WO1991006670A1

WO1991006670A1 - Methods of determining bacteria populations electrochemically

Info

Publication number: WO1991006670A1
Application number: PCT/US1990/006507
Authority: WO
Inventors: Gilson H. Rohrback; Elmond A. Holmes
Original assignee: Infometrix, Incorporated
Priority date: 1989-11-07
Filing date: 1990-11-02
Publication date: 1991-05-16
Also published as: EP0499624A1; JPH05500311A; EP0499624A4

Abstract

A method of determining populations of live, whole bacteria electrochemically. The bacteria are filtered, and the filtrate is employed in association with an electrochemical measuring unit (15) to determine the bacteria count density. In accordance with a flow-through method, the average signal over the predetermined time period of the test is employed, in conjunction with a constant, to determine the population. In accordance with the bypass method, reagent is passed through a bypass line (18) to the electrochemical measuring unit (15), and the resulting signal is subtracted from the signal resulting from the filtrate, a constant being employed to correlate the resulting remainder with bacteria count. A changing-concentration method employs a changing concentration of bacteria in the same fluid to determine populations of bacteria. A saved-sample method employs a second test of the same filtrate, after a predetermined time period, to compensate for any contaminants that may be present.

Description

METHODS OF DETERMINING BACTERIA POPULATIONS ELECTROCHEMICALLY

Prior Art and Its Limitations

Over a period of decades, various electrochemical methods have been proposed for determination of populations of bacteria. A basic method is disclosed in U.S. Patent 3,506,544, entitled, "Method of Determining Microbial Populations, Enzyme Activities, and Substrate Concentrations by Electrochemical Analysis", inventors H. P. Silverman and J. M. Brake. Said patent 3,506,544 is hereby incorporated by reference herein.

The cited Silverman/Brake patent describes a two-step reaction process by which: first, a bacteria or enzyme- catalyzed oxidation/reduction reaction occurred, and second, an electrochemical oxidation reaction of the reduced species (reduced form, state or substance) , that was produced in the first step, was carried out to produce a measurable current. Stated more specifically, the bacteria or enzyme sample was mixed with a special water-based reagent which contained both a suitable substrate and the oxidized species of an oxidation/reduction (redox) couple. This reagent/sample mixture was placed in a vessel containing a three electrode system, namely a power electrode, a reference electrode, and a current-measuring electrode. A control and measuring system (a) maintained the measuring electrode at a selected potential relative to the reference electrode, (b) caused the reduced redox species produced by the bacterial or enzyme action to be oxidized back to its higher oxidation state, and (c) measured the current resulting from this electrochemical reaction. Because the enzymatic-activated reaction (first step) occurred throughout the entire fluid volume, whereas the electrochemical reaction (second step) occurred only at the surface of the measuring electrode, it was assumed that the depletion of the enzyme-produced reduced species by the electrochemical reaction was negligible compared to the build-up of concentration of the reduced species. Therefore, the concentration of the reduced species continually increased and the electrochemically produced current increased proportionately. It was "key" to the Silverman/Brake process that it was the rate of increase of the electrochemically produced current which was determined. Except for important variations and problems, this rate of current increase was proportional to the rate of increase in the concentration of the reduced species, which in turn was proportional to the rate of the enzyme catalyzed reaction, which was proportional to the concentration of the bacteria or enzyme in the original sample.

Thus, the indicated electrochemical method of prior art was to determine bacteria concentration by measuring a current that was related to the sample, and computing the bacteria population density by using the assumed relationship:

Bacteria = K- di dT

K was a calibration factor (or proportionality factor^ . The prior art assumed that this K was constant, but applicants believe that in many instances it was not actually constant.

In addition to the major limitation of the prior art relative to the lack of constancy of the calibration or proportionality factor, there were other serious drawbacks of prior art systems. These include the following: (1) Many bacteria species have a propensity to attach themselves to surfaces , and may even find surfaces having impressed potentials ( as on electrodes ) particularly attractive sites for establishing colonies . Accordingly , in prior systems the electrodes which were exposed directly to bacteria samples needed to be cleaned and sterilized after each test. This was a time consuming and expensive task . Single use , disposable electrodes were developed but these were too expensive , causing test costs to become excessive . Even more troublesome was the action of the bacteria on the measuring electrode. The bacteria could alter the characteristics of this electrode and cause its electrochemical activity to change significantly, even during the period of a test . Stated otherwise, part of the difficulty in prior art systems was caused by changes in the activity of the me a sur ing el ectrode re lat ive to the electroactive substance in the fluid , and by changes in the effective area of such electrode.

(2 ) There were important problems relative to contaminants. In the context of the present patent application, a contaminant is defined as any substance in the original test sample (other than the bacteria themselves) which will produce a current at the measuring electrode . This may result from a contaminant promoting the reaction in the above-mentioned "first step" (generally this contaminant is a free enzyme which came from the bacteria prior to commencement of the test) . This may al so ( or alternatively ) result i f the contaminant is a substance which will itself be directly oxidized at the measuring electrode at the potential value used. Either action will produce a spurious current and an incorrect bacteria count. In addition to the basic problem of having a contaminant present in the sample, often there is also a variation in contaminant concentration from sample to sample and thus a variation in the current caused by the contaminant. An exactly constant contaminant current from sample to sample, from a given system, would be compensated for in the calibration factor derived from independent plate counts of the sample. But this is rarely the case.

(3) Many prior inventors working in the field of electrochemical measurement of bacteria population have not realized that care must be taken to prevent damage to the bacteria by mechanical abuse. If bacteria are subjected to sufficient mechanical stress, their membranes may be ruptured, releasing a vast number of enzymes which will markedly alter the catalytic ability of the solution to produce the reduced species of the redox couple (first step) . Even seemingly mild disturbances can produce significant effects on the bacteria. It is believed by applicants that the rapidly spinning, rotating electrodes generally used in prior systems may rupture bacteria membranes, as would other abrasive or destructive mechanical methods that have been used.

(4) Another characteristic of the prior art was that there was often widely fluctuating current. Such fluctuating current made it difficult to interpret results, or to be sure of the bacteria count. The various drawbacks of the prior art were such that electrochemical systems for determining populations of bacteria have not proved to be of major practical value and have not had major commercial importance. This was especially true relative to tests of small populations of bacteria.

General Objects of the Present Invention

Objects of the present invention include (without limitation) the following: (a) Assuring that the bacteria will not be subjected during the test procedures to harsh mechanical treatment which could rupture their cell membranes and thus produce free enzymes, and will not be subjected to less violent disturbances which could alter the bacteria's ability to catalyze a chemical reaction. (b) Making electrochemical measurements without any bacterial contamination of electrode surfaces. (c) Determining or compensating for that portion of the current measure which is caused by background or by any contaminant present in the sample. (d) Making an initial reduction in the amount of contaminant in the original sample, thereby producing a "purified" sample before testing. (e) Achieving accurately, relatively rapidly, and practically, the bacteria count densities desired by many companies, scientists, and professionals.

The above and other objects are achieved in several ways. These will now be described.

Summary of the Invention

The first new and improved method employs a filter cartridge which has collected the bacteria from the sample to be counted. A reagent fluid is moved through such cartridge while an electrochemical measurement of the reduced species caused by the bacteria is made in the filtrate from the cartridge. The bacteria count is then determined as a function of the fluid flow, electrochemical signal, and time. It is emphasized that (in accordance with one major aspect of the invention) the procedure is such as to make the direct response of the electrochemical signal a characteristic to the bacteria while nulling the contaminant.

The second new and improved method comprises means to move fluid sample from a vessel through a filter while electrochemically measuring the reduced species caused in the filtrate stream and then determining the bacteria count as a function of changing bacteria concentration, electrochemical signal, and time. An important aspect of this method is that the procedure is such as to make the second derivative of the electrochemical signal characteristic to bacteria while nulling the contaminant.

