EP0499624A1 - Methods of determining bacteria populations electrochemically - Google Patents
Methods of determining bacteria populations electrochemicallyInfo
- Publication number
- EP0499624A1 EP0499624A1 EP90917636A EP90917636A EP0499624A1 EP 0499624 A1 EP0499624 A1 EP 0499624A1 EP 90917636 A EP90917636 A EP 90917636A EP 90917636 A EP90917636 A EP 90917636A EP 0499624 A1 EP0499624 A1 EP 0499624A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- bacteria
- fluid
- reagent
- filter
- electrochemical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
Definitions
- 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.
- 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.
- 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:
- K was a calibration factor (or proportionality factor ⁇ .
- 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.
- 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.
- 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 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.
- 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.
- electrochemical measurement of reduced species 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.
- 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.
- 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.
- 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.
- 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.
- the reagent is passed out of the cartridge to the electrochemical measuring unit, and a measurement is made in the filtrate.
- 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.
- 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
- 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.
- the two different concentrations are achieved by filtering liquid out of the above-indicated vessel, the electrochemical measurements then being made in the filtrates.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 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.
- 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.
- fluid from housing 19 passes out through the power electrode 21 to waste.
- 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.
- a known bacteria the population of which is to be determined.
- 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.
- 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.
- 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.
- the bypass-caused current is compared to the cartridge-caused current.
- 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 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.
- K- j _ constant or proportionality factor
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the filter cartridge 10 is connected to the unit 15, the cartridge being removable because of the presence of the couplings 16 and 17.
- 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.
- the voltage application and current measurement unit P is also present in the apparatus of Fig. 2 either directly or by equivalency.
- the box labeled "electrochemical measuring unit” in Fig. 2 may also incorporate the "voltage application and current measurement” box of Fig. 1.
- the picoameter may be provided in the data process block 28.
- 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) .
- deter ⁇ mination of bacteria count is effected by making measurements at two different flow rates through the cartridge.
- 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 (K 2 ) 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.
- 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.
- 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.
- 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.
- 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.
- 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 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.
- 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.
- K 2 is a proportionality factor derived using an independent method of counting (such as plate count) bacteria in an aliquot of the original sample.
- Fig. 3 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.
- 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.
- 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.
- 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) .
- 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 V 2 ) .
- V ⁇ initial volume
- V 2 substan ⁇ tially smaller volume
- the electrochemical measuring unit 15 is employed to read the electrochemical signal at each concentration.
- 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.
- 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.
- 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
- K 3 is a proportionality constant which is derived (in the manner described above) by using a plate count, etc.
- 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 V 2 to be present. Current I 2 is then measured. At a time interval equal to that recorded between the I and I 2 , and while the volume remains at V 2 , measurement of current I 3 is made.
- K 4 K 4 (I 4 + I 2 - 2I 3 )
- the constant K 4 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.
- the apparatus employed in the saved-sample method may be substantially the same as that described above relative to the changing-concentration method.
- 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.
- 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.
- measurements are made by the electrochemical measuring unit.
- 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.
- a second-pass current signal equal to the originally measured current demonstrates that no contaminant was present.
- an increased current relative to the saved sample of fluid indicates the presence of a contaminant..
- the quantity of any such current increase is employed as a correction factor.
- 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
- 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:
- 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) .
- the reagent flow was shifted from the bypass route to flow through the container holding the reagent/soy broth mixture.
- a peak current over zeroed background or base line
- 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.
- 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.
- 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.
- 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.
- 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.
- rotation paddles wire loops, dropping liquid metals, capillary tubes, flat plates, rods, etc. Throw away units have also been employed (as stated above) .
- 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) .
- 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.
- 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 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.
- 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.
