of the organic redox couple and the substrate material available to the bacteria being measured are providing a reagent, which is a fluid containing the oxidizing species, to induce a catalytic chemical reaction between the oxidizing species and the substrate, producing a reduced species of the redox couple described above and providing the bacteria to be measured in the reagent; Although the bacteria are prevented from coming into contact with the electrochemical measurement device, the presence of the bacteria contacting the catalyzed reduced species with the electrochemical measurement device and the resulting signal using the measuring device to determine the population of bacteria in the sample. Law. 39. A method for determining bacterial population, the method comprising: determining the bacterial population from a predetermined amount of a sample; is collected upstream of the filter in a small-capacity filter cartridge at an industrial site. retaining the bacteria in the cartridge in a live, undisturbed state; and the contained bacteria to the test chamber, and the reagent fluid is directly passed through the cartridge. from the upstream side of the contact filter to the downstream side thereof, and The population of bacteria in said predetermined volume of sample is determined by the last mentioned procedure. By performing electrochemical measurements on the filtrate produced by the process, The aforementioned method comprising determining. 40. The method includes, prior to said transfer of the reagent through the cartridge, 40. The method of claim 39, further comprising washing the germs. 41. A method for electrochemically determining bacterial population, the method comprising: providing a filter container with bacteria collected therein, directing a reagent fluid through the container and The reduced species produced by the bacteria in the container are transferred through the filter therein. An electrochemical measurement is carried out in the filtrate from the vessel and Determine bacterial counts as a function of reagent flow rate, electrochemical signal and time The steps mentioned above involve direct reaction of the electrochemical signal. Performed to characterize against bacteria, but nullify the presence of any contaminants The method as described above. 42. A method for electrochemically determining the bacterial population, including the bacteria being counted. providing a fluid sample in a container and moving the fluid sample from the container through a filter; At the same time, reducing species present in the filtrate stream are measured electrochemically and a bacteriometer is used. Determine the number as a function of changing bacterial concentration, electrochemical signal and time The steps mentioned above include that the second derivative of the electrochemical signal is carried out to be characteristic of The method described above. 43. A method for electrochemically determining the bacterial population, the method in which the bacterial population is determined. providing a fluid containing a sample of bacteria; providing a filter; provide electrochemical measurement means; The fluid is passed through the filter to the electrochemical measuring means, and the filtrate is passed through the electrochemical measuring means. Move at a speed fast enough to produce only a baseline signal in the measurement means. move it, Next, the fluid is passed through the filter to the electrochemical measurement means. The obtained signal produced by the measuring means is related to the population of bacteria in the fluid. moving at a sufficiently slow average speed such that the first mentioned signal used as a baseline for the second mentioned signal, and said method comprising determining the population of said bacteria as a function of both said signals. .