In accordance with one major aspect of the present invention, the electrochemical measurements are performed in the filtrate, without the presence of bacteria, as distinguished from measurements made in a chamber containing both fluid and bacteria. Because the measurements are made in the filtrate, there can be no mechanical abuse of the bacteria at the measuring electrode or elsewhere. Further¬ more, there is no possibility of bacterial contamination of the measuring electrode or of other parts of the apparatus beyond the filter. Stated in another manner, applicants have discovered that marked improvements are achieved by placing the entire electrode assembly in the filtrate, such filtrate having been produced either by a filter cartridge, which has previously been used to collect bacteria from the original sample, or by a filter positioned at or downstream from the outlet of a vessel containing the bacteria. The "electrochemical" measurement of reduced species, as referred to herein, includes the standard procedures for electrochemical measurement, including measure of electrode current at constant potential, and/or electrode potential at constant current, and/or other volta etric or amperometric methods to determine the concentration of an electrochemi¬ cally active substance.

The measuring electrode may be made from a variety of chemically inert and electrically conductive materials, such as platinum, palladium, gold, carbon, graphite, etc., and in any of many standard configurations such as rotating, stationary, capillary, rods, wire, etc. In a two electrode instrumentation, the reference and power electrodes may be combined to be a standard type of reversible electrode used both to sense potential and to supply current. The three electrode system contains a measuring electrode, a reference electrode, and a power electrode; whereas a four electrode system comprises a measuring electrode, a power electrode and two reference electrodes.

There are different forms of the aspect of the present invention by which the electrochemical measurements are made in the filtrate, and each form has variations. In accordance with one method, bacteria are collected in a filter cartridge at a test site, and the cartridge is then taken to a laboratory where the bacteria count density is determined. The determination is effected by passing a reagent solution into and through the bacteria-containing filter cartridge, in such manner that bacteria present in the cartridge will cause the production of substantial reduced species in the filtrate. The reagent, with its reduced species, then flows to the electrochemical measuring unit, where the current produced by electrochemical action generated by the reduced species is determined. This current is adjusted for contaminants, for background current, and for electrochemical unit calibration, to produce the bacteria count density of the original sample.

The average flow rate of reagent solution through the filter cartridge is caused to be sufficiently low that the bacteria will produce significant reduced species. Preferably, instead of using a constant flow rate, the method is so performed that there is intermittent flow. This is done by causing the flow rate to be zero for a predetermined period of time, and then using a specific flow rate for a period of time, with electrochemical measurements being made during the entire procedure.

To amplify upon portions of the above, and to state additional aspects of the invention, reagent is introduced into a container—very preferably a small-volume filter cartridge—which contains the bacteria from a predetermined volume of original sample. The reagent is held in the cartridge for a predetermined time period which is sufficiently long that any bacteria therein will cause the production of sufficient reduced species to generate measurable current in the electrochemical measuring unit. Then, the reagent is passed out of the cartridge to the electrochemical measuring unit, and a measurement is made in the filtrate. Furthermore, the same reagent, but reagent which was not introduced into the cartridge, is passed to the electrochemical measuring unit independently of the cartridge. Another measurement is made relative to this second-mentioned reagent. The difference between the measurements is multiplied by a calibrating constant, such constant having been determined by an independent method on an aliquot of the original sample. Later, the procedure is repeated relative to other samples, using the same steps as those stated, using the previously-determined constant. In another form of the method, the reagent is passed through the bacteria-containing cartridge at different low flow rates, and measurements are made in the electrochemical measuring unit relative to each rate. The effects of background are thus compensated.

A further method of determining bacteria count density normally makes use of much larger volumes than do the above- indicated "cartridge" methods. The original sample (preferably a purified sample created by prefiltering) is placed in a vessel containing a filter at the chamber exit path. After a period of environmental adjustment for the bacteria in the chamber, during which period reduced species is produced, the reduced species is drawn from the chamber and passed through the electrochemical measuring unit. Measurements are made relative to two substantially different concentrations of bacteria in the (one and the same) fluid. These measurements are then employed to determine bacteria population, there being no errors introduced because of the presence of any contaminants in the fluid. Very preferably, the two different concentrations are achieved by filtering liquid out of the above-indicated vessel, the electrochemical measurements then being made in the filtrates.

In accordance with an additional form, the determination of bacteria count involves saving part of the fluid after it is filtered and made the subject of a measurement in the electrochemical measuring unit. Later, the electrical signal produced by the saved filtrate is determined and is compared to the signal it first produced in the original pass through the electrochemical unit. It is then known whether or not contaminant is present, and a correction is made relative to the measured bacteria count. Description of the Drawings

Fig. 1 is a schematic or diagrammatic view of apparatus for performing what may be called the bypass method of the present invention, a central region of the filter housing being broken away to show the filter;

Fig. 2 is a schematic or diagrammatic view of apparatus for performing what may be called the flow through method of the present invention; and

Fig. 3 is a schematic or diagrammatic view of apparatus for performing what may be called the changing-concentration and saved-sample methods of the present invention.

Forming of a Purified Sample

Throughout this specification and claims, unless the words "coarse filter" are used, the word "filter" means micro filter. This is one of the many commercially available micro filters which are suitable to retain bacteria in filtration. These filters are sterile and neutral biologically, being neither cytotoxic, bactereostatic nor bactericidal.

It is preferred to perform, separately, an initial filtration step before the below-described methods are commenced. This is because of the vast quantities and types of contaminants that are present in many industrial systems. For example, commercial oxidants are commonly used for bac¬ teria and algae control. Chlorine, chlorine dioxide and acrolein are all electrochemically active and will affect the electrochemical signals produced by the electrochemical measuring unit. Many other biocides, herbicides, and al- gecides will introduce errors, as will various water con¬ ditioning agents.

Even when chemical treating agents are not present in the original sample, free enzymes will normally be present. These will have been formed from the bacteria themselves through the normal process of excretion of enzymes by the bacteria through their membrane walls. The presence of these enzymes will cause a high reading of bacteria count density.

A purified sample is preferably made by, first, passing the raw sample through a coarse filter, then discarding the coarse filter, thereafter filtering again (by a micro filter) the filtrate that passed through the coarse filter, and collecting the bacteria on such second-mentioned filter. The raw filtrate that results from such procedure is discarded, and below-described procedures are .commenced.

It is emphasized that the bacteria are kept alive and uninjured. Thus, for example, they are not allowed to become dry on a filter, and they are not subjected to mechanical or other abuse. After initial filtration, a purified sample is made by taking the filtered original sample, washing it, and reconstituting it with reagent fluid.

A purified sample may in many cases be made by use of the reagent itself, without the separate steps stated in the preceding paragraphs.

A filter cartridge may be positioned at an appropriate point in a commercial plant, for example at a test spigot. A predetermined volume of fluid from the plant is passed through the spigot and filter cartridge. Then, the cartridge is filled with reagent fluid so as to keep the bacteria alive, following which the cartridge is plugged at each end. The cartridge is taken to a test laboratory where the present method is performed, or the method may be performed at the plant itself.

As a specific example, milk at a dairy may be passed through a spigot having a coarse filter therein, and subsequently flowed slowly through a filter cartridge. Then, after a suitable predetermined amount of such flow, the cartridge is removed and taken back to the test laboratory for testing to determine bacteria count density.

Detailed Description of the Bypass Method

As an aid to description of what may be termed the bypass method, there will first be described, with reference to schematic or diagrammatic Fig. 1, an apparatus for use in performing that method. It is, however, pointed out that although the apparatus of Fig. 1 measures current, other electrochemical signals (such as potential) may also be measured. Preferably, though not necessarily, the bypass method makes use of a filter cartridge such as is indicated at 10. This is a small-volume closed container across which is extended, in sealing relationship, a filter F typically formed of synthetic resin and adapted to prevent passage of bacteria while permitting passage of liquid. An exemplary volume of the cartridge is of the order of 1 cc.