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Abstract
Selon un procédé électrochimique de quantification de populations de bactéries entières vivantes, on filtre les bactéries, puis on utilise le filtrat en association avec une unité de mesure électrochimique (15) afin de déterminer la densité de comptage des bactéries. Selon un procédé d'écoulement, on utilise la moyenne des signaux pendant la période prédéterminée de durée du test, en conjonction avec une constante, afin de quantifier la population de bactéries. Selon un procédé de déviation, on transmet un réactif par une ligne de déviation (18) jusqu'à l'unité de mesure électrochimique (15) et on soustrait le signal obtenu du signal généré par le filtrat, une constante étant utilisée pour mettre en corrélation le reste ainsi obtenu avec la numération des bactéries. Selon un procédé à concentrations variables, on utilise une concentration variable de bactéries dans un même fluide afin de quantifier des populations de bactéries. Selon un procédé à échantillon récupéré, ou soumet le filtrat à un deuxième test après un intervalle de temps prédéterminé, ce qui permet de compenser la présence éventuelle de contaminants.According to an electrochemical method for quantifying populations of whole living bacteria, the bacteria are filtered and then the filtrate is used in combination with an electrochemical measuring unit (15) in order to determine the counting density of the bacteria. According to a flow method, the average of the signals is used during the predetermined period of the test duration, in conjunction with a constant, in order to quantify the population of bacteria. According to a deflection method, a reagent is transmitted by a deflection line (18) to the electrochemical measuring unit (15) and the signal obtained is subtracted from the signal generated by the filtrate, a constant being used to set correlation of the remainder thus obtained with the bacteria count. According to a process with variable concentrations, a variable concentration of bacteria is used in the same fluid in order to quantify populations of bacteria. According to a recovered sample process, or subjects the filtrate to a second test after a predetermined time interval, which makes it possible to compensate for the possible presence of contaminants.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US43360789A | 1989-11-07 | 1989-11-07 | |
US433607 | 1995-05-18 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0499624A1 true EP0499624A1 (en) | 1992-08-26 |
EP0499624A4 EP0499624A4 (en) | 1993-03-17 |
Family
ID=23720799
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900917636 Ceased EP0499624A4 (en) | 1989-11-07 | 1990-11-02 | Methods of determining bacteria populations electrochemically |
Country Status (3)
Country | Link |
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EP (1) | EP0499624A4 (en) |
JP (1) | JPH05500311A (en) |
WO (1) | WO1991006670A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU6104994A (en) * | 1993-03-18 | 1994-10-11 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture | Apparatus for the quantitative determination of particulate analytes |
US20220214302A1 (en) * | 2019-05-20 | 2022-07-07 | National Institute For Materials Science | Detection apparatus and data collection method |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4246343A (en) * | 1979-05-02 | 1981-01-20 | University Of Virginia And National Aeronautics & Space Administration | Microbial detection and enumeration method and apparatus |
US4386157A (en) * | 1981-10-26 | 1983-05-31 | Beggs James M Administrator Of | Method for detecting coliform organisms |
US4517291A (en) * | 1983-08-15 | 1985-05-14 | E. I. Du Pont De Nemours And Company | Biological detection process using polymer-coated electrodes |
-
1990
- 1990-11-02 WO PCT/US1990/006507 patent/WO1991006670A1/en not_active Application Discontinuation
- 1990-11-02 EP EP19900917636 patent/EP0499624A4/en not_active Ceased
- 1990-11-02 JP JP50053091A patent/JPH05500311A/en not_active Withdrawn
Non-Patent Citations (3)
Title |
---|
FOOD MICROBIOLOGY vol. 6, no. 3, 1989, USA pages 159 - 169 R.A. PATCHETT ET AL. 'Rapid detection of bateria by an amperometric electrode system - a comparison of some redox mediators.' * |
JOURNAL OF APPLIED BACTERIOLOGY vol. 66, no. 1, 1989, CAMBRIDGE UK pages 49 - 55 R.A. PATCHETT ET AL 'Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods.' * |
See also references of WO9106670A1 * |
Also Published As
Publication number | Publication date |
---|---|
JPH05500311A (en) | 1993-01-28 |
WO1991006670A1 (en) | 1991-05-16 |
EP0499624A4 (en) | 1993-03-17 |
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