Flow through the filter is typically slow, for example 1 cc, or less, per minute. It is to be understood that the conduits that connect to the filter cartridge 10 and to other components are preferably small in internal diameter, so that even the stated slow flow through the filter cartridge 10 does not result in excessive time periods for passage of filtrate to the electrochemical measuring unit.

A bag (or other container) 11 of reagent connects through conduits to a pump 12 and thus to one component 13 of a double pole-double throw valve. The other component 14 of such valve is connected to the first component 13 , for example by solenoid means, so that both components 13,14 work simultaneously. When the valve components 13,14 are each in one posi¬ tion, reagent from bag 11 passes through pump 12 and com¬ ponent 13 to filter cartridge 10, thence to valve component 14 and on to the electrochemical measuring unit 15. It is to be understood that the filter cartridge is connected in series with valve components 13,14 by couplings 16,17 that permit removal of the cartridge from the system when desired, and permit mounting of a new cartridge when desired.

A bypass 18 connects between valve components 13,14 to conduct reagent therebetween independently of the cartridge 10. When the valve components 13,14 are each in the other position, reagent flows from bag 11 and pump 12 through the bypass 18 to measuring unit 15.

The illustrated electrochemical measuring unit 15 has an insulating housing 19 in which is mounted a measuring electrode 20. The measuring electrode shown here is a cylindrical carbon electrode that is rotated slowly in hous¬ ing 19, by motor M, and is in relatively close-fit relation¬ ship in the housing. Suitable seals prevent leakage. The volume of the chamber within the housing (exteriorly of the electrode) is small, preferably less than 1 cc.

The electrochemical measuring unit further comprises a power electrode 21 and a reference electrode 22, the latter being substantially isolated from the chamber around measur¬ ing electrode 20 as by a conductive plug 23. The measuring, power and reference electrodes are connected to terminals of a voltage application and current measuring apparatus P well known in the art, one example being described in the above cited patent 3,506,544. A picoammeter, not shown, is provided in apparatus P to measure current flow in the measuring electrode. It is to be understood that the reference electrode 22 maintains a substantially constant potential, and monitors the potential of the measuring electrode which can thereby be itself maintained at a substantially constant and selected potential. This is done by means of an appropriate circuit and by application of current from the power electrode 21 to the measuring electrode.

In the illustrated electrochemical measuring unit 15, fluid from housing 19 passes out through the power electrode 21 to waste.

Proceeding next to a description of the method, this comprises passing reagent through a filter on the upstream face of which are collected (from a known volume of original sample) a known bacteria the population of which is to be determined. (The type of bacteria is known either from ex¬ perience in the particular industrial system, etc., or by microscopic or other analysis.) The bacteria are washed to remove contaminants, either prior to connection of the cartridge in the circuit (as described above) , or by the first amount of flow of reagent through the filter.

At least during a desired time period after such washing of contaminant from the bacteria, the pump 12 is so operated that the average flow of reagent through filter cartridge 10 is sufficiently low that the bacteria in the cartridge will generate sufficient reduced species that a measurable current will be generated in the electrochemical measuring unit 15.

Stated more definitely, a known volume of original sample is passed through the filter cartridge 10, either at an industrial plant or in the laboratory. This results in collection of bacteria on the upstream face of the filter F in the cartridge. Then, the cartridge is filled with suitable fluid and is placed in circuit by couplings 16,17, with the bacteria on the upstream face of filter F. Pump 12 and the valve components 13,14 are then operated to pump reagent from bag 11 alternately through filter cartridge 10 and bypass 18, the manner of flow and rate of flow through the filter cartridge being repeatable (and repeated) for dif¬ ferent samples.

The electrochemical measuring unit 15 is used to make current measurements, preferably at all times, and the amplitudes of the currents are compared. In other words, the bypass-caused current is compared to the cartridge-caused current. Thus, when the conditions are such that the fluid in the electrochemical measuring unit is from the cartridge, the current will be relatively high because of the effect produced by the bacteria. On the other hand, when the fluid in the electrochemical measuring unit is from the bypass 18, and which has not passed through the cartridge, the current will be relatively low because it is background current, cur¬ rent not representative of bacterial action in the cartridge.

When the amplitudes of the currents measured by the picoammeter repeat, the test may be terminated. Stated otherwise, when the same current amplitudes are produced each time the fluid is from the cartridge, and the same (lower) current amplitudes are produced each time the fluid is from the bypass, the test may be terminated. Furthermore, only one reading need be made relative to fluid from the cartridge, and one relative to fluid from the bypass, provided the prior testing for a given system has been per¬ formed sufficiently that it is known the results will repeat should the alternate readings be continued.

(The word "amplitude", as used in this specification, can mean peak, integral, least mean square, etc., so long as the same definition is employed from test to test; any varia¬ tions thus introduced are then part of the constant indicated below. ) The method further comprises subtracting the current generated when the fluid in the electrochemical measuring unit came from the bypass, from the current generated when the fluid came from the filter cartridge. This current dif¬ ference is then multiplied by a constant or proportionality factor (K-_j_) to thus read out the bacteria count density in colony forming units (cfu's) per milliliter, in the original sample. This constant (K^) is one which correlates the bac¬ teria count determined by the present method, when the proce¬ dure is repeated from sample to sample, with a count made by an independent means (for example, a plate count) on an ali¬ quot of the original sample.

If the current read (by the picoammeter) on each occa¬ sion when the fluid in the electrochemical measuring unit is from the bypass is highly constant from reading to reading, then picoammeter readings occurring when the fluid is from the cartridge—and even though such picoammeter readings are only small amounts larger than the readings when the liquid is from the bypass—are significant. On the other hand, when the "bypass" readings vary slightly, it is desired that the test be so performed that the "cartridge" readings be greatly higher than is the case relative to the "bypass" readings. In the latter situation, it is often desirable that the "cartridge" readings be caused to be at least two or three times the variations in the "bypass" readings.

The present bypass method, with intermittent flow, is one best mode contemplated by the inventors.

A constant or intermittent flow rate through the cartridge may be employed at all times when the valve 13,14 is in such position that reagent from pump 12 passes through the cartridge, depending (of course) on whether the pump is on or off. When the valve 13,14 is shifted so that it passes reagent through the bypass 18 instead of through the cartridge, flow through the cartridge is zero.

The length of time the valve 13,14 is switched to bypass position, so that there is no flow through cartridge 10, is preferably changed in a known manner—from sample to sample—that is related to the expected (or determined in the early stages of the test) bacteria count. Thus, if the bac¬ teria count is low, then the length of time that the reagent is caused to remain in the cartridge 10, without flowing, is caused to be relatively long. If, on the other hand, the bacteria count is expected to be high, the length of time the reagent is caused to remain in the cartridge 10 is caused to be relatively short. In both cases, the period is such that the bacteria generate sufficient reduced species to cause the electrochemical measuring unit to make a significant and meaningful measurement.

There are different constants to correlate the current readings with the bacteria 'count density. For example, there is one constant for a three-minute period of .zero flow in the cartridge, and another constant for a nine-minute period of zero flow in the cartridge. Both of these constants are such that both the nine-minute and three-minute periods will produce the correct bacteria count density readout. Both constants are determined, as stated above, by correlating the current readings after they are repeatable again and again, with bacteria count densities determined by (for example) a plate count of the same volume of sample.

It is to be understood that pump 12 is preferably stopped when the system is in "bypass" condition, as soon as fluid from the bypass has reached the electrochemical measur¬ ing unit and purged it of filtrate from the cartridge. This condition is known to exist as soon as the picoammeter reads minimum current. Conversely, when the system is in the "cartridge" condition, it is know that filtrate has purged the electrochemical measuring unit of reagent from the bypass, because the picoammeter will then show a maximum reading.

The specific electrochemical method described above uses the measurement of current as the output signal for deter¬ mination of bacteria population, but the method is not limited thereto. Any other method suitable for determination of reduced species, such as a method employing potentiometric instrumentation, may be substituted.

In many applications it is preferred that a data proces¬ sor be employed, as stated under the following subhead.

Detailed Description of the Flow-Through Method

The flow-through method comprises moving a reagent through a filter cartridge (or other container) containing the sample of bacteria, namely the bacteria from a predeter¬ mined volume of original sample, and measuring the reduced species in the stream of filtrate. Two sets of data, namely fluid flow versus time, and currents indicating reduced species versus time, are used in data processing to determine the bacteria count density.

The flow-through method is the other best mode contemplated by the inventors.

Referring to Fig. 2, there is shown one form of ap¬ paratus for use in performing the flow-through method. The electrochemical measuring unit 15 is identical to the unit 15 described relative to Fig. 1. As in Fig. 1, the filter cartridge 10 is connected to the unit 15, the cartridge being removable because of the presence of the couplings 16 and 17. In Fig. 2, the reagent pump is illustrated as being a syringe 25 the piston 26 of which is driven by a motor 27. Motor 27 is associated with sensing or other means that sense motor and/or piston positions, and that transmit flow data to a data processing and bacteria readout unit indicated schemati¬ cally at 28. Unit 28 is also fed current data from the measuring electrode. The transmission of flow data to unit 28 is indicated by the arrow 29, while the transmission of current data thereto is indicated by the arrow 30. It is to be understood that the voltage application and current measurement unit P, indicated relative to Fig. 1, is also present in the apparatus of Fig. 2 either directly or by equivalency. For example, the box labeled "electrochemical measuring unit" in Fig. 2 may also incorporate the "voltage application and current measurement" box of Fig. 1. Alterna¬ tively, for example, the picoameter may be provided in the data process block 28.

When there is a constant flow rate of reagent through the filter F in cartridge 10, that is to say when motor 27 is operated at a constant speed, the bacteria count density will be equal to a constant times the average current sensed by electrochemical measuring unit 15 and transmitted to the data processing unit 28. Such a steady flow rate may be employed where the concentration of bacteria is quite high, or where the pump is operated so as to create a very slow flow (distinctly slower than in the below-stated example under the present subheading) .

In a preferred form of the flow-through method, deter¬ mination of bacteria count is effected by making measurements at two different flow rates through the cartridge.

Whether the flow is constant, intermittent or variable, it is repeated from test to test relative to different samples. Furthermore, all conditions are kept the same from test to test. Then, for each type of test, that is to say for each computer program (where flows are determined by computer) , a constant (K₂) is known which correlates the average current with the actual bacteria count density. This constant is derived, for each type of test, by (for example) making a plate count. Then, for a later test (in all embodi¬ ments of the invention) , the same constant is employed time after time.

When the flow through the filter in the cartridge is caused to be zero for a predetermined time period, there will an accumulation of reduced species in the cartridge 10. Then, when the flow is started at the end of the predeter¬ mined time period, the flow will move the thus-accumulated reduced species to and into the electrochemical measuring unit 15. The integral of the current generated in the unit 15 will be the area under the current pulse (a curve of cur¬ rent versus time) that results from the transmission of the specified accumulation to the unit 15. When the flow program is exactly repeatable and is repeated from sample to sample, there is a constant proportion between the amplitude of the pulse and the area under the curve (generated, for example, in the data processor 28) . Therefore, the bacteria count density is equal to a constant times the current, where the current is the amplitude of the pulse resulting in the unit 15 because of the presence of the accumulation received from the cartridge.

A radical difference between the present flow-through method and prior art is that the flow-through method does not measure a rate of change of current, in order to determine bacteria population. Instead, the present flow-through method measures the average current flowing at the measuring electrode at a constant average flow rate.

In accordance with one aspect of a preferred form of the flow-through method, reagent is passed through the filter in cartridge 10 at a rate so fast that very little reduced species is generated, especially if the bacteria density is low. It follows that the vast majority of liquid passed to the electrochemical measuring unit from the filter is then only reagent, not reduced species. The resulting current transmitted to the data processor is properly regarded as background current, being a background current which takes into account factors including any residual contaminant that may be present in the bacteria after washing.

To state an example of the flow-through method, let it be assumed that the size of the filter is such that the first pulse of reagent (having a volume of 1 cc, and a flow rate of, for example, 1 cc/minute) washes the great majority of residual contaminant out of the filter cartridge. Then, a second pulse, also 1 cc at 1 cc/minute, produces the back¬ ground current. Again, this assumes that the bacteria count density in the cartridge is sufficiently low that the 1 cc/minute produces very little reduced species. Thereafter, after these first two pulses, the pump is stopped and there is caused to be a delay of, for example, fifteen minutes. Then, after the delay, the pump is again started to effect flow through the filter at the rate of 1 cc/minute. This propels the pulse, slug or volume of filtrate to the electrochemical measuring unit 15. The current indicated by the picoammeter or the data processor 28 then shows when such pulse containing substantial reduced species begins to enter the unit 15; the signal thereafter produced is related to the concentration of reduced species. When such slug has passed through the measuring unit, the signal returns to essentially the same background current as previously measured (this being the base line) . The bacteria count is then equal to a constant ( ₂) times the average amplitute of current produced by held slug, less the background current. The bacteria count density can be read out by the data processor, for ex¬ ample in colony forming units per milliliter (cfu's/milliliter) of original sample.

The present method comprehends making current measure¬ ments at two different fluid flow rates. Such two flow rates effectively provide the means for electrical measurement of two different bacteria concentrations in one and the same fluid, this being because the effective bacteria concentra¬ tion in a filter cartridge is inversely proportional to the flow rate. The relationships are such that the effect of contaminants is nulled out in the computed readout of bac¬ teria count.

To state the above in another manner, background current (that resulting from any source other than current produced as a result of bacterial action) is eliminated from the test results if the reagent fluid is moved through the filter cartridge alternately at two different flow rates. The two currents measured in the electrochemical measuring unit, together with the measured flow rates, are used to compute the bacteria count.

Applicants have found that the component of current ("I") produced by the bacterial action will be different at the two flow rates (F^ and F₂) , but the background current component from other sources is substantially constant at all flow rates. Thus, two equations with only two unknowns are provided and solved for B (bacteria) :

In general: I_Measured = ---Bacteria ⁺ ---Background ⁽where --•Background ^is constant⁾

Therefore, at low flow rate F 1- = K, 2_ + I Background at high flow rate F₂: I₂ = K₂2_F_ + IBackground

solving for B: B = ^ _(l/^ _ ^

where current values (I-_j_ ₂) and flow rates (F-_]_ ₂) are all measured quantities, and K₂ is a proportionality factor derived using an independent method of counting (such as plate count) bacteria in an aliquot of the original sample.

For example, when lcc/minute average flow rate causes a 12.1 nanoamp reading, and a 1/10 cc/minute average flow rate causes a 16.8 nanoamp current,

F₂ = 1.0 I₂ = 12.1

^{then: B} - ^K ₂ I^ .^_li ⁼ °'^52K2

Detailed Description of the Changing-Concentration Method

The changing-concentration method will be described relative to Fig. 3, which shows one form of apparatus that may be employed in practicing such method. The electrochemi¬ cal measuring unit 15 in Fig. 3 is shown as being identical to that of Fig. 1, and the same reference numerals are employed relative to it. The same is true of the voltage ap¬ plication and current measurement unit P.

A vessel 32, which in most applications has a volume many times that of the filter cartridge 10, is provided with a vented cap.33. A reagent container 34, for example a bag, connects to vessel 32 through a valve 35. A filter 38 is provided at the bottom of vessel 32, the top face of the fil¬ ter being preferably in direct communication with the inte- rior of such vessel. Filter 38 is contained in a filter housing 39 which connects to a pump 37, the latter being con¬ nected to the housing 19 of measuring electrode 20.

A gas injector 40 introduces an inert and oxygen-free gas into the fluid in vessel 32 in order to remove oxygen from such fluid and in order to stir it. Other elements may be associated with the vessel, for example a mechanical stir¬ rer and a means to introduce washing fluid. The gas passed through the gas injector 40 and thus into the fluid in the vessel preferably comprises oxygen-free nitrogen gas. A sample inlet, not shown, is also provided in the vessel cap or cover 33 in order to eliminate the necessity of removing such cover when sample is introduced.

The liquid that passes through the electrochemical measuring unit goes to waste; it is not recirculated to the vessel 32. Accordingly, a valve 41 is provided in the waste line, and is in open condition during performance of the changing-concentration method.

The changing-concentration method preferably includes the preliminary steps of introducing into vessel 32 a known volume of sample. While valve 35 is maintained closed, the pump 37 is operated so that all liquid is removed from the vessel and passed to waste. Thus, the bacteria in the vessel are present on the upper face of the filter 38.

Pump 37 is then stopped, valve 35 is opened, and reagent is passed from bag 34 into vessel 32 to fill it to a desired level. Valve 35 is then closed. The gas injection means 40, alone or in combination with a mechanical stirrer, is employed to purge the reagent of oxygen and (preferably) keep the reagent-bacteria mixture homogeneous. The apparatus is now ready to commence testing for bacteria count. Stated generally, the basic concept of the changing- concentration method is to change the concentration of bac¬ teria in one and the same liquid (by removing fluid from ves¬ sel 32 via the filter) , and to measure the rate of increase in concentration of reduced species (by means of a suitable electrochemical measuring unit) . There are thus generated two sets of data, one being concentration of reduced species versus time, and the other being concentration of bacteria versus time. These data are used to determine bacteria count per unit volume, there being a nulling of the effects of any contaminants that may remain after washing. Any one of a large number of concentration programs (procedures) may be employed, and each has its own data processing algorithm which uses the two sets of data in a function that is charac¬ teristic to bacteria.

The preferred method of concentration " of bacteria employs filtering, as shown in Fig. 3. Pump 37 is started to reduce the volume of bacteria-containing reagent in vessel 32 from an initial volume (which may be termed V^) to a substan¬ tially smaller volume (which may be termed V₂) . At predeter¬ mined times, for example when the volume of liquid in vessel 32 is V₁ and (subsequently) when the volume is V , the electrochemical measuring unit 15 is employed to read the electrochemical signal at each concentration. Preferably, electrical signals are transmitted to a data processor from the electrochemical measuring unit, and volume signals are transmitted to such processor from the pump 37 or from sen¬ sors associated with vessel 32.

In the preferred form of the changing-concentration method, a data processor is employed and is fed signals from the electrochemical measuring unit and from the pump or ves¬ sel, so as to receive inputs of volume versus time and signal versus time. The pump 37 is caused to commence operating shortly after reagent is introduced into vessel 32 from the reagent container 34, and the electrochemical measuring unit is caused to continuously measure current while the pump is operating.

Then, the data processor is caused to determine the second derivative of the signal present in the measuring electrode 20. Such second derivative is proportional to the bacteria count (B) . If the signal is a current measure, the general equation is:

B is the bacteria count; K₃ is a proportionality constant which is derived (in the manner described above) by using a plate count, etc. ; and I is the current in the measuring electrode.

Any desired flow program may be employed, but the program must be repeated from sample to sample. As before, all conditions must be maintained the same from test to test.

A second measure of converting the readings produced by the measuring electrode in the changing-concentration method to bacteria count density is a follows. Pump 37 is started, and three currents are measured by the electrochemical measuring unit, the currents being measured equally spaced in time. Specifically, at volume V_l7 current I is measured. Then, more fluid is passed from the vessel 32 to cause a volume V₂ to be present. Current I₂ is then measured. At a time interval equal to that recorded between the I and I₂, and while the volume remains at V₂, measurement of current I₃ is made.

The bacteria count (B) is then determined by the follow¬ ing equation:

B = K₄(I₄ + I₂ - 2I₃) As before, the constant K₄ is one which was determined in accordance with an independent counting method, such as plate count, so as to translate the results of the equation to the actual bacteria count density in cfu/milliliter.

The above-described changing-concentration method eliminates the effect of any residual contaminants. It is pointed out that although the concentration of bacteria in the reagent increases as reagent is filtered from the bottom of the vessel and pumped to waste, there is no corresponding increase in the concentration of contaminants. The con¬ centration of contaminants remains the same while the con¬ centration of bacteria increases. Thus, mathematically, the contaminants may be eliminated from the equations so that only the effects produced by the bacteria are employed to produce the bacteria count density.

Detailed Description of the Saved-Sample Method

Referring again to Fig. 3, the apparatus employed in the saved-sample method may be substantially the same as that described above relative to the changing-concentration method. However, a valve 42 is provided in a Tee that ex¬ tends to a container (such as a bag) indicated by the reference numeral 43.

The saved sample method greatly reduces or eliminates the effect of any residual contaminants in the bacteria (remaining after preparation of the purified sample as set forth above) , in the following manner.

The first part of the method is the same as that described above relative to the changing-concentration method. However, the liquid that passes through the electrochemical measuring unit during the test (during which readings are made by the electrochemical measuring unit) is not passed to waste but instead to the bag 43, the valve 42 being open and valve 41 being closed. Furthermore, during passage of the filtrate and reagent from filter housing 39 through pump 37 and the electrochemical measuring unit to valve 42, measurements are made by the electrochemical measuring unit.

Thereafter, at a substantially later time, the saved sample of liquid is again passed through the electrochemical measuring unit. This may be done, for example, by separating the bag 43 from the conduit associated with valve 42 and introducing the saved sample from such bag into pump 37 and thus to the electrochemical measuring unit. The introducing is done by means, not shown, independently of vessel 32. The saved sample is then made the subject of second current measurements.

It is thus determined whether or not contaminant was present when the original reading was made. A second-pass current signal equal to the originally measured current demonstrates that no contaminant was present. On the other hand, an increased current relative to the saved sample of fluid indicates the presence of a contaminant.. In the latter event, the quantity of any such current increase is employed as a correction factor. Stated otherwise, the difference be¬ tween the current resulting from the second pass of liquid, and the current resulting from the original pass of liquid, is computed. This difference is subtracted from the current measured when the liquid was originally passed through the electrochemical measuring unit. Then, the thus-corrected current is employed to determine the bacteria count density. (The results are employed in conjunction with a constant, determined by plate count, to achieve the density.) The test is repeated again, in identical manner, from sample to sample. Specific Examples

EXAMPLE 1; Enzyme test to show viability of bypass method.

Enzymes from tryptic soy broth are known to catalyze the reaction between certain oxidants and substrates, including methylene blue and glucose. This characteristic was used to test the viability of the bypass method, by determining cur¬ rent response to the buildup of reduced species (in this case reduced methylene blue) .

A small plastic container with a volume of about 1/2 ml was charged with a 50/50 mixture of reagent and tryptic soy broth. The cartridge had input and exit connections so that it could be used in the bypass apparatus as a substitute for the filter cartridge (Figure 1) . The small container was placed in the test loop, and a waiting period of 15 minutes was selected to allow catalytic action by the enzymes to form a concentration of reduced methylene blue. The reagent had the following composition:

H₂0 75 ml mono basic phosphate 0.4 g

K₂HP0₄ 0.8 g glucose 0.14 g methylene blue 0.005 g potassium chloride 0.35 g The tryptic soy broth had the composition:

H₂0 100 ml pancreatic digest of casein 1.7 g papaic digest of soybean meal 0.3 g NaCl 0.5

K₂HP0₄ 0.25 g dextrose 0.25 g

During the last minute of waiting period, the reagent was moved by the pump through the bypass, into the electrochemi¬ cal measuring unit, and the current zeroed (caused to appear to be zero, even though background current was flowing) . At the end of the waiting period, the reagent flow was shifted from the bypass route to flow through the container holding the reagent/soy broth mixture. When the slug of retained liquid, held in the plastic container was passed through the electrochemical measuring unit, a peak current (over zeroed background or base line) of 120 nanoamperes was recorded, which subsequently returned close to the zeroed value when the slug had passed. This test confirms that when a reduced species is formed by biological action, the concentration of reduced species can be measured easily by the peak current produced in the electrochemical measuring unit.

EXAMPLE 2; Quantitative bacteria test of the bypass method.

In this test, a syringe filter from the Nalge Company (of Rochester, New York) was used to retain the bacteria. The sterile filter membrane of cellulose acetate was 25 mm in diameter and had a pore size of 0.45 microns. The top of the filter was removed and a new top added, which increased the volume of the space above the filter surface to about 1/2 ml. A pure culture of E. Coli bacteria (about 1 ml) was filtered through the filter membrane. The filter cartridge was then filled with the methylene blue/reagent fluid used in Example (1) above, and the cartridge was placed in the apparatus loop as shown in Figure 1. A holding period of 15 minutes, after introduction of the reagent, was employed.

As in Example (1), near the end of the retention period, the reagent was moved via the bypass and through the electro¬ chemical measuring unit where the current was zeroed. Then the reagent flow was shifted to pass through the filter cartridge. When the slug of retained fluid reached the electrochemical measuring unit, a peak current of 4.5 nanoamps was observed.

Moving the retained slug out of the cartridge resulted in fresh reagent entering the filter cartridge. This was held again for a period of 15 minutes, and the determination of the change in current was repeated as above. A peak current of 4.6 nanoamps was recorded, which showed that for a specific waiting period the concentration of reduced species produced (as measured by the electrochemical measuring unit) was essentially constant.

The fresh reagent solution, which now occupied the filter chamber, was again held, this time for a period of 30 minutes. A determination of the peak current for this holding period was found to be 9.4 nanoamps, essentially twice the current found for the 15 minute holding periods. This test showed that the quantity of reduced species which is produced is proportional to the time held in the filter chamber. The slight increase in peak current over the expected value of 9.2 (2 x 4.6) may be a result of the increase in the total number of bacteria that occurred during the hour period required to carry out the three tests on the same bacteria sample in the cartridge.

EXAMPLE 3; Qualitative tests with different microorganisms and different reagent compositions.

Two additional tests were made to determine if a peak current was produced by the pulse mode of the bypass method when there were present bacteria other than E. Coli, and with a reagent having a different oxidant and substrate:

Conditions Example (1) Example (2)

Micro organic: Yeast B Globigii

Oxidant: Phenazine Methosulfate Methylene Blue

Substrate: D-Glucose Sodium Succinate

Buffer: Phosphate Phosphate

The tests, in each case, showed a readily measurable peak current after a 15 minute holding period.

The present invention has created the ability to measure the concentration of reduced species with an accuracy and sensitivity that now makes electrochemical measurement practical and useful. The preferred method directly measures the magnitude of the electric current produced in an electrochemical measuring unit, as opposed to the prior-art method of measuring the rate of change of produced current. Further, the invention completely compensates the current measurement for background contribution from the reagent, contaminants and electronic noise. This is accomplished by moving a reagent containing the oxidant (oxidized species) and the substrate through the bacteria which are retained in a filter cartridge, and then through an electrochemical measuring unit where the magnitude of the current is measured. Subsequently, moving the same reagent through a bypass to the filter cartridge provides a measure of the background current which is subtracted from total current measurement in the filter cartridge flow path to provide the true current component caused only by the bacterial action. This current component has been shown to be proportional to the concentration of the reduced species which in turn has previously been established as being proportional to the bac¬ teria population. Accordingly, it is anticipated that for the new method, bacteria population determined by the inde¬ pendent method of pour plate counting (plate count) will show good correlation with the new electrochemical method of the present invention.

Additional Disclosure

The above-cited Silverman/Brake patent describes such factors as applied voltage, pH, temperature, elimination of oxygen, etc. , so these will not be described herein (except for the brief references made above relative to some of these factors) . Other factors described in the cited patent, such as relative to the particular substances, etc., are referred to herein only in part because they are already described in the cited patent. Portions of the cited patent particularly referred to herein are those relating to determination of populations of bacteria. However, it is emphasized that applicants present methods are directed primarily toward the determination of populations of live bacteria.

The above-described washing of soluble contaminants from bacteria is preferably effected by distilled water. The picoammeter presently employed by applicants is a model 485, manufactured by Keithley Instruments, Inc. of Cleveland, Ohio. The filter cartridge presently employed by applicants is catalog number 190-2045, manufactured by Nalge Company, of Rochester, New York.

The electrochemical measuring unit now employed by applicants comprises a stainless steel tube 21 as the power electrode. The reference electrode presently employed and now preferred is a silver/silver chloride reference electrode, which may be purchased from many commercial sources. Other reference electrodes, for example the calomel electrode referred to in the cited patent, may also be employed.

The preferred measuring electrode 20 is manufactured by Quantum Electro Development of Fullerton, California. This measuring electrode comprises a rotating solid rod of graphite housed in a cylindrical chamber 19 made of polycar¬ bonate, with a clearance between rod and chamber wall of only 0.005 inch. The rod is 1-7/16 inch in length and 1/2 inch in diameter. The retention volume of the assembly is about 1/4 cc. The rod is threaded at its center to receive a stirring rod of 316 stainless steel, which passes through a sealing means to the drive shaft. The driving motor is mounted at one end of the housing chamber. The electrode assembly runs smoothly and with minimal sonic and electrical noise. All of the electrode surfaces are rotated through the fluid at ex¬ actly the same speed, and this speed is relatively low in comparison to prior art. The preferred rate of rotation of the electrode is 400 revolutions per minute. (The graphite is obtained from Stackpole Carbon Company of St. Marys, Pen¬ nsylvania. Specifications are density 1.85, and grade 1336.) It is emphasized that there are numerous designs of measuring electrodes, including rotation paddles, wire loops, dropping liquid metals, capillary tubes, flat plates, rods, etc. Throw away units have also been employed (as stated above) .

Because the present invention employs the step of preventing the bacteria from contacting the measuring electrode, changes in the measuring electrode are much less likely to occur than in prior art. However, it is preferred by applicants that the measuring electrode be tested from time to time. Thus, though the measuring electrode is protected from bacterial contamination and from frequent sterilization procedures, there is need for assurance that the measuring electrode activity is correct a d constant. The preferred, certain way to provide such assurance is to calibrate the electrode activity occasionally, and to use this calibration in the final calculation of bacteria count. Accordingly, applicants employ means to measure the electrode's sensitivity before and after a test run, or when¬ ever the procedure is deemed to be expedient. Electrochemi¬ cal unit calibration can be made all-inclusive by determining the actual current measured in the electrochemical measuring unit, which is produced by a known quantity of the particular reduced species which will be measured during a bacteria count. A "standard solution" is used for this test and may be called the electrochemical calibration fluid. The vari¬ able area, variable fluid dynamics, and surface activity thereby become combined in one single calibration factor (with units of current per moles for reduced redox species) .

It is emphasized that the electrochemical measuring unit is calibrated by moving a fluid through it, which fluid contains the reduced redox species of known concentration, and measuring the resultant current. It is also emphasized that the redox (oxidation/reduction) couple is an organic compound that can exist in equilibrium in two states of oxidation and can be changed from one state to the other either by a chemical or electrochemical reaction: for example, the methylene blue/luco methylene blue couple. The oxidized species of the chosen redox couple is present in the reagent fluid. Its function is to assume the role of whatever oxidant normally was used by the bacteria in its natural environment (0₂, S0₄=, NO₃- or C0₃=, etc.). This new oxidant (or mediator) reacts with the substrate (also furnished by the reagent) to produce a new energy cycle for the bacteria.

The new oxidant provided can (a) be used by the bacteria in the energy cycle and (b) produce a reduced species which is easily reoxidized electrochemically. Numerous substances can be used as an oxidant, or there can be cocktail mixtures of two or more. Suitable oxidants can be selected from the well known dye mediators such as methylene blue, 2,6- dichloroindophenol, indigo disulfonate, phenosafranin, phenazine methosulfate, etc. Other substances such as benzo- quinone and ferrocyanide have also been found useful.

The substrate is a substance which will combine with the chosen oxidized species of the redox couple in a reaction catalyzed by the bacteria (or its enzyme) to produce the desired reduced species. Many organic substances have been found to be useful in different applications for this pur¬ pose. These include the simple sugars such as glucose, lac¬ tose, dextrose, fructose, etc. , and other intermediate sub¬ stances in the oxidation cycle, such as pyrunate, succinate malate, citrate, -ketoglutarate, acetate, lactate, etc. As with the oxidized species, a mixed cocktail of two or more substrates can be advantageous. The reagent is a special solution which supplies the ingredients required by the bacteria for its catalyzed reac¬ tion to produce a reduced species. The reagent contains the oxidized state of an organic redox couple (e.g. methylene blue of the redox couple methylene blue/luco methylene blue) , a substrate needed for the catalyzed reaction (e.g. glucose) , and suitable buffering ingredients to maintain a desired pH level. The reagent is oxygen free and sterile.

The foregoing detailed description is to be clearly understood as given by way of illustration and example only, the spirit and scope of this invention being limited solely by the appended claims.

What is claimed is:

Claims

THE CLAIMS

1. A method of determining bacteria populations electrochemically, which comprises: providing a sample of fluid containing bacteria the population of which is to be measured, filtering said sample to obtain a filtrate, said filtering step being so performed as to prevent said bacteria from being present in said filtrate, making an electrochemical measurement relative to said filtrate by employing electrode means not contacted by said bacteria, and employing the results of said electrochemical measure¬ ment to determine the population of bacteria in said sample.

2. The method as claimed in claim 1, in which said bacteria are live bacteria that have not been subjected to substantial abuse.

3. The method as claimed in claim 2, in which said method further comprises making said electrochemical measurement by employing a redox couple and an electrochemical measuring unit comprising a measuring electrode, and exposing said measuring electrode of said unit to said filtrate and not to said bacteria.

4. The method as claimed in claim 1, in which said bacteria are live bacteria, and in which said method further comprises

(1) washing said bacteria to remove contaminants therefrom,

(2) causing said washed bacteria to be present in a reagent comprising the oxidized species of a redox couple, (3) caus- ing said bacteria to catalyze progressive conversion of said oxidized species to reduced species, (4) filtering said bacteria from said reagent, and (5) employing an electrochemical measuring unit in contact with the filtrate resulting from said preceding step (4) , and not in contact with said bacteria, to effect said electrochemical measure¬ ment.

5. The method as claimed in claim 4, in which said method further comprises maintaining said bacteria in contact with said reagent for a time period sufficiently long that sufficient of said oxidized species is converted to reduced species that a significant reading is made by said electrochemical measuring unit.

6. The method as claimed in claim 1, in which said method further comprises performing said filtering step by means of a small-volume filter cartridge containing a filter.

7. The method as claimed in claim 1, in which said method further comprises performing said filtering step by providing a filter on the outlet side of a relatively large-volume ves¬ sel that contains said bacteria in a reagent, and filtering said reagent through said filter.

8. A method as in any one of claims 1-7, in which said method further comprises repeating said recited steps in the same way, but relative to different samples containing dif¬ ferent populations of bacteria, to thus determine the bac¬ teria populations in said different samples, and further com¬ prises employing a constant to equate the electrochemical measurements with bacteria populations, said constant being determined by making a nonelectrochemical count of bacteria relative to an aliquot of said sample.

9. A method of measuring bacteria populations by: passing a first quantity of a selected fluid through a container, thence through a filter provided on the downstream side of said container, and thence through an electrochemical measuring unit, in such manner that bacteria from a sample to be measured are present in said first quantity on the upstream side of said filter, and are not present at said unit, introducing a second quantity of said fluid into said unit independently of said container and of said filter, and at a time when there is not present at said unit substantial fluid that passed through said filter, said second quantity not having bacteria therein, employing said unit to make electrochemical measurements at at least one time when at least part of said first quantity is present at said unit, and at at least one time when at least part of said second quantity is present at said unit, and equating the population of said bacteria to the dif¬ ference between the average signal measured by said unit when said fluid from said filter is present at said unit, and the average signal measured by said unit w. an said fluid not from said filter is present at said unit.

10. The method as claimed in claim 9, in which said method further comprises providing a common source of said fluid, providing said bacteria in said container, then during one time period passing said fluid from said source through said container and said filter to said unit, and then during another time period passing said fluid from said source through a bypass to said unit independently of said container and filter.

11. The method as claimed in claim 9, in which said method further comprises causing said bacteria to be present on the upstream surface of said filter prior to said passage of said first quantity of fluid through said filter.

11. The method as claimed in claim 9, in which said method further comprises exactly repeating said method relative to different bacteria samples to be measured.

12. The method as claimed in claim 9, in which said method further comprises employing as said container a small-volume cartridge containing said filter.

13. The method as claimed in claim 9, in which said method further comprises employing as said container a vessel having substantial volume.

14. The method as in any one of claims 9-13, in which said method further comprises employing as said fluid a reagent containing the oxidized species of a redox couple, in which said oxidized species is adapted to be progressively catalyzed to reduced species by said bacteria, in which said electrochemical measuring unit is adapted to generate current related to the amount of said reduced species, in which said method is so performed that said unit produces significant current when there is present therein said fluid that has passed through said container and filter, in which said unit produces background current when there is present therein said fluid which has not passed through said filter, and in which the difference in currents is correlated to bacteria count by using a constant achieved by correlating said dif¬ ference to a bacteria count that was previously made by a nonelectrical method relative to an aliquot of said sample.

15. The method as claimed in claim 9, in which said method further comprises maintaining at least part of said first quantity of fluid in said container for a substantial time period, said time period being sufficiently long that after said part of said fluid passes through said filter to said unit said unit produces a signal significantly larger than the signal produced thereby when fluid from said second quan¬ tity is present at said unit.

16. A method of determining populations of bacteria, comprising: providing a sample of bacteria, collecting the bacteria from said sample on the upstream side of the filter in a filter cartridge, providing a reagent fluid, moving said reagent fluid through said filter cartridge at a measured average flow rate, electrochemically measuring the filtrate resulting from said last-recited step to determine the results of bacterial action on said reagent fluid, and employing the electrochemical measurement resulting from said last-recited step, and said measured average flow rate, to compute the population of bacteria in said sample.

17. The method as claimed in claim 16, in which said reagent fluid is a water-based fluid comprising the oxidized species of an organic redox couple and a substrate substance, said substrate substance being usable by said bacteria to induce a catalytic chemical reaction between said oxidized species and said substrate and thereby produce the reduced species of said redox couple.

18. The method as claimed in claim 16, in which said method further comprises moving said reagent fluid through said fil¬ ter cartridge as a continuous flow.

19. The method as claimed in claim 16, in which said method further comprises moving said reagent fluid through said fil¬ ter cartridge as an intermittent flow.

20. The method as claimed in claim 16, in which said method further comprises employing a measuring electrode to make said measurement, and in which said method further comprises causing said measurement to be the integrated signal produced in said measuring electrode during a measuring period.

21. The method as claimed in claim 16, in which said method further comprises moving said reagent fluid through said fil¬ ter cartridge at different rates of flow, and wherein said measurement is made by measuring the signal amplitude in a measuring electrode during a period of relatively high flow.

22. A method of determining bacteria populations, compris¬ ing: providing a sample of bacteria, providing a reagent fluid, combining said sample of bacteria with said reagent fluid to thus make a second sample, passing said second sample through a filter which retains said bacteria, to thus provide a bacteria- free filtrate from said second sample, electrochemically measuring, in said filtrate, the results of bacterial action in said second sample, and employing said measurement obtained by said last-named step to compute the number of bacteria in said first-mentioned sample.

23. The invention as claimed in claim 22, in which said first-mentioned sample is formed by precipitating bacteria onto said filter at a commercial facility.

24. The method as claimed in claim 22, in which said method further comprises saving said filtrate, and electrochemically measuring it again at a later time, to thereby determine any effects on the electrochemical measurement resulting from the presence of contaminants in said second sample.

25. A method of determining bacteria populations, which comprises: providing a sample of bacteria, providing a reagent comprising the oxidized species of an organic redox couple, and a substrate substance usable by the bacteria to induce a catalytic chemi- cal reaction between said oxidized species and said substrate to form the reduced species of said redox couple, providing an electrochemical measuring means, bringing said reagent and said bacteria into contact with each other for a time period sufficient that there will be formed a sufficient quantity of said reduced species to generate a significant signal in said electrochemical measuring means when said reduced species and said electrochemical measuring means are in contact with each other, passing said reagent and reduced species through a fil¬ ter to form a filtrate free of said bacteria, bringing said filtrate into contact with said electrochemical measuring means, making a reading of signal generated by said electrochemical measuring means as the result of said contact thereof with said filtrate, thereafter saving said filtrate for a time period sufficient that any contaminant present in said filtrate can generate a significant additional amount of reduced species, thereafter bringing said saved filtrate into contact with said electrochemical measuring means and read¬ ing the resulting signal, correlating the first-mentioned one of said signals and the second-mentioned one of said signals, and employing the results of said correlation to determine the population of bacteria in said sample.

26. A method of determining bacteria populations, compris¬ ing: providing a bacteria sample, providing a reagent fluid, mixing said sample and said fluid with each other to provide a first mixture of bacteria and fluid and which has a first concentration of said bacteria in said fluid, changing said concentration of said bacteria in said fluid to thereby cause one and the same fluid to have a second concentration of said bacteria in said fluid, electrochemically determining the bacterial action in said fluid at said first concentration and at said second concentration, and using the results of the electrochemical determinations to compute the population of bacteria in said sample.

27. The method as claimed in claim 26, in which said reagent is a water based fluid comprising the oxidized species of an organic redox couple and a substrate substance, said sub¬ strate substance being usable by said bacteria to induce a catalytic chemical reaction between said oxidized species and said substrate substance to produce the reduced species of said redox couple, and in which said change in concentration is effected by filtration of said bacteria.

28. The method as claimed in claim 27, in which said electrochemical determinations are made in the filtrate resulting from said filtration, by electrochemical measuring means not contacted by said bacteria.

29. A flow-through method of determining bacteria popula¬ tions, comprising: providing a pump adapted to pump liquid through a fil¬ ter, providing an electrochemical measuring unit on the downstream side of said filter, providing a sample of bacteria in a reagent, said reagent being a fluid comprising the oxidized species of an organic redox couple and a substrate substance usable by said bac¬ teria to induce a catalytic chemical reaction between said oxidized species and said sub¬ strate to produce the reduced species of said redox couple, employing said pump to pump said reagent through said filter to said electrochemical measuring unit in such manner that, at least during portions of the time that said pumping is occurring, significant reduced species will be formed due to catalytic ac¬ tion resulting from the presence of. said bacteria, generating a curve of time versus signal, said signal being present at a measuring electrode forming part of said electrochemical measuring unit, and employing said curve to determine the population of bacteria in said sample.

30. The method as claimed in claim 29, in which said method further comprises generating said curve in a data processor, and determining the area under said curve in said step of determining bacteria population.

31. The method as claimed in claim 29, in which said method further comprises collecting the bacteria from said sample on the upstream side of said filter prior to contacting said bacteria with said reagent, and further comprises washing contaminants out of said bacteria prior to contacting of said bacteria by said reagent.

32. The method as claimed in claim 29, in which said method further comprises collecting said bacteria on the upstream face of said filter prior to contacting of said bacteria by said reagent, further comprises pumping an initial quantity of said reagent through said filter to wash contaminants from said bacteria, and further comprises disregarding any measurement effected by the filtrate resulting from said washing in said determination of bacteria population.

33. The method as claimed in claim 32, in which said method further comprises pumping an additional quantity of said reagent through said filter and to said electrochemical measuring unit to establish a background or base line representing background signal and the results of any residual contaminant, and subtracting said background or base line from subsequent determinations by said electrochemical measuring unit whereby the result is substantially independent of background signal or contaminant-caused signal.

34. The method as claimed in claim 29, in which said method further comprises pumping an additional quantity of said reagent through said filter and to said electrochemical measuring unit to establish a background or base line representing background current and the results of any residual contaminant, and subtracting said background or base line from subsequent determinations by said electrochemical measuring unit whereby the result is substantially independent of background signal or contaminant-caused signal.

35. The method as claimed in claim 29, in which said method comprises effecting said pumping of reagent through said filter intermittently, and leaving said reagent in contact with said bacteria through one or more time periods sufficiently long to cause the measuring electrode of said electrochemical measuring unit to have a relatively high signal thereat.

36. The method as claimed in claim 35, in which said method further comprises employing the area under the signal-time curve in said determination of bacteria population.

37. The method as claimed in claim 35, in which said method further comprises employing the amplitude of a pulse of signal to determine population of said bacteria, said pulse being the result of reagent passed to said electrochemical measuring unit following a time when said reagent has remained in contact with said bacteria for a substantial time period sufficient to cause production of significant reduced species.

38. A method of determining the population of bacteria in a bacteria sample of predetermined volume, said method comprising: providing an electrochemical measuring unit, providing a reagent that is a fluid comprising the oxidized species of an organic redox couple and a substrate substance usable by the bacteria to be measured, to induce a catalytic chemical reaction between said oxidized species and said substrate to produce the reduced species of said redox couple, providing in said reagent the bacteria to be measured, preventing said bacteria from contacting said electrochemical measuring unit, while causing reduced species catalyzed by the presence of said bacteria to contact said electrochemical measuring unit, and employing the resulting signal in said measuring unit to determine the population of bacteria in said sample.

39. A method of determining bacteria populations, which comprises: collecting bacteria from a predetermined volume of sample at an industrial site on the upstream side of a filter in a small-volume filter cartridge, maintaining said bacteria in live unabused condition in said cartridge, transporting said cartridge and contained bacteria to a test laboratory, moving a reagent fluid through said cartridge in a direction from said upstream side of said filter to the downstream side thereof, and electrochemically determining the population of bacteria in said predetermined volume of sample by making an electrochemical measurement relative to the filtrate produced by said last-recited step.

40. The method as claimed in claim 39, in which said method further comprises washing said bacteria prior to said moving of said reagent through said cartridge.

41. A method of determining bacteria populations electrochemically, which comprises: providing a filter container in which has been collected the bacteria from the sample to be counted, moving a reagent fluid through said container and through the filter therein, making an electrochemical measurement of the reduced species caused by the bacteria in said container, said electrochemical measurement being made in the filtrate from said container, and determining the bacteria count as a function of the flow of said reagent, electrochemical signal, and time, characterized in that the above-recited steps are so performed as to make the direct response of said electrochemical signal a characteristic to the bacteria while nulling any contaminant present.

42. A method of determining bacteria populations electrochemically, which comprises: providing in a vessel a fluid sample containing bacteria to be counted, moving said fluid sample from said vessel through a filter while electrochemically measuring the reduced species present in the filtrate stream, and determining the bacteria count as a function of changing bacteria concentration, electrochemical signal, and time, characterized in that the above-recited steps are so performed that the second derivative of the electrochemical signal is characteristic to bacteria while nulling the contaminant.

43. A method o f determining bacteria popul ati ons electrochemically, which comprises: providing a fluid containing the sample of bacteria the population of which is to be determined, providing a filter, providing an electrochemical measuring means, moving said fluid through said filter to said electrochemical measuring means at a rate sufficiently fast that the filtrate generates only a base-line signal in said electrochemical measuring means, thereafter moving said fluid through said filter to said electrochemical measuring means at an average rate sufficiently slow that the resulting signal generated by said electrochemical measuring means is related to the population of bacteria in said fluid, employing said first-recited signal as the base line for said second-recited signal, and determining the population of said bacteria as a function of both of said signals.